JP2011144056A - Substance-encapsulated calcium carbonate, and production method and use of the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To easily encapsulate proteins, peptides, and low molecular weight substances in calcium carbonate with low loss and efficiently, as long as the encapsulated protein or the like does not cause dissolution or destruction of the calcium carbonate part. Provides a technique for a substance-encapsulated calcium carbonate material that is not released to the outside.
The calcium carbonate particles are metastable by immersing calcium carbonate particles mainly composed of a metastable phase selected from the group consisting of a vaterite phase and an aragonite phase in a solution of a substance that can be adsorbed by the calcium carbonate particles. A method for producing calcium carbonate particles encapsulating a substance, wherein the phase is changed to a calcite phase.
[Selection] Figure 1

Description

  The present invention relates to calcium carbonate encapsulating a substance, a method for producing the same, and a method for releasing the encapsulated substance.

  Technology that encapsulates functional substances (molecules and macromolecules) inside the fine powder, and the material synthesized in this way makes it possible to use the encapsulated functional substances more effectively. Can be. As the material of the fine powder, an organic compound, an inorganic compound, or a composite compound thereof can be considered. Calcium carbonate is a material material that has excellent mechanical strength, biocompatibility, environmental compatibility, and high metabolism / degradability. Encapsulating functional substances inside this material is a highly available technology. Can provide. In order to effectively use the functional substance introduced into the powder, it is preferable that the functional substance is not released out of the powder under normal conditions, for example, room temperature air or room temperature neutral water. In order to encapsulate the functional substance in such a form in the powder material, simple adsorption or impregnation is not sufficient, and a complete encapsulation and encapsulation process is required.

A technique for encapsulating a substance in calcium carbonate and a technique for applying the capsule material are already known. A method for producing a calcium carbonate microcapsule material has also been reported (Patent Documents 1 and 2; Non-Patent Document 1). There are also reported examples (Patent Document 3 and Non-Patent Document 2) of techniques for introducing and encapsulating substances in the calcium carbonate microcapsules. In the method of encapsulating protein at the same time when preparing calcium carbonate microcapsules (Non-patent Document 2), the ratio of encapsulated protein is not high and lysozyme having a relatively small molecular weight (molecular weight: 14,388 Da) Then it is difficult to enclose. Many proteins have advanced functions such as enzyme activity and pharmacological activity, but it is not realistic to encapsulate such rare proteins with low encapsulation efficiency.

  Regarding the adsorption / encapsulation of proteins in calcium carbonate, recently, examples using porous calcite / calcium carbonate have been reported (Non-patent Documents 3 and 4). In many of these cases, substances such as proteins are easily detached from the base calcium carbonate. Therefore, there has been no technology that can confine proteins in calcium carbonate in normal solution, for example, in an aqueous solution in a neutral region without being dissolved in the solution, and simultaneously encapsulate the protein to be used at high efficiency.

Patent 1184016 Patent 1049606 JP2007-015990 JP2007-277036 JP 06-016417 JP2008-280191

J. Colloid Interface Sci. 68 (1979) 401-407. Chemical Engineering Journal 137 (2008) 14-22. Biomacromolecules 5 (2004) 1962-1972. Biotechnol. Prog. 21 (2005) 918-925.

  In the present invention, proteins, peptides, and low molecular weight substances are efficiently encapsulated in calcium carbonate with a small amount of loss, and the encapsulated protein or the like does not cause dissolution or destruction of the calcium carbonate part. The present invention provides a technique related to a calcium carbonate material containing a substance that is not easily released to the outside.

  From this point of view, regarding the encapsulation of proteins, peptides, and low molecular weight substances in calcium carbonate, we focused on the phenomenon of the phase transition of vaterite calcium carbonate to calcite type calcium carbonate in aqueous solution. By coexisting proteins, peptides, and low-molecular substances that are to be encapsulated, the proteins can be encapsulated in calcite-type calcium carbonate, and the encapsulated proteins can be easily detached and removed from the calcium carbonate. It was found that it was not released, and the present invention was reached.

