JP4569946B2 - Calcium phosphate fine particles - Google Patents

Description

  The present invention relates to particulate calcium phosphate carrying a desired molecule such as a pharmaceutical, and a method for producing the same.

  Conventionally, it has been extremely difficult to make a biopolymer (for example, protein) into a sustained-release medicinal product using only a substance in a living body as a base material. For this reason, in particular, a base material mainly composed of a synthetic compound is used for preparing a sustained-release drug for protein. Although such a substrate has been confirmed to be safe at the animal experiment level, its safety to humans remains unknown. In order to confirm safety for humans, it is necessary to apply the sustained-release drug to humans, accumulate a large number of cases, and undergo long-term observation over several years. From the point of view, it is difficult in reality. In addition, as a method for using a protein as a sustained-release drug, “bioactive peptide sustained-release drug” (Japanese Patent Laid-Open No. 2001-58955) and various liposome forming methods are known. However, liposomes based only on substances in the body lack stability, and the amount of protein taken into the liposomes is low, making it easy to make a highly practical protein sustained-release drug. Absent.

  On the other hand, apatite, 8 calcium phosphate, phosphorous having a Ca / P molar ratio (calcium / phosphoric acid molar ratio) of 1.3 or more in calcium phosphate (hereinafter sometimes abbreviated as “CaP” in the present specification). Acid calcium tricalcium and amorphous calcium phosphate are highly biocompatible CaP, are hardly soluble in neutral or alkaline regions, and are extremely safe for living organisms even if these CaP are inserted in places other than bone. It has been known. Therefore, these CaPs, including sintered apatite ceramics, have been widely studied as a base material when it is desired to artificially create a hard tissue in a place other than the original bone.

  Among CaP, calcium hydrogen phosphate anhydrous and calcium hydrogen phosphate dihydrate having a Ca / P molar ratio of 1 are used as powder components of calcium phosphate cement for living body, but apatite, 8 calcium phosphate, phosphorus It is calcium phosphate that is less biocompatible than tricalcium acid and amorphous calcium phosphate. That is, calcium hydrogen phosphate anhydride and calcium hydrogen phosphate dihydrate have slightly higher solubility, and when dissolved, the solution may be acidified to cause an inflammatory reaction.

Furthermore, calcium pyrophosphate having a Ca / P molar ratio of 1 is also less biocompatible than apatite, 8 calcium phosphate, 3 calcium phosphate, and amorphous calcium phosphate.
Among the CaPs, calcium dihydrogen phosphate having a Ca / P molar ratio of 0.5 is calcium phosphate having a lower biocompatibility than calcium hydrogen phosphate anhydrous or calcium hydrogen phosphate dihydrate, and its solubility is high. When dissolved, the solution becomes strongly acidic and cannot be used as a biomaterial.

Bone and tooth apatite and its precursor calcium phosphate not only have a Ca / P molar ratio of 1.3 or more, but also contain 3 to 6% by weight of a carbonate group CO ₃ ^2- . Bone and dental apatite contain carbonate groups in relation to body fluids and blood being sodium carbonate buffer. Artificial apatite can also be doped with carbonate group CO ₃ ^2-, and when doped with carbonate group, it approaches the physical properties of bone and tooth apatite. For example, bone apatite is absorbed (dissolved) by osteoclasts and is deposited by the action of osteoblasts after absorption. This process is called bone remodeling. However, a hydroxyapatite sintered body containing no carbonate group CO ₃ ^2- is not absorbed by osteoclasts and does not cause remodeling. As a result, the hydroxyapatite sintered body containing no carbonate group CO ₃ ²⁻ is not replaced by bone even when it is embedded in bone. On the other hand, a carbonate-containing apatite sintered body containing a few percent of carbonate-based CO ₃ ^2- is absorbed during the bone remodeling process in the same way as similar bones and autologous bones or normal bones, and eventually replaces real bones. (J. Biomed. Mater. Res., 39, pp. 603-610, 1998). Therefore, it can be said that carbonate-containing calcium phosphate, especially carbonate-containing apatite, has higher biocompatibility than calcium phosphate not containing any carbonate group CO ₃ ^2- .

Among CaP, since the specific surface area of apatite fine powder is as large as 100 m ² / g and is excellent in adsorption capacity, apatite fine powder has been used as an adsorbent for various substances. Apatite fine powder is considered to be a base material for preparing an adsorption-type sustained-release drug for various compounds, and has been shown to exhibit a sustained-release property according to a diffusion model by adsorption / desorption ( Burgos, AE et al., Biomaterials., 23, pp. 2519-26, 2002).

  It is known that CaP such as low crystalline apatite and amorphous calcium phosphate can be used as a safe sustained-release pharmaceutical base for physiologically active substances in the body such as proteins because CaP is inherently an in vivo substance. Tanaka et al.'S group showed a low crystallinity in the process of mixing calcium hydroxide suspension and phosphoric acid solution in the neutral region with fibrous protein collagen, which is an insoluble protein, and water soluble sugar chain polymer, chondroitin sulfate. Collagen / chondroitin sulfate-apatite composites containing nanometer-sized microcrystals were co-precipitated (Rhee, SH, et al., Biomaterials., 22 (21), pp.2843-7, 2001) .

