Gelatin gel microparticles and method for producing the same

Abstract

To provide a method that can produce gelatin gel microparticles that enables high-efficiency protein encapsulation while maintaining protein activity, ensuring both cost-effectiveness and ease of scale-up.SOLUTION: A method for producing gelatin gel microparticles comprises the steps for: preparing a W / W emulsion composition by emulsifying an aqueous solution containing fish gelatin with a crosslinkable functional group and a nonionic water-soluble polymer; and inducing crosslinking between at least two of the crosslinkable functional groups.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to gelatin gel microparticles and a method for producing the same. [Background technology]

[0002] Protein (enzyme)-encapsulated gel particles are used in biosensors, drug delivery systems, bio-molecular adsorbents, enzyme therapy, industrial enzyme immobilization carriers, etc. However, there are only a limited number of practical gel particles, and there is a demand for the development of novel gel particles that can utilize proteins more effectively.

[0003] Here, methods for producing protein-encapsulating gel particles are known, such as the W / O emulsion method, the W / W emulsion method, the flow reactor method, and the electron spray method (for example, Non-Patent Documents 1 to 4). However, the W / O emulsion method has the problem of protein inactivation due to organic solvents and stirring, and also has limitations on its application to living organisms due to the influence of residual organic solvents. The W / W emulsion method has the problem of protein inactivation due to stirring, as with the W / O emulsion method, and also has low protein encapsulation efficiency, which is a limitation in the use of expensive and rare proteins. On the other hand, the flow reactor method and the electron spray method do not cause inactivation due to organic solvents or stirring and have high encapsulation efficiency, but require relatively complex equipment, which results in high production costs and difficulty in scaling up.

[0004] Thus, until now, there has been no method capable of producing gel microparticles that can encapsulate proteins with high efficiency while maintaining the activity of the proteins, at low cost and with ease of scale-up. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Biomacromolecules 2016, 17, 3619-3631. [Non-Patent Document 2] Progress in Retinal and Eye Research 2014, 42, 27-43. [Non-Patent Document 3] ACS Nano 2015, 9, 6, 5772-5781. [Non-Patent Document 4] ACS Nano 2013, 7, 8, 6758-6766. Summary of the Invention [Problem to be solved by the invention]

[0006] The main object of the present invention is to provide a method for producing gelatin gel microparticles that can encapsulate proteins with high efficiency while maintaining the activity of the proteins, at low cost and with ease of scale-up. [Means for solving the problem]

[0007] The present inventors have conducted extensive research to achieve the above-mentioned object. As a result, they have found that gelatin gel microparticles suitable as a protein stabilizer can be mass-produced at low cost by emulsifying an aqueous solution containing fish gelatin having a crosslinkable functional group and a nonionic water-soluble polymer to obtain a W / W emulsion composition. Based on this finding, the present inventors have conducted further research and have completed the present invention. That is, the present invention includes the following configurations.

[0008] Item 1. Gelatin gel microparticles obtained by crosslinking fish gelatin, Gelatin gel microparticles encapsulating a protein other than the fish gelatin.

[0009] Item 2. The gelatin gel microparticles according to Item 1, having an average particle size of 10 nm to 500 μm.

[0010] Item 3. The gelatin gel microparticle according to Item 1 or 2, which is a stabilizer for the protein.

[0011] Item 4. The gelatin gel microparticle according to Item 1 or 2, which is a reaction field for the protein.

[0012] Item 5. The gelatin gel microparticles according to any one of Items 1 to 4, wherein the protein is an enzyme.

[0013] Item 6. A method for producing gelatin gel microparticles, comprising: A step of obtaining a W / W emulsion composition by emulsifying an aqueous solution containing fish gelatin having a crosslinkable functional group and a nonionic water-soluble polymer; and A method of manufacturing comprising a step of crosslinking at least two of the crosslinkable functional groups.

[0014] Item 7. The method according to item 6, wherein the crosslinkable functional group is an ethylenically unsaturated polymerizable group.

[0015] Item 8. The method according to Item 6 or 7, wherein the nonionic water-soluble polymer is polyvinylpyrrolidone.

[0016] Item 9. The production method according to any one of Items 6 to 8, further comprising a step of contacting the W / W emulsion composition with a protein.

[0017] Item 10. The method according to Item 9, wherein the protein is an enzyme.

[0018] Item 11. A dispersed phase containing fish gelatin having a crosslinkable functional group; a continuous phase comprising a non-ionic water-soluble polymer; A W / W emulsion composition comprising: Effect of the Invention

[0019] According to the method of the present invention, it is possible to provide a method for producing gelatin gel microparticles that can encapsulate proteins with high efficiency while maintaining the activity of the proteins, at low cost and with ease of scale-up.

[0020] According to the present invention, it is possible to provide novel gelatin gel microparticles that can encapsulate a protein with high efficiency while maintaining the activity of the protein. [Brief description of the drawings]

[0021] [Figure 1] FIG. 1H-NMR spectra of various gelatins used in the examples (from the top, Production Example 1 (GelMA-1), Production Example 2 (GelMA-2), Production Example 3 (GelMA-3), and the raw material cold water fish skin gelatin). [Diagram 2] 1 shows the results of size exclusion chromatography of cold water fish skin gelatins, Production Example 1 (GelMA-1), Production Example 2 (GelMA-2), and Production Example 3 (GelMA-3). [Diagram 3] FIG. 1 is a conceptual diagram illustrating a production method of the present invention. [Figure 4] (a) Fluorescence microscopic image of a W / W emulsion composition (WGelMA / WPVP) containing fluorescently labeled methacrylated gelatin (GelMA). (b) Fluorescence microscopic image of a W / W emulsion composition (WGelMA / WPVP) encapsulating FITC-BSA. (c) Histogram showing particle size distribution of gelatin gel microparticles obtained using a glass filter with a pore size of 5 μm or 10 μm. (d) Confocal laser microscope image of FITC-BSA-encapsulated gelatin gel microparticles obtained using a glass filter with a pore size of 5 μm. [Diagram 5] 1 is a histogram showing the particle size distribution of gelatin gel microparticles obtained using a glass filter with a pore size of 5 μm after 0 hours, 1 hour, 3 hours, 6 hours, and 24 hours. [Figure 6](a) Confocal laser microscope images of gelatin gel microparticles encapsulating fluorescently labeled enzymes (from the left, FITC-HRP, RhoB-GOD, or DNS-β-Gal) obtained using a glass filter with a pore size of 5 μm. The scale bar indicates 20 μm. (b) A graph showing the encapsulation efficiency when enzymes (HRP, GOD, β-Gal) are added alone or in combination of three types. (c) Confocal laser microscope images of gelatin gel microparticles encapsulating three types of enzymes (HRP, GOD, and β-Gal) obtained using a glass filter with a pore size of 10 μm (from the left, fluorescent observation images derived from FITC, rhodamine B, and dansyl in the same field of view, and finally a bright field phase contrast image). The scale bar indicates 20 μm. [Figure 7] Results of enzyme stability tests performed at (a) 4°C and (b) 37°C. [Figure 8] FIG. 1 shows a conceptual diagram of the cascade reaction in Example 7 (right side) and a graph showing the results (left side). [Figure 9] 1 is a graph showing the results of a cascade reaction carried out using Production Example 1 (GelMA-1), Production Example 2 (GelMA-2), and Production Example 3 (GelMA-3) as methacrylated gelatins. [Figure 10] FIG. 1 shows a conceptual diagram of the cascade reaction in Example 8 (right side) and a graph showing the results (left side). [Figure 11] 1 is a graph showing the results of Example 12. [Figure 12] Graphs showing the enzyme activity of (a) HRP, (b) GOD, and (c) β-Gal without UV irradiation (open circles) or with UV irradiation (filled circles). [Figure 13] 1 shows a 50 mL tube (left) in which the FITC-BSA-encapsulating gelatin gel microparticles recovered in Example 14 have settled, and a confocal laser microscope image of the microparticles (right; scale bar 20 μm). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0022] In this specification, the term "comprise" is a concept that encompasses all of "comprise," "consist essentially of," and "consist only of."

[0023] In this specification, the expression of a numerical range "A to B" means "greater than or equal to A and less than or equal to B."

[0024] In this specification, "encapsulation" is not limited to the mode in which the encapsulated substance is present inside the microparticles, but is a concept that includes all of adsorption, support, and retention. On the other hand, "encapsulation" does not include the mode in which the encapsulated substance is bound to the microparticles via a covalent bond. It should be noted that "encapsulating" does not prevent the encapsulated substance from being present not only inside the microparticles but also on the surface of the microparticles.