The present invention provides calcium carbonate including the following substances, a method for producing the same, and a method for releasing the included substance.
Item 1.
Including the substance characterized in that the calcium carbonate particles mainly composed of a vaterite phase are immersed in a solution of a substance that can be adsorbed on the calcium carbonate particles to change the vaterite phase of the calcium carbonate particles into a calcite phase. Method for producing calcium carbonate particles.
Item 2.
Item 2. The method according to Item 1, wherein the substance is a protein.
Item 3.
Item 3. The method according to Item 1 or 2, wherein the particles are hollow particles.
Item 4.
Item 4. The method according to any one of Items 1 to 3, wherein the metastable phase is a vaterite phase.
Item 5.
Item 4. The method according to any one of Items 1 to 3, wherein the metastable phase is an aragonite phase.
Item 6.
Item 5. A calcium carbonate particle encapsulating a substance produced by the method according to any one of Items 1 to 4.
Item 7.
Item 6. A sustained release method of an encapsulated substance, which comprises administering the particle according to Item 5 to a living body.

  Calcium carbonate whose crystal phase is vaterite, which can be synthesized by the interfacial reaction method or the like, is a metastable phase in terms of crystal, and in an aqueous solution, phase transitions to calcite, which is a stable crystal phase. The naturally occurring aragonite phase is also a metastable phase and similarly undergoes a phase transition to calcite. Calcite is the main component of limestone. This phase transition originates from the fact that vaterite-type calcium carbonate or aragonite-type calcium carbonate, which is a metastable phase in an aqueous solution, once dissolves and becomes calcite, which is a stable phase, when crystallized again. If other compounds coexist in the aqueous solution during the recrystallization process, crystallization / phase transition is considered to take place in the crystal. By allowing a water-soluble protein, peptide, and low molecular weight substance to coexist in this solution, the protein, peptide, and low molecular weight substance can be introduced into the calcium carbonate after the phase transition. The amount introduced can be adjusted by adjusting the concentration of protein, peptide, and low molecular weight substance in the aqueous solution. By using it, it is easy to introduce it again, and the final encapsulation efficiency can be improved. In addition, the proteins, peptides, and low-molecular substances encapsulated in this way are incorporated into the calcite crystal and are not released to the outside.

SEM image of vaterite-type calcium carbonate XRD pattern of vaterite-type calcium carbonate SEM image of calcium carbonate phase transformed to calcite type XRD pattern of calcium carbonate phase transformed to calcite type SEM image of calcium carbonate phase transformed to calcite type SEM image of calcium carbonate phase transformed to calcite type XRD pattern of calcium carbonate phase transformed to calcite type SEM image of calcium carbonate phase transformed to calcite type XRD pattern of calcium carbonate phase transformed to calcite type SEM image of insulin-encapsulated calcium carbonate phase transformed to calcite type XRD pattern of insulin-encapsulated calcium carbonate phase transformed to calcite type Diffuse reflection ultraviolet spectrum of calcium carbonate containing insulin. SEM image of lysozyme-encapsulated calcium carbonate phase transformed to calcite type XRD pattern of lysozyme-encapsulated calcium carbonate phase transformed to calcite type Diffuse reflection ultraviolet spectrum of calcium carbonate containing lysozyme. SEM image of BSA-encapsulated calcium carbonate phase transformed to calcite type XRD pattern of BSA-encapsulated calcium carbonate phase transformed to calcite type Diffuse reflection ultraviolet spectrum of calcium carbonate containing BSA SEM image of Chicken IgY-encapsulated calcium carbonate phase transformed to calcite type XRD pattern of Chicken IgY-encapsulated calcium carbonate phase transformed to calcite type Diffuse reflection ultraviolet spectrum of calcium carbonate containing Chicken IgY SEM image of calcium carbonate containing fluorescein sodium salt XRD pattern of calcium carbonate containing fluorescein sodium salt Diffuse reflection ultraviolet spectrum of calcium carbonate containing fluorescein sodium salt

  Vaterite-type calcium carbonate does not exist in nature and must be synthesized by itself. The method of giving the vaterite-type calcium carbonate with a good selectivity is preferably the method based on the interfacial reaction method disclosed in Patent Documents 1 and 2, but it has a good yield as described in Patent Documents 4-6. There is no particular limitation as long as it can be obtained at a rate and selectivity. Aragonite-type calcium carbonate is contained in natural limestone and the like, and a commercially available product may be used. In this patent, the proportion of metastable phases such as a vaterite phase and an aragonite phase in calcium carbonate as a raw material is preferably high, and is at least 60% or more, preferably 80% or more, more preferably 90% or more. In the above-described method using the interfacial reaction method, 90% or more of vaterite-type calcium carbonate can be produced with a yield of 95% or more. The calcium carbonate having a metastable phase as a main component is preferably a vaterite type calcium carbonate.

