JP4900646B2 - Calcium phosphate composite material and method for producing the same - Google Patents

Description

  The present invention relates to a calcium phosphate composite material and a method for producing the same.

Calcium phosphates such as hydroxyapatite are the main components of human teeth and bones, and are the most important bioinorganic materials with high biocompatibility. Bone such organism is a complex of the calcium phosphate and protein, etc., can be artificially synthesizing such materials, dental, orthopedic, in the medical and pharmaceutical fields such as regenerative medicine, it is particularly important . In recent years, researches on compounding with compounds that have been actively conducted are considered to be important for compounding with proteins and the like at a micro level. In addition, since calcium phosphate has high biocompatibility, attempts have been made to improve the biocompatibility of materials by depositing it on the surface of various materials.

  There are various methods for synthesizing complexes of calcium phosphates with other compounds. The simplest method includes a method in which a hydroxyapatite having a space for taking in a compound is produced in advance, and a compound such as a protein is added to or supported on the hydroxyapatite thus produced. For example, in Patent Document 1, porous hydroxyapatite fine particles are filled with a drug. Patent Document 2 reports a method of synthesizing needle-shaped crystalline hydroxyapatite by steam treatment and filling the pores with protein. A method (Patent Document 3 and Patent Document 4) for supporting a nucleic acid on a finished porous hydroxyapatite has also been reported. There is also an example of mixing by synthesizing plate-like hydroxyapatite in which the a-plane of hydroxyapatite is grown (Patent Document 5). Patent Documents 6 to 11 are also methods in which porous hydroxyapatite is once synthesized, and a compound such as protein is filled and kneaded therein. Other mixing methods include a method in which fine hydrogel and protein are mixed and solidified (Patent Document 12), and a method reported in Patent Documents 13 to 15.

  A typical method for producing a composite by forming hydroxyapatites from a solution is to immerse the composite material in a simulated body fluid (SBF) and from the ions in the simulated body fluid to the surface, hydroxyapatite, etc. There is a method of precipitating the phases. As typical examples, Non-Patent Document 1, Non-Patent Document 2, and Patent Document 16 can be cited. Usually, in this method, the time for immersing the material in the simulated body fluid (SBF) is several weeks or more. Moreover, although preparation of a pseudo body fluid (SBF) is not so difficult, the amount of coexisting ions must be finely adjusted, and the solution cannot be stored for a long time. That is, complicated raw material preparation and a long synthesis time are required. On the other hand, as reported in Patent Document 17, Non-Patent Document 3, Non-Patent Document 4, etc., the surface of the material is obtained by alternately immersing a material having a hydrophilic surface in a calcium ion solution and a phosphoric acid solution. In this method, hydroxyapatite is produced and fixed. In this method, the synthesis time is considerably shortened to about one day compared with the case of using simulated body fluid (SBF). However, a complicated operation of taking out the material and immersing it in a calcium ion solution and a phosphoric acid solution is required. Furthermore, there is a method in which calcium phosphate is coated on the surface of a spherical biodegradable polymer or polymer bead (Patent Document 18, Patent Document 19). A small amount of calcium phosphate is precipitated on the surface, and calcium phosphate is not the main component. Regarding calcium phosphate containing nucleic acid, a method for producing a precipitate of calcium phosphate from a solution containing nucleic acid is well known, and there are also examples of transfection using this (Patent Document 20, Patent Document 21). There is a limit to the amount of nucleic acid that can be introduced into calcium phosphate.