[0025] 1. Gelatin gel particles The gelatin gel microparticles of the present invention include crosslinked fish gelatin. Here, crosslinked fish gelatin means crosslinked fish gelatin (hereinafter, this may be simply referred to as "fish gelatin"). That is, the present invention relates to gelatin gel microparticles formed by crosslinking fish gelatin. It should be noted just for the sake of clarity that the gelatin gel microparticles of the present invention may contain other components than fish gelatin as their constituents. By using fish gelatin as a raw material, the present invention can avoid problems (such as contamination with infectious viruses or infectious peptides) that occur with raw materials derived from mammals such as cows and pigs, and can also be applied to people who cannot touch mammals such as cows and pigs for religious reasons. Furthermore, by using this raw material, the melting point is lower than gelatins derived from other raw materials (such as raw materials derived from mammals such as cows and pigs), so there is an advantage that it is easier to solize at a lower temperature and a W / W emulsion can be formed at a temperature that does not impair the activity of the protein.

[0026] The fish gelatin used in the present invention is a protein constituting collagen obtained from fish and shellfish connective tissues such as fish bones, fish skin, and fish scales. Fish gelatin can be obtained by treating these collagens with known methods such as alkali treatment and acid treatment. Alternatively, commercially available fish gelatin can be used. There is no particular limitation on the fish gelatin, and gelatin derived from any fish and shellfish can be used, such as gelatin derived from mackerel, horse mackerel, cod, sardine, herring, salmon, tilapia, shark, etc., and among them, gelatin derived from cod is preferred from the viewpoint of its low gelling temperature.

[0027] The fish gelatin may be used alone or in combination of two or more kinds.

[0028] The gelling temperature of fish gelatin is not particularly limited and a wide range can be used, but from the viewpoint of the stability and production efficiency of the gelatin gel microparticles or W / W emulsion composition, it is preferably 25° C. or lower, more preferably −5° C. to 20° C., even more preferably 0° C. to 15° C., and particularly preferably 0° C. to 10° C. In such a case, it is advantageous in that gelatin gel microparticles can be produced while maintaining a low temperature that does not damage proteins, and proteins can be encapsulated at the same time, and gelatin gel microparticles that can encapsulate proteins can be used while maintaining a low temperature.

[0029] The number average molecular weight of fish gelatin is not particularly limited, but from the viewpoint of the stability and production efficiency of gelatin gel fine particles or W / W emulsion composition, it is preferably 1,000 to 1,000,000, more preferably 10,000 to 500,000, and even more preferably 50,000 to 100,000. The number average molecular weight is determined by size exclusion chromatography.

[0030] The gelatin gel microparticles of the present invention preferably contain crosslinked fish gelatin via a covalent bond. The crosslinked fish gelatin is not particularly limited as long as it is crosslinked fish gelatin, and may be a chemically crosslinked gel or a physically crosslinked gel. Among them, from the viewpoint of stability, a chemically crosslinked gel is more preferable.

[0031] The crosslinked fish gelatin preferably has a crosslinkable functional group and / or a group formed by crosslinking the crosslinkable functional group. When the fish gelatin has a crosslinkable functional group, crosslinked fish gelatin can be obtained by crosslinking the fish gelatin, and the gelatin gel microparticles of the present invention can be obtained. The fish gelatin may be used by introducing a crosslinkable functional group into the above-mentioned fish gelatin, or a commercially available product into which a crosslinkable functional group has been introduced may be used. Alternatively, a group derived from the fish gelatin itself may be used as the crosslinkable functional group.

[0032] The crosslinkable functional group can be introduced into fish gelatin through a bond with a side chain (e.g., a hydroxy group, an amino group, a carboxy group, a carbonyl group, etc.) of the fish gelatin. The crosslinkable functional group may be directly bonded to the side chain, or may be bonded to the side chain via a linker or a spacer.

[0033] The crosslinkable functional group is not particularly limited as long as it is a group capable of forming a chemical bond between a plurality of crosslinkable functional groups. The crosslinkable functional group may be a group capable of directly forming a chemical bond with another crosslinkable functional group, or a group capable of forming a chemical bond via a crosslinking agent.

[0034] Examples of the crosslinkable functional group include polymerizable groups such as vinyl, allyl, styrene, acrylic, and methacrylic groups, functional groups such as hydroxy, amino, carboxy, thiol, azide, alkynyl, boronic acid, and cholesterol, hydrogen-bonding functional groups such as adenine, thymine, uracil, cytosine, guanine, amide, urea, and thiourea, and metal-bonding functional groups such as pyridine and bipyridyl groups. Among these, the crosslinkable functional group is preferably an ethylenically unsaturated polymerizable group such as vinyl, allyl, acrylic, or methacrylic group, and more preferably an acrylic or methacrylic group.

[0035] The crosslinkable functional group may be used alone or in combination of two or more kinds.

[0036] The introduction rate of the crosslinkable functional group is not particularly limited, and can be appropriately selected from a wide range depending on the desired crosslink density. When introducing a crosslinkable functional group into an amino group that fish gelatin originally has, the introduction rate is preferably 1 mol% to 99 mol%, more preferably 10 mol% to 90 mol%, and even more preferably 50 mol% to 80 mol%, assuming the amount of the amino group to be 100 mol%. The same applies to the case where a side chain other than an amino group (e.g., a hydroxyl group, a carboxyl group, a carbonyl group, etc.) is used.

[0037] The crosslinking for obtaining gelatin gel particles, its mode, conditions and effects will be understood from the explanation of the method for producing gelatin gel particles of the present invention described below.

[0038] The gelatin gel microparticles can also be fluorescently labeled. Fluorescent labeling can be performed using a known fluorescent dye. For example, the fluorescent dye may be introduced into fish gelatin through binding with a side chain such as a hydroxyl group, an amino group, a carboxyl group, or a thiol group of fish gelatin, as with the above-mentioned crosslinkable functional group, or may be introduced through binding with the above-mentioned crosslinkable functional group introduced into fish gelatin.

[0039] The average particle size of the gelatin gel microparticles is not particularly limited, but from the viewpoint of handling, it is preferably 10 nm to 500 μm, more preferably 50 nm to 100 μm, and even more preferably 100 nm to 10 μm. The average particle size is determined by calculating the area of ​​particles with a sphericity (true sphericity) > 0.80 using image analysis software for a microscope image containing 10 or more gel microparticles in one field of view, dividing this by pi, multiplying the square root of the value to obtain the particle size, and calculating the arithmetic average of 100 or more particles. Here, the sphericity (true sphericity) is the value obtained by dividing B by A when the maximum value of a straight line drawn so as to maximize the length passing through the gel microparticles in the microscope image is A, and the maximum value of a straight line drawn so as to maximize the length passing through the gel microparticles among the straight lines perpendicular to the straight line is B.

[0040] The sphericity of the gelatin gel microparticles is not particularly limited, but from the viewpoint of improving strength, it is preferably 0.80 or more, more preferably 0.85 or more, even more preferably 0.90 or more, particularly preferably 0.95 or more, and most preferably 0.98 or more. By definition, the sphericity (sphericity) is a value of 0 or more and 1.0 or less.

[0041] The gelatin gel microparticles of the present invention encapsulate proteins other than fish gelatin. However, in the gelatin gel microparticles of the present invention, in addition to proteins, free fish gelatin may be present inside and / or on the surface of the microparticles.

[0042] In this specification, the term "protein" is not limited by the number of amino acid residues or the presence or absence of a higher-order structure, and is a concept that includes both so-called "oligopeptides" and "polypeptides". The protein used in the present invention is not particularly limited, and any known protein can be adopted. Examples of proteins include enzymes, receptors, antibodies, antigens, vaccines, cytokines such as interferons and interleukins, chemokines, and transport proteins, and among these, enzymes are preferable. In addition, the protein may be complexed by binding with lipids, sugars, nucleic acids, metals, etc. One type of protein may be used alone, or two or more types may be used in combination.

[0043] The mass of the protein is not particularly limited, but from the viewpoint of encapsulation efficiency, it is preferably 1 / kDa to 1,000 / kDa, more preferably 5 / kDa to 500 / kDa, and even more preferably 10 / kDa to 100 / kDa.

[0044] The isoelectric point of the protein is not particularly limited, and proteins having a wide range of isoelectric points can be used.

[0045] The enzyme is not particularly limited, and any known enzyme can be used, for example, hydrolases, transferases, oxidoreductases, lyases, isomerases, synthetases, translocases, etc. Specific examples of the enzyme include horseradish peroxidase, glucose oxidase, β-galactosidase, carbonic anhydrase, malate dehydrogenase, acetylcholinesterase, etc., among which horseradish peroxidase, glucose oxidase, β-galactosidase, and acetylcholinesterase are preferred. Enzymes may be used alone or in combination of two or more.