The particle size of calcium carbonate is about 0.1 to 100 μm, preferably about 1 to 10 μm.

  The calcium carbonate particles may be hollow particles or solid particles as long as the metastable phase is a main component.

In this specification, “encapsulation” means that the substance may be present inside or on the surface of the calcium carbonate particles, and the substance is encapsulated when it is not easily eluted by washing with neutral water. . While the substance is present on the surface of the calcium carbonate particles or adsorbed, the metastable phase calcium carbonate repeatedly dissolves and precipitates, and at that time, part or all of the substance is taken into the calcium carbonate phase. The state is “inclusive”. Thus, large molecules such as proteins are “encapsulated” even if only a portion is present in the calcium carbonate phase.

  Substances to be included are proteins, peptides, low molecular weight substances and the like.

Proteins include enzymes, antibodies, receptors, proteins that form biological structures (collagen, keratin, etc.), hormones, muscle constituent proteins (actin, myosin, etc.), nutritional proteins (proteins contained in eggs, soybeans, milk, etc.) And albumin. Specific examples of enzymes that can be encapsulated include amino acid-related enzymes, carbohydrate hydrolyzing enzymes (glucosidases, endoglucanases), lipid-related enzymes, DNA-related enzymes, etc., or hydrolysis of proteases, lipases, amylases, esterases, glycosidases, etc. Examples include enzymes, isomerases, oxidoreductases, transferases, lyases, ligases and the like.

  The peptide may be any of a degradation product obtained by degrading a protein with an appropriate protease, a physiologically active peptide, and the like.

Examples of low molecular weight substances include organic compounds having a physiological activity of a molecular weight of 1000 or less, such as vitamins, amino acids, monosaccharides, disaccharides, oligosaccharides, phospholipids, glycolipids, supplements, plants, animals, microorganisms, and the like. Examples include extracts and medicines. If these substances are sparingly soluble, use a solubilizer or dissolve the substance using an organic solvent such as alcohol (methanol, ethanol, isopropanol, etc.), pH adjuster / buffer, etc. Encapsulated in calcium particles.

  The above-mentioned vaterite-type calcium carbonate / aragonite-type calcium carbonate is immersed in an aqueous solution in which proteins, peptides and low-molecular substances are dissolved. The ratio between the weight of calcium carbonate and the volume of the aqueous solution at this time (weight g of calcium carbonate / volume of the aqueous solution mL) is not particularly limited, but a smaller ratio is better when a substance such as protein is to be encapsulated with high efficiency. , Preferably 0.1 to 10, more preferably 0.1 to 2. Further, the concentration of a substance such as protein in the aqueous solution is not particularly limited, but is preferably 0.1 to 10 mg / mL, more preferably 1 to 10 mg / mL when it is desired to enclose the substance with high efficiency. When protein is used as a substance, the aqueous solution used is not particularly limited as long as it does not denature or decompose proteins and does not easily dissolve calcium carbonate, but various concentrations of sodium chloride aqueous solution, physiological saline, calcium chloride An aqueous solution, a Tris buffer, etc. can be illustrated. Examples of the aqueous solution in which calcium carbonate is easily dissolved include hydrochloric acid aqueous solution, EDTA sodium salt aqueous solution, citric acid aqueous solution and the like. An aqueous solution in which proteins, peptides, and low molecular weight substances in which vaterite-type calcium carbonate / aragonite-type calcium carbonate is immersed may be allowed to stand. The temperature is not particularly limited as long as the proteins do not denature or decompose, but is preferably 5 to 30 ° C. The time is not particularly limited as long as a sufficient phase transition occurs, but about 1 to 100 hours are exemplified. A substance such as a protein to be encapsulated is not particularly limited as long as it is soluble in water at room temperature.