  As another method for retaining proteins in CaP, a method of physically retaining growth factors in pores of hydroxyapatite or β-tricalcium phosphate porous sintered body (US Pat. No. 6,346,123, Clin. Orthop , 187, pp.277-280, 1983; J. Biomed. Mater. Res., 49, pp.415-421, 2000), a method of holding in contact with tricalcium phosphate or gypsum (JP-A-2001- 131086, JP-A-2001-122800), a method of adsorbing to a hydroxyapatite porous material or granules (Biochem. Biophys. Res. Commun., 193, pp.509-517, 1993; Biomaterials, 17, pp. 703-709, 1996; Clin. Orthop. 268, pp. 303-312, 1991; J. Mater Sci .: Mater in Med., 12, pp. 293-298, 2001; J. Biomed. Mater Res., 41 , pp.405-411, 1998), adding collagen gel to hydroxyapatite granules to fix bone formation factor (Biomaterials, 22, pp.1643-1651, 2001), hydroxyapatite and biodegradability Physiological activity in porous composites containing polymers A composition further added with a chemical substance (US Pat. No. 5,766,618), a method in which a calcium phosphate coating formed on a metal by MOCVD is immersed in a protein solution and adsorbed (US Pat. No. 6,113,993), sputter coating In this method, the calcium phosphate coating formed on titanium is immersed in a bone morphogenetic solution and adsorbed (Plastic Recon. Surgery 108, pp.434-443, 2001), and the hydroxyapatite-α calcium phosphate composite sintered body is used as a protein solution. A method of immersing and adsorbing (J. Mater Sci .; Mater in Med., 12, pp. 761-766, 2001), A method of immersing and adsorbing a porous calcium calcium phosphate in a protein solution (J. Biomed. Mater Res , 59, pp.422-428, 2002), a method in which octacalcium phosphate granules are immersed in an osteogenic factor solution and then freeze-dried (J. Biomed. Mater Res., 57, pp.175-182 , 2001), amorphous calcium phosphate, chondrogenic cells or A method of combining a precursor cell, water or water containing a physiologically active substance and curing to retain the physiologically active substance (US Pat. No. 6,277,151), mixing a bioabsorbable polymer or bioabsorbable calcium phosphate with a growth factor, A method of retaining a protein, a physiologically active substance, or a drug (US Pat. No. 5,947,893), a method of retaining a protein, a physiologically active substance, or a drug by mixing the calcium phosphate cement with a protein, a physiologically active substance, or a drug. 5,782,971, JP-A-9-225020, JP-A-4-248774, JP-A-62-228011, JP-A-7-31673, Biomed. Mater Eng., 4, pp.291-307, 1994 ; Clin. Ortho Related Res., 367S, pp. S396-S405, 1999; J. Periodont., 71, pp. 8-13, 2000; Biomaterials, 23, pp. 1261-1268, 2002; J. Biomed. Mater Res., 59, pp.265-271, 2002), a method of absorption in a calcium phosphate cement after hardening (J. Biomed. Mater Res., 44 , pp.168-175, 1999), a method of selectively adsorbing on polarized calcium phosphate (Japanese Patent Laid-Open No. 2001-187133), mixing with a paste containing a calcium component and a thickener, A fixing method (JP 2001-106638 A) and the like are known.

  As the CaP fine particles holding protein, CaP fine particles containing 50% or more of particles having a particle size of 90-225 microns and holding a drug such as protein inside the powder fine particles (US Patent No. 5,958,458, Danish Patent No. 0695/94). No.) is disclosed. However, the preferred manufacturing method is not disclosed. There is no restriction on the Ca / P molar ratio of CaP. Further, there is no disclosure of CaP microparticles that contain a carbonate group to make it more similar to biological CaP and have improved biocompatibility, absorbability, and adsorption capacity.

  Daculsi et al. Also proposed a method of adding sintered apatite powder to a phosphate buffer (PBS) containing fibronectin, an adhesion protein (Daculsi, G., et al., J. Biomed. Mater Res. 47 (2), pp.228-33, 1999). According to this report, since PBS is unsaturated in calcium phosphate, the sintered apatite powder is slightly dissolved, and eventually the PBS becomes saturated with respect to the low crystalline apatite in the vicinity of the powder particles. From this state, low crystalline apatite microcrystals are precipitated in the vicinity of the particles. At this time, fibronectin is adsorbed to the low crystalline apatite crystal plane. The adsorbed fibronectin functions as a precipitation site for low crystalline apatite microcrystals, and low crystalline apatite precipitates on fibronectin. As a result of repeating this process, fibronectin is not simply adsorbed on the surface of the low crystalline apatite, but is also supported in a captured state inside the low crystalline apatite particles. However, in this method, there is a problem that it takes 2 hours for the loading to wait for the sintered apatite powder to dissolve in the unsaturated solution. If the loading takes a long time, the production efficiency is lowered and the manufacturing cost is increased, and the bioactive substance and / or the drug cannot be carried on the biomaterial or the tissue during the operation. Further, there is a problem that low crystalline apatite does not grow around albumin, which is a particulate protein used as a comparative control of fibrous protein fibronectin, and albumin is hardly supported on low crystalline apatite. Further, calcium phosphate other than the low crystalline apatite is not disclosed as a carrier.

  A method of coprecipitation of CaP and protein from an aqueous solution is an effective loading method, and a method of precipitation on a polymer or metal surface (US Pat. Nos. 6,136,369, 6,143,948, 6,344,061, EP96201293) is disclosed. However, these methods are loading methods accompanied by a significant change in pH, and there are problems that a low molecular compound that is an active ingredient of a medicine is broken or proteins are deactivated.

The CaP microparticles holding the protein, and the CaP microparticles holding the protein by coprecipitation of CaP and protein from the aqueous solution are calcium phosphate particles having a diameter of 300 nm to 4000 nm, essentially spherical, essentially Fine particles having a smooth surface are disclosed (WO00046147). However, there is no limitation on the Ca / P molar ratio of the CaP fine particles. Further, there is no disclosure of CaP microparticles that contain a carbonate group to make it more similar to biological CaP and have improved biocompatibility, absorbability, and adsorption capacity. Further, although amorphous calcium phosphate retaining cytokines and interleukins is disclosed (US 20030082232), CaP microparticles that are closer to biological CaP and have improved biocompatibility, absorbability, and adsorption ability are disclosed. Absent.
JP 2001-58955 A US Pat. No. 6,346,123 JP 2001-131086 A JP 2001-122800 A US Pat. No. 5,766,618 US Pat. No. 6,113,993 US Pat. No. 6,277,151 US Pat. No. 5,958,458 German Patent No. 0695/94 US Pat. No. 5,947,893 US Pat. No. 5,782,971 JP 9-2225020 A JP-A-4-248774 JP-A-6-228011 JP-A-7-31673 JP 2001-187133 A JP-A-2001-106638 US Pat. No. 6,136,369 US Pat. No. 6,143,948 US Pat. No. 6,344,061 European Patent No. 96201293 WO00046147 US Patent Publication No. 20030082232 J. Biomed. Mater. Res., 39, pp.603-610, 1998 Biomaterials. 23 (12), pp.2519-26, 2002 Biomaterials. 22 (21), pp.2843-7, 2001 Clin. Orthop., 187, pp.277-280, 1983 J. Biomed. Mater Res. 49, pp.415-421, 2000 Biochem. Biophys. Res. Commun., 193, pp.509-517, 1993 Biomaterials, 17, pp.703-709, 1996 Clin. Orthop. 268, pp. 303-312, 1991 J. Mater Sci .: Mater in Med., 12, pp.293-298, 2001 J. Biomed. Mater Res., 41, pp.405-411, 1998 Biomaterials, 22, pp.1643-1651, 2001 Plastic Recon. Surgery 108, pp.434-443, 2001 J. Mater Sci .; Mater in Med, 12, pp.761-766, 2001 J. Biomed. Mater Res., 57, pp.175-182, 2001 Biomed. Mater Eng., 4, pp.291-307, 1994 Clin. Ortho. Related Res., 367S, pp.S396-S405, 1999 J. Periodont, 71, pp.8-13, 2000 Biomaterials, 23, pp.1261-1268, 2002 J. Biomed. Mater Res., 59, pp.265-271, 2002 J. Biomed. Mater Res., 44, pp.168-175, 1999 J. Biomed. Mater Res., 47 (2), pp.228-33, 1999