[0046] The gelatin gel microparticles of the present invention can encapsulate (enclose) a larger amount of protein. This is because when a protein is added to a W / W emulsion composition containing gelatin gel microparticles and a nonionic water-soluble polymer, a larger amount of protein is easily distributed in the gelatin gel microparticles. The amount of protein that can be encapsulated in the gelatin gel microparticles is not particularly limited, but is preferably 0.01% to 90% by mass, more preferably 0.1% to 80% by mass, and even more preferably 1% to 50% by mass, with the mass of the gelatin gel microparticles being 100% by mass. For this reason, the gelatin gel microparticles and W / W emulsion composition of the present invention can be excellent as a protein concentrator (separation and purification agent).

[0047] The gelatin gel microparticles of the present invention can inhibit protein aggregation, inactivation, and denaturation caused by heat, acid, base, light irradiation, and the like, by isolating the encapsulated protein in the nanospace within the microparticles, and can also promote protein refolding. Furthermore, by containing fish gelatin, the gelatin gel microparticles can be produced or used at relatively low temperatures. From the above, the gelatin gel microparticles of the present invention can be excellent as a protein stabilizer (particularly an enzyme stabilizer).

[0048] Furthermore, since the gelatin gel microparticles of the present invention allow the substrate to penetrate to the inside, it is possible to utilize the reaction caused by the protein (particularly, the enzyme) held within the microparticles. In addition, the gelatin gel microparticles of the present invention can encapsulate more protein, can maintain the activity of the protein for a longer period of time, and can easily separate the reaction product. From the above, the gelatin gel microparticles of the present invention can be excellent as a protein reaction field (particularly, an enzyme reaction field). In this case, the reaction caused by the protein may be a single type of reaction, or may be a multi-step continuous reaction (cascade reaction) combining two or more types of proteins.

[0049] The gelatin gel microparticles of the present invention can encapsulate cells as well as biologically active substances such as nucleic acids and drugs in addition to proteins. In this case, the gelatin gel microparticles of the present invention can stabilize the encapsulated substances and maintain their activity for a long period of time for the same reasons as for stabilizing proteins. Therefore, the gelatin gel microparticles of the present invention can also be excellent as drug carriers used for drug delivery.

[0050] The nucleic acid is not particularly limited, and known nucleic acids can be widely used, for example, DNA, RNA, chimeric nucleic acid of DNA and RNA, DNA / RNA hybrid, etc. According to the gelatin gel microparticle of the present invention, even nucleic acids with short half-lives such as siRNA and shRNA can be stabilized. The nucleic acid may be used alone or in combination of two or more kinds.

[0051] The drug (medicine) is not particularly limited, and a wide variety of known drugs can be used. Examples of such drugs include antitumor agents, antihypertensive agents, antihypertensive agents, antipsychotic agents, analgesics, antidepressants, antimanic agents, antianxiety agents, sedatives, hypnotics, antiepileptic agents, opioid agonists, asthma treatment agents, anesthetics, antiarrhythmic agents, arthritis treatment agents, antispasmodics, ACE inhibitors, decongestants, antibiotics, antiangina agents, diuretics, antiParkinson's disease agents, bronchodilators, antidiuretics, diuretics, antihyperlipidemic agents, immunosuppressants, immunomodulators, antiemetics, Examples of the drug include those that can act as an anti-infective agent, antineoplastic agent, an antifungal agent, an antiviral agent, an antidiabetic agent, an antiallergic agent, an antipyretic, an antigout agent, an antihistamine agent, an antipruritic agent, a bone regulator, a cardiovascular agent, a cholesterol lowering agent, an antimalarial agent, an antitussive, an expectorant, a mucolytic agent, an anti-congestive agent, a dopaminergic agent, a gastrointestinal agent, a muscle relaxant, a neuromuscular blocking agent, a parasympathomimetic agent, a prostaglandin, a stimulant, an appetite suppressant, a thyroid agent or an antithyroid agent, a hormone, an antimigraine agent, an anti-obesity agent, an anti-inflammatory agent, etc. One drug (medicine) may be used alone, or two or more drugs may be used in combination.

[0052] The cells are not particularly limited and a wide variety of known cells can be used, including, for example, chondrocytes, osteoblasts, fibroblasts, myoblasts, ligament cells, adipocytes, nerve cells, vascular endothelial cells, smooth muscle cells, cardiac muscle cells, epithelial cells, hepatocytes, pancreatic β cells, kidney cells, bone marrow-derived mesenchymal stem cells, adipose-derived mesenchymal stem cells, neural stem cells, embryonic stem cells, induced pluripotent stem cells, etc. One type of cell may be used alone, or two or more types may be used in combination.

[0053] 2. Manufacturing method of gelatin gel microparticles The method for producing gelatin gel microparticles of the present invention comprises the steps of: (1) a step of obtaining a W / W emulsion composition by emulsifying an aqueous solution containing fish gelatin having a crosslinkable functional group and a nonionic water-soluble polymer; and (2) A step of crosslinking at least two of the crosslinkable functional groups contained in the W / W emulsion composition. The production method of the present invention has such characteristics, and therefore can produce gelatin gel microparticles that can encapsulate a protein with high efficiency while maintaining the activity of the protein, at low cost and with ease of scale-up.

[0054] (1) Emulsification process The aqueous solution containing fish gelatin having a crosslinkable functional group and a nonionic water-soluble polymer can be obtained by mixing an aqueous solution obtained by dissolving fish gelatin having a crosslinkable functional group in an aqueous solvent with an aqueous solution obtained by dissolving a nonionic water-soluble polymer in an aqueous solvent. Alternatively, the aqueous solution can be obtained by dissolving each component (solute) in an aqueous solvent simultaneously or sequentially.

[0055] The fish gelatin used in the method of the present invention can be understood from the above explanation of the "gelatin gel microparticles" of the present invention.

[0056] The nonionic water-soluble polymer used in the method of the present invention is not particularly limited, and a wide variety of known nonionic water-soluble polymers can be used. Examples of nonionic water-soluble polymers include synthetic polymers such as polyvinylpyrrolidone (PVP), polyvinyl alcohol (PVA), polyethylene glycol (PEG), polyacrylamide (PAA), polydimethylacrylamide, and polyvinyl methyl ether (PVME), and natural polymers such as polysaccharides (e.g., tamarind seed gum, guar gum, locust bean gum, starch, pullulan, mannan, dextran, and dextrin).

[0057] The number average molecular weight of the nonionic water-soluble polymer is not particularly limited, but from the viewpoint of the stability and production efficiency of the gelatin gel microparticles or W / W emulsion composition, it is preferably 1,000 to 1,000,000, more preferably 5,000 to 100,000, and even more preferably 10,000 to 100,000. The number average molecular weight is determined by size exclusion chromatography.

[0058] The nonionic water-soluble polymer may be used alone or in combination of two or more kinds.

[0059] The aqueous solvent is not particularly limited as long as it contains water and can dissolve fish gelatin, a nonionic water-soluble polymer, and optionally a protein, and examples thereof include known solvents such as water, saline, Good's buffer (MES, ADA, PIPES, ACES, collamine hydrochloride, BES, TES, HEPES, acetamidoglycine, tricine, glycineamide, bicine), PBS solution (phosphate buffered saline), citrate buffered saline, acetate buffer, etc. Among these, from the viewpoint of maintaining the stability of the protein, water, PBS solution, and citrate buffered saline are preferred, and PBS solution is more preferred.

[0060] The aqueous solution is not particularly limited as long as it does not impair the object of the present invention, and may contain a solvent other than water. However, from the viewpoint of the efficiency of producing the W / W emulsion composition, it is preferable that the aqueous solution does not contain an organic solvent.

[0061] The aqueous solution is not particularly limited as long as it does not impair the object of the present invention, and may contain additives contained in known W / W emulsion compositions. From the viewpoint of the efficiency of producing the W / W emulsion composition, however, it is preferable that the aqueous solution does not contain amphiphilic molecules (e.g., emulsifiers, surfactants, dispersants, etc.) other than the nonionic water-soluble polymer.

[0062] The content of fish gelatin is not particularly limited, and is usually 0.1% by mass or more, with the mass of the aqueous solution being 100% by mass. Among them, the content of fish gelatin is preferably 0.5% by mass to 40% by mass, more preferably 1% by mass to 30% by mass, and even more preferably 1.5% by mass to 20% by mass, with the mass of the aqueous solution being 100% by mass, from the viewpoint of the production efficiency of the W / W emulsion composition.