  The calcium carbonate solids rearranged into the calcite phase can be separated and recovered from the solution by decantation or filtration. The drying process is not particularly limited as long as the protein, peptide, and low molecular weight substance are not denatured or decomposed, but a drying process at room temperature to about 30 ° C. in air is preferable. The drying time is not particularly limited, but is preferably about 1 to 20 hours. However, when a special drying process is not required, it may not be performed. If an aqueous solution in which proteins, peptides, and low-molecular substances are dissolved is recovered by decantation, it can be used in another process as it is, and the final encapsulation efficiency can be improved.

  In addition, the phase transition from vaterite / aragonite (metastable phase) to calcite does not necessarily proceed completely. In the middle of the phase transition from vaterite to calcite, even when the crystal form is almost the vaterite type, the coexisting proteins, peptides and low-molecular substances are beginning to be incorporated into the crystal. In particular, if the substance to be included is not relatively valuable and can be used in large quantities, it is not always necessary to wait for a complete phase transition.

  Examples are given below, but are not limited thereto.

Example 1 Synthesis of Vaterite Type Calcium Carbonate Vaterite type calcium carbonate was synthesized by improving the method described in Non-Patent Document 1. An aqueous solution (32 mL) of ammonium carbonate (9.23 g, 96 mmol) and a hexane solution (48 mL) of Tween 85 (1.0 g) were stirred at 8000 rpm for 1 minute using a homogenizer to form an emulsion. This emulsion was added to an aqueous solution (640 mL) of 28.2 g (192 mmol) of calcium chloride dihydrate heated to 30 ° C. at a time while stirring, and allowed to react for 5 minutes. The resulting white precipitate was filtered off, washed with 1 L of ion exchange water and 100 mL of methanol, and dried at 80 ° C. for 12 hours to obtain a vaterite-type calcium carbonate (yield: 8.1 g). The fact that this calcium carbonate is a vaterite was confirmed by SEM observation as spherical particles and by XRD measurement (FIGS. 1 and 2).

Example 2 Phase transition of vaterite-type calcium carbonate to calcite-type calcium carbonate 0.3 g of vaterite-type calcium carbonate obtained in Example-1 was added at 0.05 mol / l Tris-HCl buffer (pH 7.6). It was immersed in 6 mL and allowed to stand at room temperature for 140 hours. This solid was separated by filtration and dried at 100 ° C. for 12 hours (yield: 0.28 g). The fact that this calcium carbonate is calcite was confirmed by SEM observation as cubic particles and by XRD measurement (FIGS. 3 and 4).

Example-3 Phase transition of vaterite-type calcium carbonate to calcite-type calcium carbonate 0.1 g of vaterite-type calcium carbonate obtained in Example-1 was used as an ion exchange aqueous solution of calcium chloride dihydrate (0.2 mol / L). It was immersed in 10 mL and allowed to stand at room temperature for 96 hours. This solid was separated by filtration and dried at 100 ° C. for 12 hours (yield: 0.28 g). The fact that this calcium carbonate is calcite was confirmed by SEM observation to be cubic particles (FIG. 5) and by XRD measurement.

Example 4 Phase transition of vaterite-type calcium carbonate to calcite-type calcium carbonate In the same manner as in Example-2, 0.3 g of vaterite-type calcium carbonate was immersed in physiological saline (0.6 mL), Vaterite-type calcium carbonate was phase-transferred (yield: 0.28 g). The fact that this calcium carbonate is calcite was confirmed by SEM observation as cubic particles and by XRD measurement (FIGS. 6 and 7).

Example-5 Phase transition of vaterite-type calcium carbonate to calcite-type calcium carbonate In the same manner as in Example-2, 0.3 g of vaterite-type calcium carbonate was immersed in a 1M aqueous sodium chloride solution (0.6 mL). Then, the phase transition of the vaterite type calcium carbonate was made (yield: 0.28 g). The fact that the calcium carbonate is calcite was confirmed by SEM observation to be cubic particles and by XRD measurement (FIGS. 8 and 9).