  By controlling the CaP particles to have a Ca / P molar ratio of 1.3 or more and capturing the molecules within the CaP grain boundaries and within the grains, the molecules can be supported more stably than by simple adsorption. Such molecular-supported CaP can be expected to be used as a safe sustained-release medicine. However, the Ca / P molar ratio of the CaP particles is controlled so as to be 1.3 or more, more preferably a carbonate group is added to improve biocompatibility, absorbability, and adsorption capacity, and drastically change pH. A method for efficiently supporting CaP fine particles on proteins in a short time without accompanying proteins or low molecular organic compounds is not provided.

  Therefore, an object of the present invention is to control the Ca / P molar ratio of CaP particles to 1.3 or more, and more preferably to control the carbonic acid content to the same extent as bone CaP. An object of the present invention is to provide a means for binding a hydrophobic low-molecular organic compound or the like to CaP easily and in a short time, and firmly and in large quantities without modifying these molecules. Another object of the present invention is to provide a sustained-release medicament containing an active ingredient of the medicament using the above means.

Recently, when the highly biocompatible CaP is deposited on a substrate using an unstable calcium phosphate supersaturated aqueous solution as a starting material, the present inventors have captured the protein component dissolved in advance in the deposited CaP layer, Developed the technology to be supported on the surface (Japanese Patent Application No. 2002-341464). In general, when creating a crystal of a specific molecule, create a solution in which the molecule is as pure as possible, and concentrate it to create a supersaturated state of the solute, which does not contain impurities enough to grow the crystal slowly. Pure crystals are obtained. The invention according to the above patent application reverses this principle and attempts to trap as many impurities as possible in the crystals by precipitating the crystals as quickly as possible from a supersaturated solution to which as many impurities as possible are added.
As a result of further intensive studies to solve the above-mentioned problems, the present inventors have mixed a calcium phosphate supersaturated aqueous solution with a solvent having a relative dielectric constant lower than that of water, thereby rapidly increasing the degree of supersaturation without significant change in pH. Thus, the inventors have found that the above problem can be solved more efficiently by rapidly precipitating CaP having a Ca / P molar ratio of 1.3 or more, and the present invention has been completed.

  That is, according to the present invention, a method for producing calcium phosphate fine particles having a Ca / P molar ratio of 1.3 or more carrying one or more supported molecules, wherein a solvent having a relative dielectric constant of 80 or less is mixed with a calcium phosphate supersaturated aqueous solution. A step of rapidly precipitating fine calcium phosphate from a calcium phosphate supersaturated aqueous solution, wherein either or both of the calcium phosphate supersaturated aqueous solution and the solvent having a relative dielectric constant of 80 or less contain a supported molecule. A manufacturing method is provided.

According to a preferred aspect of the present invention, there is provided the above method, wherein the calcium phosphate fine particles are calcium phosphate fine particles containing 1 to 15% by weight, preferably 2 to 8% by weight, more preferably 3 to 6% by weight of carbonate group CO ₃ ^2-. Provided. According to a more preferred embodiment, the calcium phosphate fine particles are carbonate-containing apatite fine particles containing 1 to 15 wt%, preferably 2 to 8 wt%, more preferably 3 to 6 wt% of carbonate group CO ₃ ^2- .

  According to another preferred embodiment of the present invention, the above-mentioned method, wherein the calcium phosphate supersaturated aqueous solution is a solution containing a hydrophilic supported molecule; the solvent having a relative dielectric constant of 80 or less contains a hydrophobic supported molecule The above method which is a solution; the above method wherein the calcium phosphate supersaturated aqueous solution is a solution containing a hydrophilic supported molecule, and the solvent having a relative dielectric constant of 80 or less is a solution containing a hydrophobic supported molecule The above-mentioned method comprising a step of mixing a calcium phosphate fine particle nucleus solution for precipitation of the fine particle calcium phosphate; and depositing the calcium phosphate fine particles on a substrate to form a highly biocompatible calcium phosphate fine particle coating on the base surface. There is provided a method as described above comprising the step of forming.

Further, according to a further preferred aspect of the present invention, the above method wherein the supported molecule is a physiologically active substance; the above method wherein the physiologically active molecule is a protein or a low molecular organic compound; the hydrophilic supported molecule is a protein. And the above method wherein the hydrophobic supported molecule is a low molecular weight organic compound; the hydrophilic supported molecule is albumin and the hydrophobic supported molecule is an anticancer agent; a relative dielectric constant of 80 or less The above method is provided wherein the solvent is one or more solvents selected from the group consisting of ethanol, methanol, and dimethyl sulfoxide .

  From another viewpoint, the present invention provides calcium phosphate fine particles having a high biocompatibility and a Ca / P molar ratio of 1.3 or more produced by the above method. Moreover, the pharmaceutical containing said calcium phosphate microparticles | fine-particles is also provided by this invention. Examples of the medicament include an immune adjuvant or an anticancer agent.

  According to the method of the present invention, highly biocompatible calcium phosphate fine particles having a Ca / P molar ratio of 1.3 or more loaded with a physiologically active substance as an active ingredient of a medicine can be produced easily and in a short time. The supported molecule can be supported inside calcium phosphate without deactivating the desired physiological activity.

In the present specification, “calcium phosphate having a Ca / P molar ratio of 1.3 or more” refers to calcium phosphate having a Ca / P molar ratio of 1.3 or more of Ca content and P content, and the types thereof are particularly limited. It may be any of an anhydride, an anhydrous salt or a hydrated salt. It may be either crystalline or amorphous. Furthermore, a part of Ca or a part of P may be substituted with another atom or atomic group. Specific examples of such calcium phosphate include apatite (Ca ₁₀ (PO ₄ ) ₆ (OH) ₂ ), calcium deficient apatite, apatite in which metal ions are partially substituted for Ca, and sulfate ions are phosphate ions. Substituted apatite, amorphous calcium phosphate, amorphous calcium phosphate partially substituted with metal ions, octacalcium phosphate (Ca ₈ H ₂ (PO ₄ ) ₆ · 5H ₂ O), and tricalcium phosphate (Ca ₃ (PO ₄ ) ₂ ), tricalcium phosphate in which metal ions are partially substituted for Ca can be mentioned, but is not limited thereto.