[0063] The content of the nonionic water-soluble polymer is not particularly limited, and is usually 0.1% by mass or more, with the mass of the aqueous solution being 100% by mass. Among them, the content of the nonionic water-soluble polymer is preferably 0.5% by mass to 20% by mass, more preferably 1% by mass to 15% by mass, and even more preferably 1.5% by mass to 10% by mass, with the mass of the aqueous solution being 100% by mass, from the viewpoint of the production efficiency of the W / W emulsion composition.

[0064] The total content of the fish gelatin and the nonionic water-soluble polymer is not particularly limited and is usually 1% by mass or more, with the mass of the aqueous solution being 100% by mass. Among them, the content of these two components is preferably 3% by mass to 30% by mass, more preferably 5% by mass to 25% by mass, and even more preferably 10% by mass to 20% by mass, with the mass of the aqueous solution being 100% by mass, from the viewpoint of the production efficiency of the W / W emulsion composition.

[0065] The ratio of the content of fish gelatin to the content of nonionic water-soluble polymer (fish gelatin:nonionic water-soluble polymer) is usually 99.9:0.1 to 0.1:99.9 by mass. Among them, the ratio of the content of these two components is preferably 2:1 to 1:10 by mass, more preferably 1:1 to 1:5, and even more preferably 1:2 to 1:3, from the viewpoint of making fish gelatin a dispersed phase.

[0066] The W / W emulsion composition can be obtained by emulsifying the aqueous solution. The emulsification step is not particularly limited and can be carried out by a known emulsification process used for producing W / W emulsion compositions. The emulsification step can be carried out using, for example, a vortex mixer, a stirring or ultrasonic homogenizer, a membrane emulsifier, or a flow reactor, and is preferably carried out by a mild method (for example, a vortex mixer) from the viewpoint of not impairing the properties of gelatin.

[0067] The W / W emulsion composition has the above-mentioned characteristics, and is a two-phase polymer blend (W / W emulsion composition) including a phase (particularly, a dispersed phase) mainly containing fish gelatin having a crosslinkable functional group and a phase (particularly, a continuous phase) mainly containing a nonionic water-soluble polymer. GelMA / W PVP ) can be obtained. In this case, it is advantageous in that gelatin gel microparticles can be produced at low cost while being easy to scale up. In this case, by adding a protein to the composition, a large amount of the protein is distributed in the gelatin phase (dispersed phase). Therefore, according to the method of the present invention, a protein can be encapsulated in the gelatin gel microparticles with high encapsulation efficiency.

[0068] The average particle size of the gelatin phase (dispersed phase) is not particularly limited, but from the viewpoint of handling, it is preferably 10 nm to 500 μm, more preferably 50 nm to 100 μm, and even more preferably 100 nm to 10 μm. In this case, gelatin gel microparticles having the same average particle size can be provided. The method for evaluating the average particle size can be understood from the above explanation of the "gelatin gel microparticles" of the present invention.

[0069] Furthermore, the W / W emulsion composition is highly stable and can maintain its state for a long period of time (preferably 24 hours or more).

[0070] The W / W emulsion composition contains fish gelatin and a nonionic water-soluble polymer in the same content (composition ratio) as the aqueous solution before emulsification. The preferred content range of each component is also the same as that of the aqueous solution.

[0071] The W / W emulsion composition can be homogenized by passing it through a filter such as a ceramic filter (e.g., a glass filter) or a membrane filter. In this case, the particle size of the target gelatin gel microparticles can be made uniform. In addition, the particle size of the target gelatin gel microparticles can be adjusted by appropriately selecting the pore size of the filter.

[0072] By contacting the W / W emulsion composition with a protein, the protein can be encapsulated in the gelatin gel microparticles, which are the target product. The contact may be performed by mixing the W / W emulsion composition obtained after the emulsification treatment with the protein using a known mixing method, as long as the W / W emulsion composition and the protein are ultimately present in the same system. The contact may be performed, for example, by mixing the aqueous solution and the protein using a known mixing method before subjecting the aqueous solution to the emulsification treatment, and emulsifying the resulting mixture. From the viewpoint of maintaining the activity of the protein, the contact is preferably performed after the emulsification treatment.

[0073] The protein may be contacted with the W / W emulsion composition in the form of a dry powder or an aqueous solution.

[0074] The proteins that can be used in the method of the present invention can be understood from the above explanation of the "gelatin gel microparticles" of the present invention.

[0075] The amount of protein added is not particularly limited, and is usually 0.001% by mass to 1000% by mass, preferably 0.005% by mass to 100% by mass, more preferably 0.01% by mass to 30% by mass, and even more preferably 0.1% by mass to 10% by mass, assuming the amount of fish gelatin in the W / W emulsion composition to be 100% by mass.

[0076] The encapsulation efficiency is not particularly limited, and is usually 40% by mass or more, preferably 60% by mass or more, more preferably 70% by mass or more, even more preferably 80% by mass or more, and particularly preferably 90% by mass or more, assuming the amount of protein added to be 100% by mass.

[0077] According to the method of the present invention, in addition to proteins, physiologically active substances such as nucleic acids and drugs, as well as cells, can be encapsulated in gelatin gel microparticles.

[0078] (2) Crosslinking process The gelatin gel particles can be obtained by crosslinking at least two crosslinkable functional groups contained in the W / W emulsion composition. The gelatin gel particles can be gelled by the crosslinking.

[0079] The crosslinkable functional group used in the method of the present invention can be understood from the above explanation of the "gelatin gel fine particles" of the present invention.

[0080] The crosslinking step is not particularly limited, and can be carried out, for example, by using a crosslinking agent. The crosslinking agent is not particularly limited as long as it has a functional group reactive with the crosslinkable functional group, and can be, for example, a known polyfunctional compound having two or more reactive groups such as a hydroxy group, an amino group, a carboxy group, a thiol group, an azide group, an alkynyl group, an acryl group, or a methacryl group.

[0081] In addition, when the crosslinkable functional group is an anionic functional group such as a hydroxyl group or a carboxyl group, Pb 2+ , Cu 2+ , Cd 2+, B.A. 2+ , Sr 2+ , Ca 2+ , Co 2+ , Ni 2+ , Zn 2+ , Mn 2+ , or Mg 2+ By using a divalent metal ion such as, for example, 1,2-divalent 1,4-diaminetetraacetic acid, 1,5-diaminetetraacetic acid, 1,6-diaminetetraacetic acid, 1,7-diaminetetraacetic acid, 1,8-diaminetetraacetic acid, 1,9 ...

[0082] The crosslinking step is not particularly limited, and can be carried out, for example, by thermal crosslinking, photocrosslinking, or by adding a condensing agent. From the viewpoint of the stability of the microparticles themselves and the protein that can be encapsulated in the microparticles, it is preferable to carry out the crosslinking by photocrosslinking. In this case, a known polymerization initiator according to the type of crosslinkable functional group can be widely used. When the crosslinkable functional group is an unsaturated polymerizable group such as an acrylic group, a methacrylic group, a vinyl group, or an allyl group, the polymerization initiator is preferably, for example, a radical polymerization initiator (particularly, a photopolymerization initiator).

[0083] Alternatively, when the crosslinkable functional groups are thiol, azide, vinyl and / or alkynyl groups, crosslinking may be carried out by a click reaction.

[0084] The form of crosslinking is not particularly limited, and may be either intramolecular crosslinking or intermolecular crosslinking.

[0085] The above W / W emulsion composition and the above aqueous solution, which is its precursor, can contain a crosslinking agent, a radical polymerization initiator (particularly a photopolymerization initiator), and a polymerization aid at any timing depending on the form of crosslinking.

[0086] The photopolymerization initiator is not particularly limited as long as it is a water-soluble compound that absorbs energy rays such as light and generates radicals. Examples of the photopolymerization initiator include alkylphenone-based photopolymerization initiators such as α-hydroxyalkylphenone and lithium phenyl (2,4,6-trimethylbenzoyl) phosphinate, acylphosphine oxide-based photopolymerization initiators, and titanocene-based photopolymerization initiators. Among them, alkylphenone-based photopolymerization initiators are preferred, 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone and lithium phenyl (2,4,6-trimethylbenzoyl) phosphinate are more preferred, and lithium phenyl (2,4,6-trimethylbenzoyl) phosphinate is most preferred from the viewpoint of biocompatibility. As the photopolymerization initiator, a commercially available product Irgacure 2959 (alkylphenone-based photopolymerization initiator, manufactured by BASF Japan Ltd.) can also be used. The photopolymerization initiator may be used alone or in combination of two or more types.