Example 6 Insulin Encapsulation Using Phase Transition of Vaterite-type Calcium Carbonate to Calcite-type Calcium Carbonate 1 g of vaterite-type calcium carbonate obtained in Example-1 was added at 0.05 mol / l Tris-HCl buffer (pH
7.6) It was immersed in a mixed solution of 0.5 mL and an insulin solution (10 mg / mL, Sigma Insulin solution from bovine pancreas) and allowed to stand at room temperature for 160 hours. This solid was separated by filtration and thoroughly dried at room temperature (yield: 0.96 g). The fact that this calcium carbonate is calcite was confirmed by SEM observation as cubic particles and by XRD measurement (FIGS. 10 and 11). Absorption at about 280 nm was observed from the diffuse reflection ultraviolet spectrum of this solid, and it was confirmed that insulin was contained (FIG. 12). In addition, from the amount of insulin remaining in the collected filtrate, the insulin content was 0.3 wt%, and the encapsulation efficiency of the insulin used (encapsulation / usage × 100) was 29.1%.

Example-7 Encapsulation of lysozyme using phase transition of vaterite-type calcium carbonate to calcite-type calcium carbonate 0.05 g / l Tris-HCl buffer (3.0 g of vaterite-type calcium carbonate obtained in Example-1) It was immersed in a mixed solution of 1 mL of a solution in which 4.5 mL of pH 7.6) and 0.045 g of lysozyme were dissolved, and allowed to stand at room temperature for 168 hours. This solid was separated by filtration and thoroughly dried at room temperature (yield: 2.90 g). The fact that this calcium carbonate is calcite was confirmed by SEM observation as cubic particles and by XRD measurement (FIGS. 13 and 14). Absorption at about 280 nm was observed from the diffuse reflection ultraviolet spectrum of this solid, and it was confirmed that lysozyme was contained (FIG. 15). From the amount of lysozyme remaining in the collected filtrate, the content of lysozyme was 0.84 wt%, and the encapsulation efficiency of the lysozyme used (amount of encapsulation / amount used × 100) was 53.5%.

Example-8 Encapsulation of bovine serum albumin (BSA) using phase transition of vaterite-type calcium carbonate to calcite-type calcium carbonate 3.0 g of vaterite-type calcium carbonate obtained in Example-1 and 0.045 g of BSA It was immersed in a solution dissolved in 4.5 mL of 0.05 mol / l Tris-HCl buffer (pH 7.6) and allowed to stand at room temperature for 168 hours. This solid was separated by filtration and thoroughly dried at room temperature (yield: 2.84 g). The fact that this calcium carbonate is calcite was confirmed by SEM observation as cubic particles and by XRD measurement (FIGS. 16 and 17). Absorption at about 280 nm was observed from the diffuse reflection ultraviolet spectrum of this solid, and it was confirmed that BSA was contained.
Further, from the amount of BSA remaining in the collected filtrate, the content of BSA was 0.047 wt%, and the encapsulation efficiency of BSA used (encapsulation amount / use amount × 100) was 38.3%.

Example-9 Encapsulation of Chicken IgY Using Phase Transition of Vaterite-type Calcium Carbonate to Calcite-type Calcium Carbonate 1 g of vaterite-type calcium carbonate obtained in Example-1 and physiological saline in which 0.01 g of Chicken IgY was dissolved It was immersed in 5 mL of aqueous solution and allowed to stand at room temperature for 264 hours. This solid was separated by filtration and thoroughly dried at room temperature (yield: 0.92 g). The fact that this calcium carbonate is calcite was confirmed by SEM observation as cubic particles and by XRD measurement (FIGS. 19 and 20). Absorption at about 280 nm was observed from the diffuse reflection ultraviolet spectrum of this solid, and it was confirmed that IgY was contained (FIG. 21). From the amount of IgY remaining in the collected filtrate, the IgY content was 0.19 wt%, and the encapsulation efficiency of IgY used (encapsulation / usage × 100) was 17.3%.