In the present specification, the kind of carbonic acid-containing calcium phosphate having a Ca / P molar ratio of 1.3 or more and preferably containing 1 to 15% by weight of a carbonate group CO ₃ ^2- is not particularly limited. Either a salt or a hydrated salt may be used. It may be either crystalline or amorphous. Examples of the carbonate-containing calcium phosphate include carbonate-containing apatite and carbonate-containing amorphous calcium phosphate. The seat occupied by the carbonate group is not particularly limited, and may be, for example, a phosphate site, an OH site, a so-called non-apatitic site, or a surface adsorption site.

  The lower limit of the carbonic acid group content is that the carbonic acid-containing calcium phosphate having a carbonic acid content of less than 1.0% by weight is dissolved in the solution of carbon dioxide in the air without intentionally introducing carbonate ions into the supersaturated solution or crystal nucleus Is preferably 1.0% by weight because it forms naturally. Further, when the carbonate group content is less than 1.0% by weight, there is an advantage that the effect is small because the carbonate group is small. Since the present invention is characterized in that carbonate ions are intentionally introduced into the reaction system, the lower limit of the carbonate group content is preferably 1.0% by weight. The upper limit of the carbonate group content is preferably 15% by weight. In order to obtain this level of carbonic acid content, the carbonate ion concentration in the supersaturated solution needs to be 14 mM or more, and the amount of calcium phosphate fine particles produced from such a high concentration carbonate ion supersaturated solution is small. This is because calcium production increases and there are few practical advantages.

  When carbonate ions are intentionally introduced into the reaction system, calcium carbonate may also be produced when producing carbonate-containing calcium phosphate depending on temperature and pH conditions. However, since the biocompatibility of calcium carbonate is not extremely low, calcium carbonate may be allowed to coexist if the content is 10% by weight or less, preferably 5% by weight or less, more preferably 1% by weight or less. it can.

The carbonic acid group content of the carbonic acid-containing calcium phosphate having a Ca / P molar ratio of 1.3 or more and preferably containing 1% by weight to 15% by weight of the carbonic acid group CO ₃ ^2- is the carbonic acid contained in the supersaturated solution. It can be controlled by the concentration of the base CO ₃ ^2- . For example, from a supersaturated solution of Ca component 3.25 mM phosphate component 1.3 mM Na component 182.06 mM K component 2.6 mM HCO ₃ component 4.2 mM Cl component 184.36 mM, pH 7.4, carbonate group CO in an equilibrium state. ₃ ^2- Carbonate-containing apatite with a content of 5.06% by weight is formed. The carbonate group CO ₃ ^2- replaces the PO ₄ group of the apatite. From the supersaturated solution in which the HCO ₃ component in the above solution was reduced to 2.1 mM, and the Na component was 179.96 mM, the carbonate group CO ₃ ^2- content was 3.68% by weight in an equilibrium state. Carbonate-containing apatite is produced.

The carbonic acid group content of the carbonic acid-containing calcium phosphate fine particles having a Ca / P molar ratio of 1.3 or more and preferably containing 1% to 15% by weight of the carbonic acid group CO ₃ ^2- is 1400 to 1550 cm in the infrared absorption spectrum. The absorption intensity of the carbonate group absorption band appearing at ^-1 can be quantified by comparing with the carbonic acid-containing calcium phosphate having a known carbonic acid content. However, when the solvent having a dielectric constant of 80 or less or the supported molecule has an absorption band at 1400 to 1550 cm ⁻¹ (for example, a molecule having a carboxyl group), the solvent or the supported molecule having a dielectric constant of 80 or less is mixed. It is desirable to produce carbonic acid-containing calcium phosphate particles without quantifying the carbonic acid content with an infrared absorption spectrum. In addition to the infrared absorption spectrum, the carbonate group content can also be quantified by various methods known to those skilled in the art such as gas chromatography, coulometric titration, and microdiffusion method. When the fine particles contain calcium carbonate, the carbonate group content of the CaP fine particles can be accurately determined by previously quantifying the calcium carbonate content by powder X-ray diffraction.

According to the method of the present invention, a solvent having a relative dielectric constant of 80 or less is mixed with a calcium phosphate supersaturated solution, thereby rapidly reducing the dielectric constant of the mixture to rapidly reduce the solubility of calcium phosphate without causing a significant change in pH. The supersaturation degree of the solution can be rapidly increased. In the present invention, the solvent having a relative dielectric constant of 80 or less is a solvent having a relative dielectric constant of 80 or less, preferably 50 or less, measured at a frequency of 3 × 10 ⁸ Hz or more and 6 × 10 ⁸ Hz or less. It is. The relative dielectric constant of water is 80.1 at 20 ° C., and in a solvent having a lower relative dielectric constant than this, many electrolytes including calcium phosphate are prevented from dissociating. As a result, the supersaturation degree of calcium phosphate increases. Among solvents having a relative dielectric constant of 80 or less, proton-accepting solvents, amphoteric solvents, and aprotic solvents have no effect on reducing pH or are extremely small, and therefore can be suitably used in the method of the present invention. Examples of the solvent having a relative dielectric constant of 80 or less include alcohols such as methanol and ethanol, and hydrophilic organic solvents such as acetone, pyridine, dimethyl sulfoxide , and polyethylene glycol. Water and / or a surfactant may be mixed in the hydrophilic organic solvent.