[0087] The polymerization aid is not particularly limited, and a wide variety of known polymerization aids can be used. Examples of the polymerization aid include mercaptans, alcohols such as methanol, isopropanol, and glycerin, and amines such as triethanolamine. The polymerization aid may be used alone or in combination of two or more kinds.

[0088] The contents of the crosslinking agent, radical polymerization initiator (particularly, photopolymerization initiator), and polymerization aid are not particularly limited and can be appropriately set. Among them, the contents of these additives are, for example, usually 0% by mass to 5% by mass, preferably 0% by mass to 1% by mass, and more preferably 0% by mass to 0.5% by mass, relative to 100% by mass of the W / W emulsion composition.

[0089] Photocrosslinking (particularly, photopolymerization) can be carried out by, for example, irradiating the W / W emulsion composition with visible light or ultraviolet light using an ultraviolet light irradiation device. Photocrosslinking (particularly, photopolymerization) can be carried out by, for example, irradiating ultraviolet light at 0.32000 mW / cm 2 ~2000mW / cm 2From the viewpoint of not impairing the activity of the protein, it is preferable that the light irradiation time and light intensity are as short as possible within a range in which gelation can be achieved.

[0090] After crosslinking, the obtained gelatin gel microparticles can be washed and the nonionic water-soluble polymer can be removed. Washing (removal) can be performed by recovering the gelatin gel microparticles using a known separation and washing method such as centrifugation, filtration, dialysis, etc. In this case, in addition to the nonionic water-soluble polymer, optional components such as polymerization initiators, polymerization aids, and metal ions can be removed, and therefore the gelatin gel microparticles can be isolated.

[0091] If necessary, the gel particles may be classified by a method such as filtration or centrifugation in order to make the particle size of the gel particles uniform.

[0092] The yield of the method of the present invention is not particularly limited, and is preferably 90% or more, more preferably 95% or more, even more preferably 99% or more, and particularly preferably 100%. According to the method of the present invention, gelatin gel microparticles can be produced in a high yield by using a W / W emulsion composition.

[0093] From the viewpoint of stability, the method of the present invention is preferably carried out at a low temperature, more preferably the step using the protein is carried out at a low temperature, and even more preferably all steps are carried out at a low temperature. The low temperature is preferably 30°C or lower, more preferably 0 to 25°C, even more preferably 2 to 20°C, and particularly preferably 2 to 10°C. In the conventional method, it is necessary to carry out the method at a relatively high temperature in order to obtain an emulsion composition, which leads to a decrease in the activity of the gel particles and the proteins contained therein. On the other hand, in the method of the present invention, by combining fish gelatin having a low gelling temperature with a mild operation, it is possible to produce and use gelatin gel particles while maintaining the activity of the gel particles and the proteins that can be contained therein.

[0094] According to the method of the present invention, gelatin gel microparticles can be produced in a short time. The production time is not particularly limited, but is usually less than 24 hours, preferably less than 1 hour, and more preferably less than 30 minutes.

[0095] 3. W / W Emulsion Composition The W / W emulsion composition of the present invention comprises a dispersed phase containing fish gelatin having crosslinkable functional groups and a continuous phase containing a nonionic water-soluble polymer. That is, in the W / W emulsion composition of the present invention, an aqueous solution containing fish gelatin having crosslinkable functional groups (dispersed phase) is dispersed in an aqueous solution containing a nonionic water-soluble polymer (continuous phase).

[0096] The composition, its aspects, its conditions, and its effects can be understood from the above explanation of the production method for the "gelatin gel fine particles of the present invention."

[0097] The content of fish gelatin is not particularly limited, and is usually 0.1% by mass or more, with the mass of the W / W emulsion composition being 100% by mass. Among them, the content of fish gelatin is preferably 0.5% by mass to 40% by mass, more preferably 1% by mass to 30% by mass, and even more preferably 1.5% by mass to 20% by mass, with the mass of the W / W emulsion composition being 100% by mass, from the viewpoint of the production efficiency of the W / W emulsion composition.

[0098] The content of the nonionic water-soluble polymer is not particularly limited, and is usually 0.1% by mass or more, with the mass of the W / W emulsion composition being 100% by mass. Among them, the content of the nonionic water-soluble polymer is preferably 0.5% by mass to 20% by mass, more preferably 1% by mass to 15% by mass, and even more preferably 1.5% by mass to 10% by mass, with the mass of the W / W emulsion composition being 100% by mass, from the viewpoint of the production efficiency of the W / W emulsion composition.

[0099] The total content of fish gelatin and nonionic water-soluble polymer is not particularly limited and is usually 1% by mass or more, with the mass of the W / W emulsion composition being 100% by mass. Among them, the content of these two components is preferably 3% by mass to 30% by mass, more preferably 5% by mass to 25% by mass, and even more preferably 10% by mass to 20% by mass, with the mass of the W / W emulsion composition being 100% by mass, from the viewpoint of the production efficiency of the W / W emulsion composition.

[0100] The ratio of the content of fish gelatin to the content of nonionic water-soluble polymer (fish gelatin:nonionic water-soluble polymer) is usually 99.9:0.1 to 0.1:99.9 by mass. Among them, the ratio of the content of these two components is preferably 2:1 to 1:10 by mass, more preferably 1:1 to 1:5, and even more preferably 1:2 to 1:3, from the viewpoint of making fish gelatin a dispersed phase.

[0101] The W / W emulsion composition of the present invention can be obtained, for example, by emulsifying an aqueous solution containing fish gelatin having a crosslinkable functional group and a nonionic water-soluble polymer.

[0102] The fish gelatin, nonionic water-soluble polymer, and aqueous solvent used in the method of the present invention can be understood from the above explanation of the "gelatin gel microparticles" and "method for producing gelatin gel microparticles" of the present invention.

[0103] The W / W emulsion composition of the present invention may contain, in addition to the above-mentioned components, the above-mentioned encapsulated substances (for example, proteins, nucleic acids, drugs, cells, etc.), solvents, and additives.

[0104] The average particle size of the dispersed phase is not particularly limited and can be appropriately adjusted by emulsification treatment in the production process or homogenization treatment after production. From the viewpoint of handleability, it is preferably 10 nm to 500 μm, more preferably 50 nm to 100 μm, and even more preferably 100 nm to 10 μm.

[0105] When the W / W emulsion composition of the present invention comes into contact with a protein, it can distribute a large amount of the protein into the gelatin phase (dispersed phase). Therefore, the protein can be concentrated by contacting the W / W emulsion composition of the present invention with the protein. When the W / W emulsion composition of the present invention coexists with a protein, the proportion of the protein present in the dispersed phase is usually 40% by mass or more, preferably 60% by mass or more, more preferably 70% by mass or more, even more preferably 80% by mass or more, and particularly preferably 90% by mass or more, based on the total amount of protein in the composition being 100% by mass.

[0106] When the W / W emulsion composition of the present invention coexists with a protein, it can isolate the protein in the nanospace within fish gelatin, thereby suppressing protein aggregation, inactivation, and denaturation caused by heat, acid, base, light irradiation, etc., and can also promote protein refolding. Therefore, by allowing the W / W emulsion composition of the present invention to coexist with a protein, the protein can be stabilized.

[0107] The concentration and stabilization are preferably carried out at 30°C or lower, more preferably at 0°C to 25°C, even more preferably at 2°C to 20°C, and particularly preferably at 2°C to 10°C.

[0108] 4. Protein Concentration Methods The method for concentrating (purifying) a protein of the present invention comprises the step of contacting the above-mentioned W / W emulsion composition with a protein.

[0109] The concentration method, its aspects, its conditions, and its effects can be understood from the above explanation of the "W / W emulsion composition" of the present invention.

[0110] The protein concentration method of the present invention may be carried out in combination with a known protein concentration method.

[0111] 5. Protein stabilization methods The method for stabilizing (preserving) a protein of the present invention comprises the step of causing the above-mentioned W / W emulsion composition to coexist with a protein.

[0112] The stabilization method, its aspects, its conditions, and its effects can be understood from the above explanation of the "W / W emulsion composition" of the present invention.

[0113] The method for stabilizing a protein of the present invention may be carried out in combination with a known method for stabilizing a protein. EXAMPLES

[0114] EXAMPLES Hereinafter, examples and comparative examples will be shown to more clearly illustrate the features of the present invention, but the present invention is not limited to the following examples.

[0115] To simplify the description in the specification, the following abbreviations may be used in the examples: Abbreviations: FITC: fluorescein isothiocyanate, RhoB: rhodamine B, DNS: dansyl group, NHS: N-hydroxysuccinimide, BSA: bovine serum albumin, HRP: horseradish peroxidase, GOD: glucose oxidase, β-Gal: β-D-galactosidase, PVP: polyvinylpyrrolidone.