On the other hand, a method of coprecipitation using a supersaturated solution of calcium phosphate and adding a compound such as protein to this is also known (Patent Documents 22 to 24). In this method as well, a solution with precise preparation conditions such as a supersaturated solution of calcium phosphate must be used. In general, calcium phosphate is obtained by mixing a solution containing phosphoric acid with a solution containing calcium ions. For example, in the case of hydroxyapatite, the ratio of calcium hydroxide and phosphoric acid (or salt) is adjusted to the ratio of phosphorus to calcium. It can be easily obtained by reacting. At this time, if a protein such as collagen coexists in a solution of phosphoric acid to precipitate hydroxyapatite, it is considered that collagen and the like are also included in the hydroxyapatite, but the mixing conditions of the solution, reaction temperature and pH It has been reported that collagen is not included in hydroxyapatite unless the above is optimized (Non-patent Document 5). As described above, there is known a general and versatile method that can produce a complex of calcium phosphate, particularly hydroxyapatite and other compounds in a short time by a simple method and is not greatly affected by the characteristics of the compound to be included. There wasn't.
JP2004-075662 JP2004-073401 JP2004-154431 JP2004-123610 JP 10-45405 JP2001-133459 JP-A-11-347112 JP2004-154431 JP2004-123610 JP2004-330113 JP 06-298621 JP2004-141359 JP 04-244014 JP 04-244013 JP 02-041171 JP 06-335520 JP2000-327314 JP2002-241312 JP2000-336174 Special Table 2002-533131 Special table flat 10-508462 JP2004-168739 JP2004-173795 JP2005-021208 K. Onuma et al., Chem. Mater.vol-10, p-3346 (1998) K. Onuma et al., J. Phys. Chem. B, vol-102, p-7833 (1998) T. Taguchi et al., Biomaterials, vol-22, p-53 (2001) T. Serizawa et al., J. Biomater. Sci. Polym. Edn. Vol-12, p-1293 (2001) M. Kikuchi et al., Composites Science and Technology, 64, 819 (2004)

  The present invention provides a technique for encapsulating various core compounds (ie, compounds encapsulated in the core part) in a shell composed of calcium phosphate, and a technique relating to the material synthesized in this way. is there.

As a result of studying the direct encapsulation of various core compounds into calcium phosphate particles, the present inventors have found that an emulsion of a compound that can be dissolved or dispersed in an inner aqueous phase (aqueous phase 1) containing phosphoric acid (or phosphate). The compound was directly encapsulated by adding a W / O emulsion to an oil phase containing a surfactant that stabilizes water and adding it to the aqueous phase 2 in which a calcium compound is present under appropriate stirring conditions. The present inventors have succeeded in synthesizing calcium phosphate particles and have arrived at the present invention (conceptual diagram is shown in FIG. 1).

The present invention relates to the following microcapsules and methods for producing the same.
1. A calcium phosphate composite material in which a core compound is encapsulated in a shell made of a calcium phosphate material.
2. Item 2. The composite material according to Item 1, wherein the core compound is a physiologically active substance.
3. Item 3. The composite material according to Item 1 or 2, wherein the composite material is a microcapsule.
4). A core compound characterized in that an aqueous solution containing calcium ions or phosphate ions is allowed to act on a W / O emulsion obtained by dispersing first aqueous phase particles containing phosphate ions or calcium ions and a core compound in an oil phase. A method for producing a calcium phosphate composite material in which is encapsulated in a shell composed of a calcium phosphate material.

  In FIG. 1, the aqueous phase 1 is a phosphate compound (a compound capable of supplying phosphate ions in an aqueous solution such as phosphoric acid and phosphate), and the aqueous phase 2 is a calcium compound (calcium ions in an aqueous solution such as a calcium salt). An example of a compound capable of supplying a calcium phosphate composite material can be obtained in the same manner even when the aqueous phase 2 is a phosphate compound and the aqueous phase 1 is a calcium compound.

  The present invention is a method for encapsulating various compounds in calcium phosphate particles by a simple method. By combining various core compounds and calcium phosphate materials, dental treatment, orthopedic treatment, regenerative medicine, drug delivery system, gene Materials that can contribute to delivery, cosmetics, use of food additives, and the like can be provided. Calcium phosphates, particularly hydroxyapatite, are known as highly biocompatible and non-toxic materials, and are considered to have a particularly high potential for practical use.

  In the present specification, the “calcium phosphate material” is a general term for compounds mainly composed of calcium and phosphorus. The “core compound” means a compound included in a core portion (a portion surrounded by a shell made of a calcium phosphate material).