  There are no particular restrictions on the type of base material on which calcium phosphate fine particles are deposited to form a film, and metal materials, synthetic polymer materials, inorganic materials, biopolymer materials, biotissue materials, or composite materials thereof are used. can do. Among these, the base material which promotes the heterogeneous nucleus of CaP can be used conveniently. Examples of such a base material include titanium, titanium alloys, tantalum, zirconium, and alloys thereof as long as they are metal materials. Furthermore, an oxide film, OH group, phosphorous is formed on the surface of these metals. Examples include, but are not limited to, a metal complex in which one or more functional groups or atoms are introduced among an acid group, a sulfate group, a silanol group, or an alkaline earth metal. The introduction of the functional group can be achieved by various known methods such as alkali treatment and hydrogen peroxide treatment, and the introduction of alkaline earth metal is introduced by methods such as ion implantation and boiling in a calcium ion solution. be able to. Synthetic polymer materials include, for example, polymers containing polylactic acid, polymers containing ethylene vinyl alcohol, polyester, oxide film on the surface, OH groups, phosphate groups, sulfate groups, silanol groups, or alkaline earth metals. Among them, one or two or more functional groups and synthetic polymers into which atoms are introduced can be exemplified. Examples of the inorganic material include calcium phosphate, silica-containing glass and ceramic, phosphoric acid-containing glass and ceramic, calcium sulfate, and inorganic materials containing these, zirconium oxide, titanium oxide, and an oxide film, OH group, phosphorus on the surface. Examples thereof include an inorganic material in which one or two or more functional groups or atoms are introduced among an acid group, a sulfuric acid group, a silanol group, or an alkaline earth metal. Examples of the biopolymer material or the tissue material include collagen, fibrin, gelatin, chitin, chitosan, bone, tendon, ligament, phosphoprotein, tooth, keratin, and hair. But the kind of base material is not limited to what was illustrated above.

  The type of the molecule to be supported is not particularly limited and may be any of a high molecular compound, a low molecular organic compound, an inorganic substance, etc., but preferably a protein (including serum protein or anatomical protein), polysaccharide, etc. Examples include high molecular compounds, sugar compounds, lipid compounds, nucleic acid compounds, natural product organic compounds, synthetic low molecular weight organic compounds, etc., including mixtures of two or more molecules, crude extracts from animal tissues, plants, etc. Also good. Albumin, which has been conventionally difficult to be supported on CaP, can also be used as a supported molecule according to the method of the present invention. The supported substance is preferably a physiologically active substance. In the present specification, the term “bioactive substance” means a substance having some biological activity with respect to an organism, but this term should not be interpreted in a limited way in any sense, but in the broadest sense. There is a need to. For example, albumin and the like are also included in the physiologically active substance. The physiologically active substance includes, for example, physiologically active substances such as cytokines and hormones that can change the regulation of the living body and the functions of the living body, and examples thereof include growth factors and cell adhesion factors. The physiologically active substance also includes a low molecular weight organic compound used as an active ingredient of a medicine. The physiologically active substance may be either a hydrophilic substance or a hydrophobic substance. Examples of hydrophilic physiologically active substances include water-soluble physiologically active substances. Examples of water-soluble bioactive substances include water-insoluble or hydrophobic bioactive substances such as water-soluble carrier proteins such as albumin or polyethylene glycol, ethylene glycol / propylene glycol copolymers, carboxymethyl cellulose, dextran, polyvinyl alcohol, and polypinyl. Water soluble by binding to water soluble polymers such as pyrrolidone, poly-1,3-dioxolane, poly 1,3,6-trioxane, ethylene / maleic anhydride copolymer, or polyamino acids (homopolymer or random copolymer) Also included are physiologically active substances. The water-insoluble physiologically active material and the water-soluble carrier protein or water-soluble polymer may be bound by using the functional groups of both materials and can be bound by various known methods. Examples of hydrophobic physiologically active substances include, but are not limited to, steroids and prostaglandins.

  Examples of physiologically active substances include basic fibroblast growth factor, IL-1 (interleukin 1), IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL -8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-15, IL-17, IL-18, GM-CSF (granulocyte macrophage colony stimulating factor), G-CSF (Granulocyte colony stimulating factor), erythropoietin, CSF-1 (colony stimulating factor), SCF (stem cell factor), thrombopoietin, EGF (epidermal growth factor), TGF-α (transforming growth factor-α), HB-EGF ( Heparin-binding EGF-like growth factor), epiregulin, neuregulin 1, 2, 3, PDGF (platelet-derived growth factor), insulin, HGF (hepatocyte growth factor), VEGF (vascular endothelial growth factor), NGF (nerve growth) Factor), GDNF (glial cell line-derived neurotrophic factor), midkine, TGF-β (transforming) Growth factor-β), beta glycan, activin, BMP (bone morphogenetic factor), TNF (tumor necrosis factor), IFN-α / β (interferon-α / β), IFN-γ (interferon-γ), fibronectin, laminin , Cadherin, integrin, selectin and the like, but are not limited thereto. Among the physiologically active substances, examples of the hydrophilic compound used as an active ingredient of a medicine include adriamycin, and the hydrophobic compound used as an active ingredient of a medicine includes, for example, a poorly water-soluble drug. An example of a compound that is easily soluble in an organic solvent is mitomycin C. But the compound used as an active ingredient of a medicine is not limited to these.

  In this specification, the term “supported” means not only simple adsorption on the surface of CaP fine particles but also that supported molecules are held by CaP by being trapped in CaP grain boundaries and grains. Without being bound to any particular theory, according to the method of the present invention, when CaP grows from a supersaturated solution, the supported molecules are adsorbed on the surface of CaP and further on the adsorbed supported molecules. CaP precipitates and this process is repeated. As a result, the supported molecules are trapped in the interstices of CaP and are trapped in CaP grain boundaries and grains by being sandwiched between a plurality of CaPs.

  In this specification, the “calcium phosphate supersaturated aqueous solution” is a solution in which calcium and phosphorus are dissolved in excess of the solubility of calcium phosphate (however, the Ca / P molar ratio is 1.3 or more), and the Ca / P molar ratio is 1.3. This is a supersaturated solution in which the above calcium phosphate is deposited. Such a calcium phosphate supersaturated aqueous solution may be any supersaturated state with respect to at least one calcium phosphate having a Ca / P molar ratio of 1.3 or more, preferably at least a Ca component of 0.1 to An aqueous solution containing 5.0 mM and a phosphoric acid component of 0.1 to 20 mM and having a pH of 5.0 to 9.0 can be used. When pH is less than 5.0, calcium hydrogen phosphate anhydride or calcium hydrogen phosphate dihydrate is thermodynamically more stable than calcium phosphate having a Ca / P molar ratio of 1.3 or more. , Calcium hydrogen phosphate anhydride or calcium hydrogen phosphate dihydrate is precipitated from a solution of less than 5.0. When precipitating CaP particles, mixing of a phosphoric acid solution and a calcium hydroxide suspension, mixing of a calcium chloride solution and a water-soluble phosphate solution, as conventionally performed, Since the pH is low, even if the average pH of the mixed solution is neutral, a locally low pH portion is temporarily formed, and calcium hydrogen phosphate anhydride or calcium hydrogen phosphate dihydrate is formed. Precipitates and often mixes into the composite. In the present invention, phosphorus and calcium are dissolved in a supersaturated state in advance and the pH is adjusted to 5.0 to 9.0, whereby only calcium phosphate particles having a Ca / P molar ratio of 1.3 or more can be precipitated.