Example-10 Fluorescein / sodium salt-encapsulated calcium carbonate 1 g of vaterite-type calcium carbonate obtained in Example-1 was immersed in a solution of 0.5 g (1.33 mmol) of fluorescein / sodium salt dissolved in 50 mL of ion-exchanged water. And left to stand at room temperature for 480 hours. This solid was separated by filtration and thoroughly dried at room temperature (yield: 0.77 g). This calcium carbonate was still a vaterite by SEM observation and XRD measurement (FIGS. 22 and 23). Even after this solid was washed twice with 5 L of ion-exchanged water, absorption from a fluorescein sodium salt of about 500 nm could be observed from the diffuse reflection ultraviolet spectrum, so that its inclusion could be confirmed ( FIG. 24).

Example-11 Dissolution of calcium carbonate-1
In a solution of 0.50 g of ethylenediamine-N, N, N ′, N′-tetraacetic acid tetrasodium salt tetrahydrate (Dojindo Laboratories: EDTA · 4Na) dissolved in 10 mL of ion-exchanged water, 0.08 g of site-type calcium carbonate (manufactured by Kanto Chemical Co., Inc.) was added and stirred. After about 20 minutes, the powder particles disappeared, and the aqueous solution became completely transparent.

Example-12 Dissolution of calcium carbonate-2
Example 1 In a solution of 1.23 g of ethylenediamine-N, N, N ′, N′-tetraacetic acid tetrasodium salt tetrahydrate (Dojindo Laboratories: EDTA · 4Na) dissolved in 10 mL of ion-exchanged water, Example 1 0.08 g of vaterite-type calcium carbonate synthesized in (1) was added and stirred. Although the solution was translucent for about 3 hours, the powder particles of calcium carbonate disappeared completely. After about 90 hours, the aqueous solution became completely transparent.

Example-13 Dissolution of calcium carbonate-3
Example 1 To a solution of 0.50 g of ethylenediamine-N, N, N ′, N′-tetraacetic acid tetrasodium salt tetrahydrate (Dojindo Laboratories: EDTA · 4Na) dissolved in 10 mL of ion-exchanged water, Example 1 0.08 g of vaterite-type calcium carbonate synthesized in (1) was added and stirred. After about 3 hours, the powder particles of calcium carbonate remained, but after about 90 hours, the powder particles disappeared and the aqueous solution became completely transparent.

-Example-14 Release of protein etc. by dissolution of calcium carbonate 10 mL of ethylenediamine-N, N, N ', N'-tetraacetic acid tetrasodium salt tetrahydrate (Dojindo Laboratories: EDTA.4Na) 0.50 g 0.08 g of lysozyme-encapsulated calcium carbonate synthesized in Example 7 was added to the solution dissolved in ion-exchanged water and stirred. After about 90 hours, the powder particles disappeared, and the aqueous solution became completely transparent. In the ultraviolet spectrum of the solution, absorption derived from 280 nm lysozyme was observed, and it was confirmed that the inclusion substance was released by dissolution of calcium carbonate.

  Calcium carbonate is a main ingredient such as limestone and eggshell, and is easily degradable in vivo. Therefore, calcium carbonate is a material material friendly to the human body and the environment, and it can be expected to be applied in various fields in the future. In this patented technology, proteins are efficiently encapsulated in the calcium carbonate, and the encapsulated protein is not easily released. Therefore, it can be said that it is a stable protein immobilization technique. Therefore, the application of the material newly prepared and found in this patent can be considered as follows, for example. As an immobilized enzyme, it can be expected to be applied in many fields of the enzyme industry. Proteins with a sensing function can be applied to sensors. Furthermore, due to the dissolution performance of calcium carbonate, it can be expected to be applied to a drug delivery system that releases protein by its dissolution action.

Claims (7)

  Calcium carbonate particles mainly composed of a metastable phase selected from the group consisting of a vaterite phase and an aragonite phase are immersed in a solution of a substance that can be adsorbed on the calcium carbonate particles, and the metastable phase of the calcium carbonate particles is converted to calcite. A method for producing calcium carbonate particles encapsulating a substance, wherein the phase is changed to a phase.   The method of claim 1, wherein the substance is a protein.   The method according to claim 1, wherein the particles are hollow particles.   The method according to claim 1, wherein the metastable phase is a vaterite phase.   The method according to claim 1, wherein the metastable phase is an aragonite phase.   The calcium carbonate particle which included the substance manufactured by the method in any one of Claims 1-5.   A method for sustained release of an encapsulated substance, comprising administering the particles according to claim 6 to a living body.

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