A phosphoric acid compound means the compound which can supply a phosphate ion, for example, phosphoric acid and a phosphate are mentioned.
The calcium phosphate composite material including the core compound can be produced by improving an existing method for synthesizing microcapsules. In the case of particles such as silica as described above, hollow spherical particles can be synthesized, but in the case of calcium phosphate, spherical particles cannot be obtained by the prior art method, and the particle size is an irregular shape with a micron size. Powdered calcium phosphate is obtained. Calcium phosphate materials include hydroxyapatite, calcium hydrogen phosphate (dicalcium phosphate / die hydrate, DCPD), tricalcium phosphate (tricalcium phosphate), calcium dihydrogen phosphate, octacalcium phosphate, tetracalcium phosphate, etc. . The formation of this calcium phosphate can be controlled by the ratio of the raw material phosphorus and calcium. Further, other ions such as sodium, potassium, and fluorine may be mixed in a range that does not adversely affect the properties of the calcium phosphate material.
Examples of phosphate ion raw materials (compounds capable of supplying phosphate ions in an aqueous solution) used in the aqueous phase 1 or 2 of FIG. 1 include phosphoric acid, phosphates (ammonium dihydrogen phosphate, diammonium hydrogen phosphate, And sodium dihydrogen phosphate, disodium hydrogen phosphate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, and the like. Examples of calcium ion raw materials (compounds that can supply calcium ions in an aqueous solution) used in the aqueous phase 2 or 1 in FIG. 1 include calcium hydroxide, calcium oxide, calcium carbonate, calcium halide, calcium nitrate, calcium sulfate, and the like. I can give you.

At this time, the concentration / volume of the solution in which phosphoric acid is dissolved, the concentration / volume of the solution in which calcium is mixed, and the concentration / volume of the organic solution in which the surfactant is dissolved are not particularly limited. The concentration of the aqueous solution is preferably 0.5 to 20M, particularly 1 to 5M. The surfactant is not particularly required as long as the emulsion is stable. For example, the total weight concentration of the surfactant is preferably 5 to 50 g / L, and particularly preferably 10 to 30 g / L. The concentration of the calcium compound is preferably 0.1 to 5M and particularly preferably 0.2 to 2M in both cases of dissolution or suspension. The volume ratio of the aqueous solution (water phases 1 and 2) to be used and the oil phase is the volume ratio of the oil phase / water phase 1 and is preferably 0.3 to 20, particularly 0.5 to 10. In addition, the volume ratio of aqueous phase 2 / aqueous phase 1 is preferably 1 to 20, particularly 3 to 12.
Examples of compounds that can be encapsulated in a shell composed of a calcium phosphate material include physiologically active substances such as inorganic powders, organic polymers, biomolecules, nucleic acids, and drugs. Examples of the inorganic powder include various metal oxides, various metal carbonates, phosphates, metal powders and nonmetal powders. Examples of the metal oxide include alumina, titania, zirconia, copper oxide, iron oxide, and zinc oxide. Examples of the metal carbonate include calcium carbonate, magnesium carbonate, cobalt carbonate, strontium carbonate, iron carbonate, and copper carbonate. Examples of the phosphate include calcium phosphate, iron phosphate, and aluminum phosphate. Examples of the metal powder or non-metal powder include carbon, iron powder, and copper powder. Examples of the organic polymer include hydrophilic polymers such as polyacrylamide, sodium polyacrylate, and sodium polymethacrylate that are soluble in water or insoluble in water. Specific examples of bioactive substances such as biomolecules, nucleic acids, and drugs that can be encapsulated include proteins, DNA, hormones, carbohydrates, and various pharmaceuticals. These biomolecules are not particularly limited, but those that can exist relatively stably in the above-described phosphoric acid or phosphate solution are preferable. The minimum time for mixing the physiologically active substance to be included in the phosphoric acid or phosphate aqueous solution is the time for forming an emulsion of the aqueous phase and the oil phase, and is expected to be about several minutes. The nucleic acid is not particularly limited, but deoxyribonucleic acid / DNA is considered good because of its stability in an acidic aqueous solution. The DNA is not particularly limited with respect to the base sequence and molecular weight. At this time, it is necessary to appropriately select an aqueous solution of phosphoric acid to be used. The relative amount of the compound to be encapsulated with respect to calcium phosphate is not particularly limited as long as it can be dissolved or sufficiently dispersed. The phosphoric acid solution to be used is a strong acid having a pH value of about 1, the ammonium dihydrogen phosphate solution has a pH value of about 4, the diammonium hydrogen phosphate solution has a pH value of about 8, It is necessary to appropriately select a solution that does not react.

  The aqueous phase 1 in which the phosphoric acid or phosphate containing the compound to be encapsulated is dissolved is added to the oil phase containing a nonionic surfactant that stabilizes the emulsion and emulsified. The nonionic surfactant used in this case is not particularly limited as long as it can stabilize the emulsion, and examples thereof include Tweens and Spans such as Tween80 and Span80. The oil phase includes hydrocarbons such as n-hexane, n-pentane, n-heptane and cyclohexane, aromatic hydrocarbons such as benzene and toluene, and halogenated hydrocarbons such as chloroform, methylene chloride and carbon tetrachloride. And solvents having low solubility in water, such as esters such as ethyl acetate and ethers such as diethyl ether and diisopropyl ether, and a preferable oil phase is composed of n-hexane, benzene and the like.