[0116] Cold water fish skin gelatin, FITC (purity 95%), RhoB-isothiocyanate (purity ~100%), DNS chloride (purity 95%), FITC-BSA (quality 200), and BSA (purity 95%) were purchased from Sigma-Aldrich Co. LLC (USA). Methacrylic anhydride (purity 97.0%) and deuterium oxide (purity 99.8%) were purchased from Tokyo Chemical Industry Co., Ltd. HRP (100 units / mg or more), GOD from Aspergillus niger (150-250 units / mg), β-Gal from Escherichia coli (500 units / mg or more), boronic acid (99.5% purity), potassium chloride (99.5% purity), hydrogen chloride, lithium acetate (98.0% purity), ninhydrin (98.0% purity), hydrindantin (~100% purity), and sodium hydroxide (97.0% purity) were purchased from Wako Pure Chemical Industries, Ltd. PD-10 columns were purchased from Global Life Sciences Solutions USA LLC (Cytiva). All starting materials and solvents were used without further purification.

[0117] (Nuclear Magnetic Resonance (NMR) Spectroscopy) ECZ400S (JEOL Ltd.) 1 H-NMR spectra were obtained using DO as the solvent and chemical shifts are reported in ppm relative to the residual solvent signal.

[0118] (molecular weight measurement) Molecular weight distribution (MWD) curve, number average molecular weight (M n ) and polydispersity index (D M ) was analyzed by elution through two linear PVA gel columns (Shodex SB-803HQ: exclusion limit 1 × 10) at 25 °C (flow rate = 0.65 mL / min) in acetate buffered saline containing NaOAc (100 mM), NaCl (300 mM), and EDTA·2Na (1 mM). 5 g / mol, particle size 6 μm, pore size N / A, inner diameter 8.0 mm x 30 cm; Shodex SB-806M HQ: exclusion limit 2 x 10 7The measurements were performed by size exclusion chromatography using an HPLC system (JASCO Corporation) equipped with a column (M g / mol, particle size 13 μm, pore size N / A, inner diameter 8.0 mm × 30 cm), a pump (PU-2080Plus), a column oven (CO-2060Plus), and a differential refractive index detector. w =7.50×10 2 ~5.80×10 6 Calibration was performed using six polyethylene glycol standards in the g / mol range.

[0119] (Measurement of crosslinking functional group introduction rate) The introduction rate of crosslinkable functional groups into gelatin was evaluated by the amount of amino groups remaining in gelatin. Ninhydrin (334 mg, 2.24 mmol) and hydrindantin (60.6 mg, 0.188 mmol) were dissolved in dimethyl sulfoxide (15 mL; deoxygenated). Aqueous lithium acetate solution (5 mL, 4 M) was added to the ninhydrin solution. Cold water fish skin gelatin was dissolved in ultrapure water at concentrations of 1.4, 1.2, 1.0, 0.8, 0.6, and 0.4 g / L to prepare a calibration curve. 200 μL of gelatin solution and 200 μL of ninhydrin solution were mixed and kept at 95 °C for 10 minutes, after which a mixed solvent of ethanol and water (volume ratio of ethanol:water = 1:1) was added and cooled in an ice bath. The absorbance at 570 nm was measured using a UV-Vis spectrophotometer (V-730, JASCO Corporation), and a calibration curve of gelatin concentration and absorbance was created. Aqueous gelatin solutions were also evaluated in the same manner, and the remaining amino groups in the gelatin were calculated from the calibration curve.

[0120] [Production Example 1: Methacrylated gelatin (GelMA-1)] Cold water fish skin gelatin (6.00 g) was completely dissolved in 20 mL of pure water. While cooling in an ice bath at 0°C, methacrylic anhydride (5.16 g, 33.4 mmol) was added dropwise, and the resulting solution was vigorously stirred at room temperature for 1 hour. The resulting solution was sealed in a dialysis membrane with a molecular weight cut-off of 3500 Da, and dialyzed against pure water to remove excess methacrylic anhydride and by-product methacrylic acid. After freeze-drying, solid methacrylated gelatin (GelMA-1) was obtained. The introduction rate of methacrylic groups was 72% relative to the amino groups in the gelatin. As shown in Figure 1, 1 The presence of vinyl groups was confirmed from the H-NMR spectrum. The results of size exclusion chromatography shown in Figure 2 show that the weight average molecular weight M w It was also confirmed that the molecular weight was maintained at 46,000. These results were also the same for Production Examples 2 and 3 described below.

[0121] [ka]

[0122] [Production Example 2: Methacrylated gelatin (GelMA-2)] Except for changing the amount of methacrylic anhydride to 2.52 g (16.3 mmol), methacrylated gelatin (GelMA-2) was obtained in the same manner as in Production Example 1. The introduction rate of methacryl groups was 57%. 1 The H-NMR spectrum is shown in FIG. 1, and the results of size exclusion chromatography are shown in FIG.

[0123] [Production Example 3: Methacrylated gelatin (GelMA-3)] Except for changing the amount of methacrylic anhydride to 1.26 g (8.16 mmol), methacrylated gelatin (GelMA-3) was obtained in the same manner as in Production Example 1. The introduction rate of methacryl groups was 40%. 1 The H-NMR spectrum is shown in FIG. 1, and the results of size exclusion chromatography are shown in FIG.

[0124] [Manufacturing example 4: FITC-HRP] HRP (10.0 mg) and FITC (1.2 mg, 3.1 μmol) were dissolved in a solution (50 mM, 1.5 mL, pH = 8.2), and the mixture was stirred for 1 h at 25 °C. Then, the FITC-labeled HRP was purified with a PD-10 column and stored at 4 °C.

[0125] [Manufacture example 5: RhoB-GOD] GOD (10.0 mg) and RhoB-isothiocyanate (1.4 mg, 2.9 μmol) were dissolved in a solution (50 mM, 1.5 mL, pH = 8.2), and the mixture was stirred for 1 h at 25 °C. Then, Rhodamine B-labeled GOD was purified on a PD-10 column and stored at 4 °C.

[0126] [Manufacture example 6: DNS-β-Gal] β-Gal (10.0 mg) and dansyl chloride (1.0 mg, 1.3 μmol) were dissolved in a solution (50 mM, 1.5 mL, pH = 8.2), and the mixture was stirred for 1 h at 25 °C. Then, the DNS-labeled β-Gal was purified on a PD-10 column and stored at 4 °C.

[0127] (Particle size measurement) The average particle size of gelatin gel microparticles was determined by image analysis using fluorescence microscope images. Image analysis was performed using ImageJ software by converting the original images to 8-bit black and white images, adjusting the auto-threshold, and running a particle analysis program for particles with a sphericity of >0.90. The particle size was calculated using the following formula:

[0128]

number

[0129] [In the formula, D represents the diameter of the microparticle, and A represents the spherical area of ​​the microparticle.] Furthermore, a particle size (diameter) histogram of the gelatin gel microparticles was created using MATLAB (registered trademark) (version: R2022a).

[0130] (Fluorescence microscopy) The measurement sample was applied to a glass dish (Iwaki Co., Ltd.). The observation image of the gelatin gel microparticles was obtained using a fluorescence microscope (BZ-X810, Keyence Co., Ltd.) with the excitation wavelength set to FITC and the objective lens magnification set to 40x or 100x.

[0131] (Confocal Laser Scanning Microscope (CLSM) Observation) Gelatin gel microparticles encapsulating fluorescent enzymes (FITC-HRP, Rhodamine B-GOD, and / or DNS-β-Gal) were placed on a glass dish. Images of the gelatin gel microparticles were obtained using a confocal laser microscope (Eclipse Ti; Nikon Corporation) with an excitation wavelength set to 488, 561, or 640 nm and an objective lens magnification set to 60x.

[0132] (Enzyme entrapment efficiency measurement) In the method for producing gelatin gel microparticles described later, the emulsion composition before passing through a glass filter was centrifuged at 10°C to separate the gelatin phase and the PVP phase. The amount of enzyme present in each phase was evaluated by measuring the fluorescence intensity of each phase using a spectrofluorophotometer (FP-8350, JASCO Corporation). The enzyme encapsulation (encapsulation) efficiency was defined as the ratio of the amount of enzyme present in the gelatin phase to the total amount of enzyme present in each phase (i.e., the amount of enzyme added). FITC-BSA and FITC-HRP were measured at an excitation wavelength (Ex): 494 nm and a fluorescence wavelength (Em): 420 nm. RhoB-GOD was measured at Ex: 550 nm and Em: 580 nm. DNS-β-Gal was measured at Ex: 485 nm and Em: 340 nm. The slit width was set to 5 nm for the excitation wavelength and 5 nm for the fluorescence wavelength.