Furthermore, when it is assumed that the supported molecule is administered in vivo, a calcium phosphate supersaturated aqueous solution containing salts having a composition close to the biological salt composition is preferable. As such a solution, Ca-P-Na-K -Cl-based Ca component 0.1 to 2.5 mM, phosphoric acid component 1.0 to 20 mM, K component 0 to 40 mM, Na component 0 to 200 mM, Cl component 0 to 200 mM, and pH 5.0 to 9. 0 aqueous solution, Ca—P—Na—K—Mg—Cl—CO ₃ Ca component 1.2 to 2.75 mM, phosphoric acid component 0.6 to 16 mM, K component 0 to 30 mM, Na component 30 to An aqueous solution containing 150 mM, Mg component 0.1 to 3.0 mM, Cl component 30 to 150 mM, HCO 3 component 0 to 60 mM and pH 5.0 to 9.0 can be used. Various pH buffering agents for stabilizing pH can also be added to these calcium phosphate supersaturated aqueous solutions.

A supersaturated solution that does not contain carbonate ions, such as the above Ca-P-Na-K-Cl-based supersaturated solution, can be used, but even if the supersaturated solution does not contain carbonate ions, carbonic acid-containing CaP fine particles can be used. When used as a nucleus for forming CaP fine particles, the resulting CaP fine particles as a whole have a Ca / P molar ratio of 1.3 or more, preferably carbonic acid containing 1 to 15% by weight of carbonate group CO ₃ ^2- It can be set as contained CaP fine particles or carbonate-containing apatite.

  In the method of the present invention, for example, the calcium phosphate supersaturated solution can be divided into two, a stable calcium phosphate supersaturated solution and an unstable calcium phosphate supersaturated solution. The stable calcium phosphate supersaturated solution is a supersaturated calcium phosphate solution that requires 8 days or more from the last solution mixing after the completion of solution preparation until the precipitation of calcium phosphate due to spontaneous nucleation. Such stable calcium phosphate supersaturated solutions include Hank's solution and 1-fold concentration simulated body fluid. An unstable calcium phosphate supersaturated solution is a solution in which calcium phosphate precipitation occurs spontaneously by spontaneous nucleation within 7 days after solution preparation. The induction time until spontaneous nucleation can be controlled by selecting the chemical composition and temperature of the aqueous solution at optimum values. Spontaneous nucleation means that the components of the solution gather spontaneously without the help of the substrate, foreign substances, or the walls of the container, and the solution becomes visually cloudy, or in this way, fine particles are formed in the solution. That is. The particle size of the particles in the solution can be measured by a light scattering method.

  A calcium phosphate supersaturated solution can be prepared by sequentially dissolving a reagent powder containing at least calcium, a reagent powder containing at least phosphorus, and, if necessary, a pH buffer in water. Alternatively, a solution containing at least a calcium component, a solution containing at least a phosphoric acid component, a solution containing both a calcium component and a phosphoric acid component, and a solution containing a pH buffering agent if necessary can also be prepared in order. . In the case of an unstable supersaturated solution, a solution containing at least a calcium component, a solution containing at least a phosphoric acid component, or a solution containing both a calcium component and a phosphoric acid component further contains a component for controlling the spontaneous nucleation induction time. One or two or more solutions for controlling the induction time until spontaneous nucleation may be further mixed. The calcium component and the phosphate component are preferably dissolved separately in different containers before mixing.

  The kind of powder containing at least a calcium component or a solution thereof, powder containing at least a phosphoric acid component or a solution thereof, or a solution containing a calcium component and a phosphoric acid component is not particularly limited. Examples of the powder containing a phosphate component or a solution thereof include, for example, phosphate buffered saline, phosphate solution, dipotassium hydrogen phosphate powder or solution thereof, potassium dihydrogen phosphate powder or solution thereof, dihydrogen phosphate. Examples thereof include sodium powder or a solution thereof, sodium dihydrogen phosphate powder or a solution thereof, and the like. Examples of the powder containing calcium component or solution thereof include calcium chloride powder or solution thereof, calcium lactate powder or solution thereof, calcium acetate powder or solution thereof, calcium gluconate powder or solution thereof, or calcium citrate powder or solution thereof. Etc. Examples of the solution containing a calcium component and a phosphoric acid component include a stable supersaturated solution such as Hank's solution and 1 × simulated body fluid, and a calcium phosphate unsaturated solution. Examples of such a calcium phosphate unsaturated solution include a solution having a calcium chloride concentration of 2.5 mM, a potassium hydrogen phosphate concentration of 1.0 mM, and a pH of less than 5.

  Unstable calcium phosphate supersaturated solutions are suitable medical infusions or preparations, for example, medical electrolyte infusions, dialysis / peritoneal perfusate, infusion correction formulations, calcium preparations, dialysis / peritoneal perfusate replenishers, correction electrolytes. Although it can also produce by mixing the 1 type (s) or 2 or more types chosen from the infusion agents, it is not limited to these. Since medical infusions are sterilized and those that can be administered into the body have exothermic substances removed, there is an advantage that the safety of the CaP fine particles and the film is increased. Moreover, it can use suitably for manufacturing the CaP microparticles | fine-particles and film | membrane of this invention in the medical field like an operating room. When preparing a calcium phosphate supersaturated solution only by a combination of medical infusions, medical infusions or preparations containing at least a calcium component, medical infusions or formulations containing at least a phosphate component, and in some cases, suitable for pH correction. An acidic infusion or alkaline infusion, and optionally a medical electrolyte infusion for controlling the induction time until spontaneous nucleation can be mixed. As an example of a medical infusion containing at least a calcium component, a commercially available Ringer's solution (Otsuka Pharmaceutical), as an example of a medical infusion containing at least a phosphoric acid component, commercially available Clinizarz B (Kobayashi Pharmaceutical), a pH correction infusion Examples include commercially available Maillon (Otsuka Pharmaceutical), alkalizing agents such as dialysis sodium bicarbonate replenisher bifill (Takeda Pharmaceutical) or acidifying agents such as Conclito A (Otsuka Pharmaceutical), induction time until spontaneous nucleation As an example of a medical infusion for controlling the blood pressure, commercially available potassium infusion, MEDECTECT K (Terumo), commercially available physiological saline (each company), 10% sodium chloride (each company), etc. can be used. .