The emulsification method is not particularly limited, but a sufficient emulsion may be formed using a homogenizer or the like. The compound to be encapsulated can also be mixed during the formation of the emulsion. This emulsion is added to the aqueous phase 2 in which the above-mentioned calcium compound is dissolved or suspended (aqueous phase 2 in FIG. 1) to obtain calcium phosphate particles. At this time, if the compound to be encapsulated in the aqueous phase 1 does not coexist, calcium phosphate particles having no inclusion can be synthesized. The calcium phosphate particles thus obtained can be analyzed and identified by powder X-ray diffraction. The encapsulation of the compound can be confirmed by an infrared spectrum, an ultraviolet spectrum or the like. The fact that the mixed compound was actually encapsulated in calcium phosphate was confirmed by the following method. Regarding the water-soluble compound, it was confirmed that albumin having a high water-solubility was hardly detached even after washing the synthesized albumin-containing hydroxyapatite with a sufficient amount of water (see Example 7). . In addition, with respect to compounds insoluble in water, the powder is embedded in the particles of hydroxyapatite and DCPD from the optical microscopic image of the transmitted light of iron oxide or copper oxide encapsulated hydroxyapatite and iron oxide or copper oxide encapsulated DCPC. This was confirmed (see Examples 10 to 13).

EXAMPLES Hereinafter, although an Example demonstrates this invention concretely, this invention is not limited only to these Examples.
Example 1 Synthesis of Hydroxyapatite by Interfacial Reaction Method-1
A solution of phosphoric acid (8.28 g, 84.5 mmol) dissolved in water to make a total volume of 36 ml (aqueous phase 1 in FIG. 1), Tween80 (1.01 g: the number of moles is unknown per mixture) and Span80 (0.50 g: number of moles) Was dissolved in n-hexane and mixed with a solution (oil phase in FIG. 1) having a total volume of 72 ml, and emulsified at a rotational speed of about 8300 using a homogenizer. After this emulsification treatment for 1 minute, 99.9% purity calcium hydroxide (Wako Pure Chemicals; 9.26 g, 125 mmol) is suspended in water to make a total volume of 250 ml (aqueous phase 2 in FIG. 1). added. After stirring for 2 hours (rotation speed: about 400 rotations), a precipitate formed by filtration was obtained (12.25 g).

The powder X-ray diffraction pattern of the obtained powder is shown in FIG. It was confirmed that hydroxyapatite was generated.
Example 2: Synthesis of hydroxyapatite by the interfacial reaction method-2
A solution of ammonium dihydrogen phosphate (9.72 g, 84.5 mmol) dissolved in water to a total volume of 36 ml, a solution of Tween 80 (1.01 g) and Span 80 (0.50 g) in n-hexane to a total volume of 72 ml, The mixture was mixed and emulsified with a homogenizer at a rotational speed of about 8300. After this emulsification treatment for 1 minute, 99.9% purity calcium hydroxide (manufactured by Wako Pure Chemicals; 9.26 g, 125 mmol) was suspended in water and added to a solution having a total volume of 250 ml. After stirring for 2 hours (rotation speed: about 400 rotations), a precipitate formed by filtration was obtained (10.22 g). The powder X-ray diffraction pattern of the obtained powder is shown in FIG. It was confirmed that hydroxyapatite was generated.
Example 3 Synthesis of Hydroxyapatite by Interfacial Reaction Method-3
Dissolve diammonium hydrogen phosphate (11.16 g, 84.5 mmol) in water to a total volume of 36 ml, dissolve Tween80 (1.01 g) and Span80 (0.50 g) in n-hexane, and total volume of 72 ml
The obtained solution was mixed and emulsified at a rotational speed of about 8300 using a homogenizer. After this emulsification treatment for 1 minute, 99.9% purity calcium hydroxide (manufactured by Wako Pure Chemicals; 9.26 g, 125 mmol) was suspended in water and added to a solution having a total volume of 250 ml. After stirring for 2 hours (rotation speed: about 400 rotations), a precipitate formed by filtration was obtained (9.52 g). The powder X-ray diffraction pattern of the obtained powder is shown in FIG. It was confirmed that hydroxyapatite was generated.
Example 4: Synthesis of DCPD (Dicalcium Phosphate Dihydrate) by Interfacial Reaction Method Phosphoric acid (16.56 g, 169.0 mmol) was dissolved in water to make a total volume of 36 ml (aqueous phase 1 in FIG. 1). Tween80 (1.01 g) and Span80 (0.50 g) were dissolved in n-hexane and mixed with a solution having a total volume of 72 ml (oil phase in FIG. 1), and emulsified with a homogenizer at about 8300 revolutions. . After 1 minute of this emulsification treatment, 99.9% pure calcium hydroxide (Wako Pure Chemicals; 9.2
(6 g, 125 mmol) was suspended in water and added to a solution having a total volume of 250 ml (aqueous phase 2 in FIG. 1).
After stirring for 2 hours (rotation speed: about 400 rotations), a precipitate formed by filtration was obtained (10.26 g).