[0133] (Enzyme stability test) The gelatin gel microparticles encapsulating the enzyme were shaken for 5 or 14 days in a PBS solution at 4° C. or 37° C. After a certain period of time had passed, the activity of the enzyme contained in the gelatin gel microparticles was evaluated.

[0134] [Example 1: Gelatin gel microparticles] Gelatin gel microparticles not encapsulating an enzyme were produced (Figure 3). A PBS solution of GelMA-1 (225 μL, 20 wt%), PVP (900 μL, 10 wt%), 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone (9 μL, 16 mM) and triethanolamine (13.5 μL, 4 M) was added to a glass tube and mixed vigorously at 25°C for 30 seconds to obtain a W / W emulsion composition (W GelMA / W PVP ) was obtained. This W / W emulsion composition was passed through a glass filter with a pore size of 5 μm or 10 μm. The W / W emulsion composition was irradiated with ultraviolet light for 20 seconds using a Xe lamp (UI-502Q, Ushio Inc.) equipped with a 360 nm (±40 nm) sharp cut filter. The PVP solution of the gelatin gel microparticles was diluted with 1 mL of pure water, and the gelatin gel microparticles were collected by centrifugation at 30,000 G at 10° C. using a centrifuge (Allegra X-30, Beckman Coulter). The collected microparticles were redispersed in pure water to wash out the PVP, and centrifuged again. This operation was repeated three times.

[0135] [Example 2: FITC-BSA-encapsulated gelatin gel microparticles] Next, enzyme-encapsulated gelatin gel microparticles were produced (Figure 3). Gelatin gel microparticles encapsulating FITC-labeled BSA were obtained in the same manner as in Example 1, except that an aqueous enzyme (FITC-BSA) solution (13.5 μL, 5 mg / mL) was added to the emulsion composition immediately after passing it through the glass filter, and the emulsion composition was mixed for 10 minutes by shaking the glass tube.

[0136] A W / W emulsion composition (W) containing fluorescently labeled methacrylated gelatin (GelMA) GelMA / W PVP ), and a W / W emulsion composition containing FITC-BSA (W GelMA / W PVP Fluorescence microscopy images of the gelatin-containing gelatin solution are shown in Figures 4(a) and 4(b). These results indicate that the enzyme is colocalized with gelatin, i.e., the enzyme is well distributed in the gelatin phase.

[0137] The particle size distribution of the FITC-BSA-encapsulated gelatin gel microparticles obtained using a glass filter and the confocal laser microscope images are shown in Figures 4(c) and 4(d). From these results, and in comparison with Figures 4(a) and 4(b), it can be seen that by passing a W / W emulsion composition through a glass filter having a specified pore size, it is possible to uniformly adjust the particle size within a desired range. The same result was obtained for the gelatin gel microparticles of Example 1.

[0138] Moreover, the particle size distribution shown in FIG. 4(c) remained stable for at least 24 hours (FIG. 5).

[0139] The encapsulation efficiency of the enzyme (FITC-BSA) was 92% (Figure 6(b)). This indicates that most of the enzyme that came into contact with the W / W emulsion composition was efficiently distributed into the gelatin phase.

[0140] [Example 3: FITC-HRP-encapsulated gelatin gel microparticles] Gelatin gel microparticles containing FITC-labeled HRP were obtained in the same manner as in Example 1, except that an aqueous enzyme (FITC-HRP) solution (13.5 μL, 5 mg / mL) was added to the emulsion composition immediately after passing it through the glass filter, and the emulsion composition was mixed for 10 minutes by shaking the glass tube.

[0141] The image observed by a fluorescent microscope is shown in Figure 6(a). The encapsulation efficiency of the enzyme was 70% (Figure 6(b)). In other words, it can be said that most of the enzyme that came into contact with the W / W emulsion composition was efficiently distributed into the gelatin phase.

[0142] [Example 4: RhoB-GOD-encapsulated gelatin gel microparticles] Gelatin gel microparticles encapsulating rhodamine B-labeled GOD were obtained in the same manner as in Example 2, except that RhoB-GOD was used as the enzyme.

[0143] The image observed by a fluorescent microscope is shown in Figure 6(a). The encapsulation efficiency of the enzyme was 66% (Figure 6(b)). In other words, it can be said that most of the enzyme that came into contact with the W / W emulsion composition was efficiently distributed into the gelatin phase.

[0144] [Example 5: DNS-β-Gal-encapsulated gelatin gel microparticles] Gelatin gel microparticles encapsulating DNS-labeled β-Gal were obtained in the same manner as in Example 2, except that DNS-β-Gal was used as the enzyme.

[0145] The image observed by a fluorescent microscope is shown in Figure 6(a). The encapsulation efficiency of the enzyme was 71% (Figure 6(b)). In other words, it can be said that most of the enzyme that came into contact with the W / W emulsion composition was efficiently distributed into the gelatin phase.

[0146] [Example 6: Gelatin gel microparticles encapsulating FITC-HRP, RhoB-GOD and DNS-β-Gal] Gelatin gel microparticles encapsulating three types of fluorescently labeled enzymes were obtained in the same manner as in Example 2, except that an FITC-HRP aqueous solution (9.0 μL, 5 mg / mL), a RhoB-GOD aqueous solution (9.0 μL, 5 mg / mL), and a DNS-β-Gal aqueous solution (9.0 μL, 5 mg / mL) were added as the enzyme aqueous solutions.

[0147] The confocal laser microscope image is shown in FIG. 6(c). From the images obtained in the same field of view, it can be seen that the three types of enzymes are colocalized with the gelatin gel microparticles, that is, the enzymes are well distributed in the gelatin phase. The enzyme encapsulation efficiency was FITC-HRP: 45%, RhoB-GOD:, and DNS-β-Gal: 95% (FIG. 6(b)). In other words, it can be said that most of the enzymes that came into contact with the W / W emulsion composition were efficiently distributed in the gelatin phase. In particular, DNS-β-Gal was encapsulated in the microparticles with a higher efficiency than when it was used alone. This suggests the possibility that some kind of interaction exists between the gelatin gel microparticles of the present invention and the enzymes.

[0148] [Example 7: Gelatin gel microparticles encapsulating HRP and GOD] Gelatin gel microparticles encapsulating two types of enzymes were obtained in the same manner as in Example 2, except that an aqueous enzyme solution was added consisting of an aqueous HRP solution (13.5 μL, 5 mg / mL) and an aqueous GOD solution (13.5 μL, 5 mg / mL).

[0149] (Cascade reaction using gelatin gel particles) The obtained enzyme-encapsulated gelatin gel microparticles were used as an enzyme reaction field. The enzyme-encapsulated gelatin gel microparticles were suspended in 1315 μL of citrate-buffered saline at pH=5.35. The final concentration of HRP was 1.17 μg / mL, and the final concentration of GOD was 1.24 μg / mL. A citrate-buffered saline solution of o-phenylenediamine (OPD) (75 μL, 2.78 mM) was added to the suspension. After the cuvette was stabilized at 30°C for 15 minutes, a D-glucose solution (100 μL, 1.00 M) was added to the cuvette. The absorbance at 440 nm was measured with a UV-Vis spectrophotometer (V-730, JASCO Corporation) to track the reaction. As a positive control, the enzyme alone was used instead of the enzyme-encapsulated gelatin gel microparticles.

[0150] The change in absorbance over time is shown in Figure 8. When gelatin gel particles not containing enzyme were used, the absorbance did not change, whereas when enzyme-encapsulated gelatin gel particles with an average particle diameter of 6.2 μm were used, the absorbance increased over time. In other words, a multi-stage continuous reaction (cascade reaction) by two types of enzymes encapsulated in the gelatin gel particles proceeded. Furthermore, when enzyme-encapsulated gelatin gel particles with an average particle diameter of 2.3 μm were used, the absorbance increased 80% faster. Considering that particles with an average particle diameter of 2.3 μm have a surface area five times that of particles with an average particle diameter of 6.2 μm, this suggests that the substrate and product of the enzyme reaction are diffused within the gelatin gel particles.

[0151] In addition, to examine the effect of the crosslink density in the gelatin gel microparticles, the methacrylated gelatin (GelMA-1) in Production Example 1 was replaced with Production Example 2 (GelMA-2) or Production Example 3 (GelMA-3), and a similar cascade reaction was carried out. These microparticles had similar properties (e.g., encapsulation efficiency) except for the different crosslink densities. As a result, it was found that the lower the crosslink density in the gelatin gel microparticles, the more the cascade reaction was promoted (Figure 9). This supports the idea that diffusion in the gelatin gel microparticles can contribute to the reaction rate.