  The calcium phosphate supersaturated solution can be prepared as a solution in which a desired supported molecule (for example, protein) is dissolved. The dissolution operation can be easily performed by methods well known to those skilled in the art. For example, an aqueous solution of the supported molecule can be added to the calcium phosphate supersaturated aqueous solution within a range in which the supersaturated state of calcium phosphate can be maintained, or the supported molecule powder can be directly dissolved in the calcium phosphate supersaturated aqueous solution. For example, the supported molecule powder may be allowed to stand on the liquid surface of the calcium phosphate supersaturated solution. The supported molecule may be a single supported molecule or a mixture of two or more supported molecules, but is preferably a hydrophilic molecule that can be dissolved in a calcium phosphate supersaturated solution. . For example, simple proteins, glycoproteins, lipid binding proteins, nucleic acids, mucopolysaccharides, lipids, synthetic organic compounds, and mixtures thereof can be dissolved in a calcium phosphate supersaturated solution by a method well known to those skilled in the art. The optimum concentration of the molecule to be supported varies depending on the type of the molecule to be supported and the amount to be supported, but this concentration can be appropriately determined by a preliminary test, for example. In the case of albumin, about 0.25 mg / ml is preferable, but it is not limited thereto.

  The supported molecules can be dissolved in a solvent having a relative dielectric constant of 80 or less, preferably a hydrophilic organic solvent. In this case, the supported molecule is preferably a hydrophobic molecule. The type of the hydrophobic molecule is not particularly limited, and for example, a physiologically active substance such as a hydrophobic peptide or lipid can be used. Two or more kinds of hydrophobic molecules may be dissolved in a solvent having a relative dielectric constant of 80 or less. In the solvent having a relative dielectric constant of 80 or less, the same supported molecule as the supported molecule dissolved in the calcium phosphate supersaturated aqueous solution can be dissolved. The conditions for dissolution in a solvent having a relative dielectric constant of 80 or less and the concentration of supported molecules are not particularly limited. If the hydrophobic molecule is an organic compound, a hydrophilic organic solvent suitable for dissolving the compound may be selected. Any hydrophilic organic solvent can be used, but it is preferable to select a hydrophilic organic solvent that does not substantially deactivate water-soluble supported molecules dissolved in a calcium phosphate supersaturated aqueous solution. The mixing ratio of the calcium phosphate supersaturated aqueous solution and the hydrophilic organic solvent can also be selected within a range in which the water-soluble supported molecules dissolved in the calcium phosphate supersaturated aqueous solution are not substantially deactivated. For example, when the polymer dissolved in the calcium phosphate supersaturated aqueous solution is albumin, ethanol is preferably used as the hydrophilic organic solvent. If the concentration of ethanol mixed in the calcium phosphate supersaturated aqueous solution is 20% (v / v) or less, albumin No protein precipitation occurs due to denaturation. It is desirable to confirm beforehand the mixing ratio that does not deactivate the supported molecules by a preliminary test.

  If the desired hydrophobic molecule is sparingly soluble in the hydrophilic organic solvent, the hydrophobic molecule can be dissolved and dissolved in advance in another organic solvent that is miscible with the hydrophilic organic solvent. The solution may be prepared by adding the solution to a hydrophilic organic solvent. As such an organic solvent, a small amount of a hydrophobic organic solvent may be used in addition to a hydrophilic organic solvent. For example, since mitomycin C dissolves in methanol better than ethanol, mitomycin C can be dissolved in methanol in advance, and the methanol solution can be added to ethanol to prepare an ethanol solution of mitomycin C.

According to the method of the present invention, by mixing a supersaturated calcium phosphate aqueous solution in which a desired supported molecule is dissolved and a hydrophilic organic solvent, CaP fine particles having a high biocompatibility Ca / P molar ratio of 1.3 or more, Carbonate-containing CaP fine particles having a Ca / P molar ratio of 1.3 or more and preferably containing 1 to 15% by weight of a carbonate group CO ₃ ^2- can be rapidly precipitated. This step can be performed, for example, as follows.

(1) It is preferable to prepare a suspension (referred to herein as “CaP fine particle nucleus liquid”) containing fine particles that serve as nuclei for forming CaP fine particles having a Ca / P molar ratio of 1.3 or more. The fine particles serving as the core of the CaP fine particles are not particularly limited as long as they have high biocompatibility and assist the crystal growth of calcium phosphate, and it is not always necessary to use calcium phosphate. As the CaP fine particle nuclear liquid of calcium phosphate, for example, a calcium phosphate liquid in which CaP having a Ca / P molar ratio of 1.3 or more has been precipitated is stirred once and allowed to stand for 10 minutes or longer (calcium phosphate microcrystals float. Or the stirred calcium phosphate solution may be used as it is. In addition, a suspension of calcium phosphate or non-calcium phosphate fine particles or powder such as hydroxyapatite and octacalcium phosphate, after reacting calcium ions and phosphate ions to form a low crystalline apatite gel-like precipitate. Qing etc. can be mentioned. Further, CaP particles or powder in which a part of the calcium component or the phosphoric acid component is partially substituted with other metal ions, an atomic group such as a carbonate group or a sulfate group can also be used. Examples of the non-calcium phosphate CaP fine particle nucleus liquid include collagen fine powder, phospholipid fine powder, titanium oxide fine powder, fine powder having a syranol group, and a liquid in which barium apatite fine powder is suspended. However, it is not limited to these. An appropriate base material may be used as the CaP nucleus instead of the fine particles, and in this case, a CaP film is formed on the surface of the base material. By selecting an appropriate CaP nucleus, the phase of the CaP fine particles to be generated can be intentionally controlled. For example, even in the case of a supersaturated calcium phosphate aqueous solution in which carbonate-containing amorphous calcium phosphate is produced as fine particles without nuclei, the formation of carbonate-containing amorphous calcium phosphate is suppressed by inserting an appropriate nucleus, and the produced fine particles are carbonated apatite. It can be made into fine particles. In addition, when the supersaturated calcium phosphate aqueous solution is an unstable calcium phosphate supersaturated solution that spontaneously generates CaP, fine particles serving as a nucleus are not necessarily required.