The powder X-ray diffraction pattern of the obtained powder is shown in FIG. It was confirmed that DCPD (dicalcium phosphate dihydrate) was produced.
Example 5: Synthesis of albumin-encapsulated hydroxyapatite by the interfacial reaction method 1 g of albumin (CALZYME Laboratories, Bovine Serum) was quickly dissolved in a solution in which phosphoric acid (8.28 g, 84.5 mmol) was dissolved in water to make a total volume of 36 ml. (Aqueous phase 1 in Fig. 1) Immediately after dissolution, Tween80 (1.01 g) and Span80 (0.50 g) were dissolved in n-hexane to make a total volume of 72 ml (oil phase in Fig. 1). The mixture was emulsified at a rotational speed of about 8300 using a kneader. After this emulsification treatment for 1 minute, 99.9% purity calcium hydroxide (Wako Pure Chemicals; 9.26 g, 125 mmol) is suspended in water to make a total volume of 250 ml (aqueous phase 2 in FIG. 1). added.
After stirring for 2 hours (rotation speed: about 400 rotations), a precipitate formed by filtration was obtained (13.48 g).

The powder X-ray diffraction pattern of the obtained powder is shown in FIG. It was confirmed that hydroxyapatite was generated. Further, absorption at 280 nm attributed to albumin was observed from the solid ultraviolet spectrum (FIG. 7), and it was confirmed that albumin was included.
Example 6: Synthesis of polyacrylamide-encapsulated hydroxyapatite by interfacial reaction method Phosphoric acid (8.28 g, 84.5 mmol) was dissolved in water, an aqueous solution of polyacrylamide was added so that the content of polyacrylamide was 2 g, and the total volume was 36 ml. Tween80 (1.01
g) and Span 80 (0.50 g) were dissolved in n-hexane and mixed with a solution (oil phase in FIG. 1) having a total volume of 72 ml, and emulsified at a rotational speed of about 8300 using a homogenizer. After this emulsification treatment for 1 minute, 99.9% purity calcium hydroxide (Wako Pure Chemicals; 9.26 g, 125 mmol) is suspended in water to make a total volume of 250 ml (aqueous phase 2 in FIG. 1). added. Stir for 2 hours (rotation speed:
After about 400 revolutions), a precipitate formed by filtration was obtained (13.08 g).

A powder X-ray diffraction pattern of the obtained powder is shown in FIG. It was confirmed that hydroxyapatite was generated.
Example 7: Synthesis of albumin-encapsulated hydroxyapatite by interfacial reaction method A solution in which phosphoric acid (8.28 g, 84.5 mmol) was dissolved in water to make a total volume of 36 ml was prepared by adding Tween80 (1.01 g) and Span80 (0.50 g) to n- Mixed with a solution of 72 ml in total volume dissolved in hexane,
The emulsion was emulsified at a rotational speed of about 8300 using a homogenizer. After performing this emulsification treatment for 20 seconds, 1 g of albumin powder was quickly added, and after emulsification with a homogenizer for 1 minute, 99.9% purity calcium hydroxide (Wako Pure Chemicals; 9.26 g, 125 mmol) was added to water. The suspension was added to a solution (aqueous phase 2 in FIG. 1) having a total volume of 250 ml. After stirring for 2 hours (rotation speed: about 400 rotations), a precipitate formed by filtration was obtained (13.50 g).