[0152] [Example 8: Gelatin gel microparticles encapsulating HRP, GOD and β-Gal] Gelatin gel microparticles encapsulating three types of enzymes were obtained in the same manner as in Example 2, except that an HRP aqueous solution (9.0 μL, 5 mg / mL), a GOD aqueous solution (9.0 μL, 5 mg / mL), and a β-Gal aqueous solution (9.0 μL, 5 mg / mL) were added as the enzyme aqueous solutions.

[0153] (Cascade reaction using gelatin gel particles) The obtained enzyme-encapsulated gelatin gel microparticles were used as an enzyme reaction field. The enzyme-encapsulated gelatin gel microparticles were suspended in 1115 μL of phosphate buffered saline at pH 6.60. The final concentrations of HRP, GOD, and β-Gal were 0.51 μg / mL, 0.65 μg / mL, and 1.08 μg / mL, respectively. A phosphate buffer solution of OPD (75 μL, 2.78 mM) was added to the suspension. After the cuvette was stabilized at 30°C for 15 minutes, a lactose solution (300 μL, 0.33 M) was added to the cuvette. The absorbance at 440 nm was measured using a UV-Vis spectrophotometer (V-730, JASCO Corporation) to track the reaction.

[0154] The results are shown in Figure 10. Although the optimum pH for HRP was 6.0 to 6.5, the optimum pH for GOD was 5.5 to 6.0, and the optimum pH for β-Gal was 6.7 to 7.2, the cascade reaction by the three types of enzymes encapsulated in the gelatin gel microparticles proceeded efficiently.

[0155] Example 9: Effect of pH The relationship between the enzyme encapsulation efficiency and pH was examined. FITC-labeled BSA, HRP, and GOD were used as enzymes. The encapsulation and encapsulation amount of each enzyme were evaluated in the same manner as in the above-mentioned Examples, except that the pH value was adjusted to 7.4 or 5.4. The enzyme encapsulation efficiency under each condition is shown in Table 1. IEP indicates the isoelectric point of each enzyme. The results in Table 1 show that the enzyme could be encapsulated with good efficiency at any pH.

[0156] [Table 1]

[0157] [Example 10: Effect of the mixing ratio of GelMA and PVP (GelMA / PVP)] The relationship between the enzyme encapsulation efficiency and the mixing ratio of GelMA and PVP was examined. FITC-labeled BSA, HRP, and GOD were used as enzymes. The encapsulation and encapsulation amount of each enzyme were evaluated in the same manner as in the above-mentioned Examples, except that the mixing ratio of GelMA and PVP (GelMA / PVP) was adjusted to 4 / 6, 3 / 7, or 2 / 8. The enzyme encapsulation efficiency under each condition is shown in Table 2. From the results in Table 2, it can be said that the encapsulation efficiency tends to improve as the proportion of GelMA in the composition decreases.

[0158] [Table 2]

[0159] [Example 11: Effect of enzyme concentration] The relationship between the enzyme encapsulation efficiency and the enzyme addition concentration was examined. FITC-labeled BSA, HRP, and GOD were used as enzymes. The encapsulation and encapsulation amount of each enzyme was evaluated in the same manner as in the above-mentioned Examples, except that an enzyme aqueous solution with an enzyme concentration of 50 μg / mL, 100 μg / mL, or 150 μg / mL was added to the emulsion composition immediately after passing through a glass filter. The enzyme encapsulation efficiency under each condition is shown in Table 3. From the results in Table 3, the enzyme could be encapsulated with high efficiency at any enzyme addition concentration.

[0160] [Table 3]

[0161] [Example 12: Enzyme stabilization against acid] Gelatin gel microparticles were used as a protein stabilizer. Enzyme gelatin gel microparticles containing β-Gal as an enzyme were shaken in citrate phosphate buffer (pH = 6.0) at 37°C for 24 hours. After a certain period of time, the activity of the enzyme contained in the gelatin gel microparticles was evaluated.

[0162] The enzyme activity of β-Gal after 0 hours and 24 hours is shown in FIG. 11. The enzyme activity of β-Gal not encapsulated in gelatin gel microparticles was lost by 59% after 24 hours. In contrast, the activity of β-Gal encapsulated in gelatin gel microparticles was almost completely maintained after 24 hours. In other words, it can be said that the enzyme was stabilized by the gelatin gel microparticles of the present invention. This suggests that the gelatin network surrounding the enzyme in the gelatin gel microparticles of the present invention suppresses the inactivation of the enzyme by acid. Since this condition is similar to the condition for producing lactose-free milk for lactose intolerant patients, it is considered that the gelatin gel microparticles encapsulating β-Gal are industrially useful.

[0163] [Example 13: Enzyme stabilization against ultraviolet irradiation] Gelatin gel particles were used as a protein stabilizer. The enzymes used were HRP, GOD, and β-Galβ-Gal before and after irradiation with ultraviolet light (UV; wavelength 360 nm; irradiation time 20 min). Enzyme activity was measured by quantifying the enzyme reaction product for each enzyme, and the enzyme reaction was monitored for 1 hour.

[0164] The results are shown in Figure 12. The enzyme activity was almost unchanged with or without UV irradiation. In other words, it can be said that the gelatin gel microparticles of the present invention stabilized the enzyme against UV irradiation.

[0165] [Example 14: Mass production of enzyme-encapsulated gelatin gel particles] Scale-up was examined. A PBS solution of GelMA-1 (10 mL, 20 wt%), PVP (40 mL, 10 wt%), 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone (400 μL, 16 mM) and triethanolamine (600 μL, 4 M) was added to a glass tube and vigorously mixed at 25° C. for 180 seconds to obtain a W / W emulsion composition (W GelMA / W PVP ) was obtained. This W / W emulsion composition was passed through a glass filter with a pore size of 10 μm. To the emulsion composition immediately after passing through the glass filter, an enzyme (FITC-BSA) aqueous solution (600 μL, 5 mg / mL) was added, and the emulsion composition was mixed for 10 minutes by shaking the glass tube. The W / W emulsion composition was irradiated with ultraviolet light for 20 seconds using a Xe lamp (UI-502Q, Ushio Inc.) equipped with a 360 nm (± 40 nm) sharp cut filter. The PVP solution of the gelatin gel microparticles was diluted with 50 mL of pure water, and the gelatin gel microparticles were collected by centrifugation at 30,000 G at 10 ° C. using a centrifuge (Allegra X-30, Beckman Coulter). The collected microparticles were redispersed in pure water to wash out the PVP, and centrifuged again. This operation was repeated three times. The enzyme encapsulation efficiency was 91%.

[0166] A 50 mL tube containing the FITC-BSA-encapsulated gelatin gel microparticles obtained by mass production and a confocal laser microscope image of the microparticles are shown in Figure 13. Even when scaled up, gelatin gel microparticles similar to those in Example 1 could be mass-produced at low cost, and the obtained gelatin gel microparticles could encapsulate proteins with high efficiency.

[0167] According to the method of the present invention, gelatin gel microparticles capable of encapsulating a protein with high efficiency while maintaining the activity of the protein can be produced at low cost and with ease of scale-up.

Claims

1. Gelatin gel microparticles formed by crosslinking fish gelatin, Gelatin gel microparticles encapsulating a protein other than the fish gelatin.

2. 2. The gelatin gel microparticles according to claim 1, wherein the average particle size is from 10 nm to 500 μm.

3. The gelatin gel microparticle according to claim 1 or 2, which is a stabilizer for the protein.

4. The gelatin gel microparticle according to claim 1 or 2, which is a reaction field for the protein.

5. The gelatin gel microparticle according to claim 1 or 2, wherein the protein is an enzyme.

6. A method for producing gelatin gel microparticles, comprising the steps of: A step of obtaining a W / W emulsion composition by emulsifying an aqueous solution containing fish gelatin having a crosslinkable functional group and a nonionic water-soluble polymer; and A method of manufacturing comprising a step of crosslinking at least two of the crosslinkable functional groups.

7. The method according to claim 6 , wherein the crosslinkable functional group is an ethylenically unsaturated polymerizable group.

8. The method according to claim 6 , wherein the nonionic water-soluble polymer is polyvinylpyrrolidone.

9. The method according to any one of claims 6 to 8, further comprising the step of contacting the W / W emulsion composition with a protein.

10. The method of claim 9 , wherein the protein is an enzyme.

11. a dispersed phase comprising fish gelatin having crosslinkable functional groups; a continuous phase comprising a non-ionic water-soluble polymer; A W / W emulsion composition comprising:

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