The powder X-ray diffraction pattern of the obtained powder is shown in FIG. It was confirmed that hydroxyapatite was generated. Further, absorption at 280 nm attributed to albumin was observed from the solid ultraviolet spectrum (FIG. 10), and it was confirmed that albumin was included. Moreover, by comparing the ultraviolet spectrum of solids before and after washing 1 g of hydroxyapatite encapsulating albumin with 1.5 liters of water (FIG. 11), albumin encapsulated in hydroxyapatite is not neutral water. Since it was hardly released, it was confirmed that albumin was included in the hydroxyapatite solid.
Example 8: Synthesis of zirconia-encapsulated hydroxyapatite by the interfacial reaction method A solution in which phosphoric acid (8.28 g, 84.5 mmol) was dissolved in water to make the total volume 36 ml, Tween80 (1.01 g) and Span80 (0.50 g) were mixed with n- Mixed with a solution of 72 ml in total volume dissolved in hexane,
The emulsion was emulsified at a rotational speed of about 8300 using a homogenizer. After performing this emulsification treatment for 20 seconds, quickly add 1 g of zirconia powder, and after emulsification with a homogenizer for 1 minute, 99.9% purity calcium hydroxide (Wako Pure Chemicals; 9.26 g, 125 mmol) is added to water. Suspend and total volume 250
The solution was added to the ml solution (aqueous phase 2 in FIG. 1). After stirring for 2 hours (rotation speed: about 400 rotations), a precipitate formed by filtration was obtained (12.99 g).

FIG. 12 shows a powder X-ray diffraction pattern of the obtained powder. It was confirmed that hydroxyapatite was generated. Further, absorption at 230 nm attributed to zirconia was observed from the solid ultraviolet spectrum (FIG. 13), and it was confirmed that zirconia was included. Example 9: Synthesis of alumina-encapsulated DCPD by interfacial reaction method A solution in which phosphoric acid (8.28 g, 84.5 mmol) was dissolved in water to make a total volume of 36 ml was prepared by adding Tween80 (1.01 g) and Span80 (0.50 g) to n-hexane. And mixed with a solution with a total volume of 72 ml,
The emulsion was emulsified at a rotational speed of about 8300 using a homogenizer. After 20 seconds of this emulsification treatment, 1 g of alumina powder was quickly added, and after further emulsification with a homogenizer for 1 minute, 99.9% purity calcium hydroxide (Wako Pure Chemicals; 9.26 g, 125 mmol) was added to water. The suspension was added to a solution (aqueous phase 2 in FIG. 1) having a total volume of 250 ml. After stirring for 2 hours (rotation speed: about 400 rotations), a precipitate formed by filtration was obtained (13.70 g).

The powder X-ray diffraction pattern of the obtained powder is shown in FIG. It was confirmed that DCPD was generated.
Example 10: Synthesis of iron oxide-encapsulated DCPD by the interfacial reaction method A solution in which phosphoric acid (8.28 g, 84.5 mmol) was dissolved in water to make a total volume of 36 ml, Tween80 (1.01 g) and Span80 (0.50 g) were added to n- Mixed with a solution of 72 ml in total volume dissolved in hexane,
The emulsion was emulsified at a rotational speed of about 8300 using a homogenizer. After 20 seconds of this emulsification treatment, 1 g of iron oxide powder was quickly added, and after 1 minute of emulsification with a homogenizer, 99.9% pure calcium hydroxide (Wako Pure Chemicals; 9.26 g, 125 mmol) was added to the water. Was added to a solution (the aqueous phase 2 in FIG. 1) having a total volume of 250 ml. After stirring for 2 hours (rotation speed: about 400 rotations), a precipitate formed by filtration was obtained (14.20 g).

FIG. 15 shows a powder X-ray diffraction pattern of the obtained powdered iron oxide-encapsulated DCPD, and FIG. 16 shows an optical microscope image by transmitted light. It was confirmed that the iron oxide powder (black part) was embedded in the solid of DCPD.
Example 11: Synthesis of copper oxide-encapsulated DCPD by the interfacial reaction method A solution in which phosphoric acid (8.28 g, 84.5 mmol) was dissolved in water to make the total volume 36 ml, Tween80 (1.01 g) and Span80 (0.50 g) were added to n- Mixed with a solution of 72 ml in total volume dissolved in hexane,
The emulsion was emulsified at a rotational speed of about 8300 using a homogenizer. After this emulsification treatment for 20 seconds, 1 g of copper oxide powder was quickly added, and after emulsification with a homogenizer for 1 minute, 99.9% purity calcium hydroxide (Wako Pure Chemicals; 9.26 g, 125 mmol) was added to water. Was added to a solution (the aqueous phase 2 in FIG. 1) having a total volume of 250 ml. After stirring for 2 hours (rotation speed: about 400 rotations), a precipitate formed by filtration was obtained (13.90 g).

FIG. 17 shows a powder X-ray diffraction pattern of the obtained powdered copper oxide-encapsulated DCPD, and FIG. 18 shows an optical microscope image by transmitted light. It was confirmed that the copper oxide powder (black part) was embedded in the solid of DCPD.
Example 12: Synthesis of DNA-encapsulated hydroxyapatite by the interfacial reaction method-1
In a solution of ammonium dihydrogen phosphate (9.72 g, 84.5 mmol) dissolved in water to a total volume of 36 ml, 0.1 g of DNA (deoxyribonucleic acid sodium salt derived from shark testis; manufactured by Wako Pure Chemical Industries, Ltd.) was dissolved (see FIG. 1). Aqueous phase 1) Immediately after dissolution, mix Tween80 (1.01 g) and Span80 (0.50 g) in n-hexane to a total volume of 72 ml (oil phase in Fig. 1), and use a homogenizer. The emulsion was emulsified at a rotational speed of about 8300. After this emulsification treatment for 1 minute, 99.9% purity calcium hydroxide (Wako Pure Chemicals; 9.26 g, 125 mmol) is suspended in water to make a total volume of 250 ml (aqueous phase 2 in FIG. 1). added. After stirring for 2 hours (rotation speed: about 400 rotations), a precipitate formed by filtration was obtained (10.58 g).

The powder X-ray diffraction pattern of the obtained powder is shown in FIG. It was confirmed that hydroxyapatite was generated. Further, absorption at 270 nm attributed to DNA was observed from the solid ultraviolet spectrum (FIG. 20), and it was confirmed that DNA was encapsulated.
Example 13: Synthesis of DNA-encapsulated hydroxyapatite by the interfacial reaction method-2
Dissolve 0.1 g of DNA (deoxyribonucleic acid sodium salt derived from testis of shark; manufactured by Wako Pure Chemical Industries, Ltd.) in a solution prepared by dissolving diammonium hydrogen phosphate (11.16 g, 84.5 mmol) in water to a total volume of 36 ml (FIG. 1) Aqueous phase 1) Immediately after dissolution, mix Tween80 (1.01 g) and Span80 (0.50 g) in n-hexane to a total volume of 72 ml (oil phase in Fig. 1), and use a homogenizer. The emulsion was emulsified at about 8300 revolutions. After this emulsification treatment for 1 minute, 99.9% purity calcium hydroxide (Wako Pure Chemicals; 9.26 g, 125 mmol) is suspended in water to make a total volume of 250 ml (aqueous phase 2 in FIG. 1). added. After stirring for 2 hours (rotation speed: about 400 rotations), a precipitate formed by filtration was obtained (11.75 g).

FIG. 21 shows a powder X-ray diffraction pattern of the obtained powder. It was confirmed that hydroxyapatite was generated. Further, absorption at 270 nm attributed to DNA was observed from the solid ultraviolet spectrum (FIG. 22), and it was confirmed that DNA was encapsulated.
Example 14: Synthesis of collagen-encapsulated hydroxyapatite by interfacial reaction method A solution of ammonium dihydrogen phosphate (9.72 g, 84.5 mmol) dissolved in water to give a total volume of 36 ml was prepared by adding Tween80 (1.01 g) and Span80 (0.50 g). Was dissolved in n-hexane and mixed with a solution having a total volume of 72 ml, and emulsified at a rotational speed of about 8300 using a homogenizer. After performing this emulsification treatment for 20 seconds, 0.2 g of collagen (Sigma; type I) powder was quickly added, and after further emulsification with a homogenizer for 1 minute, 99.9% purity calcium hydroxide (Wako Pure Chemicals; 9.26 g, 125 mmol) was added to a solution (aqueous phase 2 in FIG. 1) suspended in water to a total volume of 250 ml. After stirring for 2 hours (rotation speed: about 400 rotations), a precipitate formed by filtration was obtained (8.88 g).

The powder X-ray diffraction pattern of the obtained powder is shown in FIG. It was confirmed that hydroxyapatite was generated.
Example 15: Synthesis of collagen-encapsulated hydroxyapatite by the interfacial reaction method A solution of diammonium hydrogen phosphate (11.16 g, 84.5 mmol) dissolved in water to make a total volume of 36 ml was obtained by adding Tween80 (1.01 g) and Span80 (0.50 g). Is dissolved in n-hexane and the total volume is 72 ml.
The obtained solution was mixed and emulsified at a rotational speed of about 8300 using a homogenizer. After performing this emulsification treatment for 20 seconds, 0.2 g of collagen (Sigma, Type I) powder was quickly added, and after further emulsification with a homogenizer for 1 minute, 99.9% purity calcium hydroxide (manufactured by Wako Pure Chemical; 9.26 g, 125 mmol) was added to a solution (aqueous phase 2 in FIG. 1) suspended in water to a total volume of 250 ml. After stirring for 2 hours (rotation speed: about 400 rotations), a precipitate formed by filtration was obtained (8.50 g).
.

The powder X-ray diffraction pattern of the obtained powder is shown in FIG. It was confirmed that hydroxyapatite was generated.

  Various applications of materials newly prepared and found in this patent are envisaged. For example, the following applications are conceivable.

Since biomolecules such as proteins and nucleic acids are encapsulated in calcium phosphate, it is expected to be applied to the sustained release technology of these biomolecules. Since proteins play many roles in living bodies, drug delivery systems using them, and nucleic acids can be used for gene therapy and the like, and therefore they are particularly promising for gene delivery systems. In addition, composites of calcium phosphate, which is a component of teeth and bones, and other compounds are expected to be applied to dentistry, orthopedics, regenerative medicine, cosmetics, food additives, and the like. Furthermore, it is envisaged to develop cell culture media and substrate materials for biomarkers.

The conceptual diagram of the synthesis | combining method of compound-encapsulated calcium phosphate. X-ray powder diffraction pattern of hydroxyapatite by the interfacial reaction method. X-ray powder diffraction pattern of hydroxyapatite by the interfacial reaction method. X-ray powder diffraction pattern of hydroxyapatite by the interfacial reaction method. DCPD (Dicalcium Phosphate Dye Hydrate) powder by the interfacial reaction method. X-ray powder diffraction pattern of albumin-containing hydroxyapatite. Diffuse reflection ultraviolet spectrum of albumin-containing hydroxyapatite. X-ray powder diffraction pattern of polyacrylamide-encapsulated hydroxyapatite. X-ray powder diffraction pattern of albumin-containing hydroxyapatite. Diffuse reflection ultraviolet spectrum of albumin-containing hydroxyapatite. Desorption behavior of albumin by water washing of albumin-encapsulated hydroxyapatite. X-ray powder diffraction pattern of zirconia-encapsulated hydroxyapatite. Diffuse reflection ultraviolet spectrum of zirconia-containing hydroxyapatite. X-ray powder diffraction pattern of alumina-encapsulated DCPD. The powder X-ray-diffraction pattern of iron oxide inclusion DCPD. Optical microscope image of iron oxide-encapsulated DCPD (500x) The powder X-ray-diffraction pattern of copper oxide inclusion DCPD. The optical microscope image (500 times) of copper oxide inclusion DCPD. X-ray powder diffraction pattern of DNA-encapsulated hydroxyapatite. The diffuse reflection ultraviolet spectrum of DNA-encapsulated hydroxyapatite. X-ray powder diffraction pattern of DNA-encapsulated hydroxyapatite. The diffuse reflection ultraviolet spectrum of DNA-encapsulated hydroxyapatite. X-ray powder diffraction pattern of hydroxyapatite containing collagen. X-ray powder diffraction pattern of hydroxyapatite containing collagen.

Claims (2)

Suspension containing calcium ions or phosphate ions in a W / O emulsion in which first aqueous phase particles containing a core compound selected from the group consisting of phosphate ions or calcium ions and protein and DNA are dispersed in an oil phase. Or an aqueous solution, wherein the calcium ion raw material of the first aqueous phase particles or the calcium ion raw material to act on the W / O emulsion is selected from the group consisting of calcium hydroxide, calcium oxide and calcium carbonate, A core compound selected from the group consisting of protein and DNA, comprising a suspension in which this calcium ion raw material is suspended in water to act on the first aqueous phase particles or W / O emulsion, is formed from a crystalline calcium phosphate material. Of crystalline calcium phosphate composite material encapsulated in shell Production method. The manufacturing method of Claim 1 whose raw material of a calcium ion is calcium hydroxide.

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