(2) Preferably, the CaP fine particle nucleus liquid is added to and mixed with the supersaturated calcium phosphate aqueous solution. The mixing ratio of the CaP fine particle nucleus liquid is not particularly limited, and the mixing timing is not particularly limited, but a volume ratio of about 1/10 to 1/100 is added immediately before proceeding to the next stage. It is preferable.

(3) A solvent having a relative dielectric constant of 80 or less, preferably a hydrophilic organic solvent, is added to and mixed with the supersaturated calcium phosphate aqueous solution (2) or the supersaturated calcium phosphate aqueous solution not mixed with the CaP fine particle nucleus liquid. The addition and mixing conditions of the solvent are not particularly limited. For example, CaP fine particles may precipitate rapidly while capturing supported molecules simply by mixing and stirring at room temperature. For example, when ethanol is added as a hydrophilic organic solvent to a stable supersaturated calcium phosphate solution, the precipitation rate varies depending on the mixed concentration of ethanol, but in general, the ethanol ratio is 5% (v / v) or more. In the case of adding and mixing in such a manner, precipitation of CaP fine particles starts to occur when left at room temperature for 10 minutes, and reaches an almost equilibrium state after being left overnight. Regarding the loading amount, for example, when the supported molecule is albumin and the initial albumin concentration in the supersaturated calcium phosphate aqueous solution is 0.25 mg / ml, the loading amount of albumin may be 65% at the maximum. Further, when mitomycin C was dissolved in a methanol: ethanol mixture (volume ratio 1: 9) so as to be 50 μg / ml and mixed with a supersaturated calcium phosphate aqueous solution containing 0.25 mg / ml albumin, 3 minutes later, % Mitomycin C may be supported on CaP microparticles having a size of 0.22 μm or more. However, it is not limited to these conditions.

  The particle size of the CaP fine particles having a Ca / P molar ratio of 1.3 or more produced by the method of the present invention is not particularly limited, but is, for example, in the range of 10 nm to 1000 μm, preferably 10 nm to 500 μm, more preferably 200 nm to 10 μm. When the upper limit of the particle size of the CaP fine particles exceeds 1000 μm, the cells may not be able to be taken into the cells by phagocytosis. When the particle size lower limit of CaP fine particles having a Ca / P molar ratio of 1.3 or more is less than 10 nm, the particle dispersion may become a substantially colloidal solution and the concentration step may be difficult. The particle size of CaP fine particles having a Ca / P molar ratio of 1.3 or more can be measured by a sedimentation method or a light scattering method based on the Stokes principle. The shape of the fine particles is not particularly limited, and any shape such as a spherical shape, a polyhedron, a plate shape, or a rod shape is allowed.

  The thickness of the calcium phosphate film produced by the method of the present invention is not particularly limited. In this specification, the term “film” means, for example, a state in which an area of 5% or more of the substrate surface area is coated, and includes a case where the substrate surface is not completely coated. . The “film” means a thin film formed on the surface of the base material by the deposition of calcium phosphate carrying supported molecules. Usually, the thickness of the film is about 10 nm to 100 μm. The area covered by the film formed on the substrate surface or the thickness of the film can be measured, for example, with an optical microscope or an electron microscope.

  The phase of CaP microparticles can be identified by methods known to those skilled in the art. For example, fine particles may be separated from the solution, dried, more preferably freeze-dried, and phase identification may be performed by powder X-ray diffraction. If the fine particles are crystalline, the morphology of the self-shaped crystal particles is observed by a method known to those skilled in the art such as a scanning electron microscope and an optical microscope, and the crystal plane uniquely determined by the lattice constant and the crystal point group The crystal phase can be identified with an angle between.

  The Ca / P molar ratio of the CaP fine particles can be obtained by chemically analyzing the solution after the fine particles are filtered by various methods known to those skilled in the art and calculating the Ca decrease amount and the P decrease amount of the solution. . More preferably, the fine particles may be directly chemically analyzed. When calcium carbonate coexists, the amount of calcium carbonate can be quantified and corrected by powder X-ray diffraction. The Ca / P molar ratio of the fine particles can also be determined by identifying the phase by powder X-ray diffraction or the like and specifying the Ca / P molar ratio specific to the phase. For example, if the fine particles are identified as carbonate-containing apatite, the Ca / P molar ratio inherent to the carbonate-containing apatite is 1.5 or more, and thus the Ca / P molar ratio of the fine particles is found to be 1.5 or more.

  Whether the supported molecules are supported on the CaP microparticles is determined by using a method of measuring the protein concentration of the supernatant after centrifugation of the microparticles at 282 nm or a protein staining solution when the supported molecules are proteins. It can be confirmed by colorimetric measurement or fluorescence measurement. When the non-supported substance is a colored substance, it can be confirmed by colorimetrically measuring the concentration of the specific support substance in the supernatant after centrifugal sedimentation of the fine particles. Whether the supported molecule is supported on the CaP film can be confirmed in the same manner as described above by removing the substrate having the CaP film from the liquid when the supported molecule is a protein. Further, after separating the film from the base material, the calcium phosphate of the film is dissolved with an acid to form a solution, and the protein concentration or colored substance concentration of this solution may be measured by colorimetry or fluorescence.

  By the method of the present invention, the supported molecules are not simply adsorbed on the surface of the fine particles, but are trapped in the CaP grain boundaries and in the grains. This is because the CaP fine particles are prepared in advance and the substance is adsorbed to them. By using it as a comparative control, it can be easily confirmed. Further, for example, in addition to observation with a transmission electron microscope, it can also be confirmed by examining the release behavior of the supported molecules from the fine particles. If the supported molecules are simply adsorbed on the surface of the fine particles, all the supported molecules can be released without decomposing or dissolving the fine particles, but the supported molecules are trapped in the CaP grain boundaries and within the grains. In such a case, all supported molecules cannot be released unless the entire fine particles are decomposed or dissolved. Therefore, using a solution saturated with calcium phosphate or a solution in which calcium phosphate hardly dissolves and a solution in which calcium phosphate dissolves (comparative control), CaP fine particles are introduced into these solutions to reduce the amount of supported molecules released. By comparing, it can be confirmed that the supported molecules are trapped in the CaP grain boundaries or grains.

  After mixing, the solute can be rapidly concentrated by physical means to accelerate the precipitation of CaP fine particles. For that purpose, the following means can be used in appropriate combination. 1) A step of concentrating solutes by means including freezing, heat transpiration, microwave irradiation, laser irradiation, vacuum transpiration, or a combination thereof, 2) using a dehydrating agent and / or a specific absorbent of the hydrophilic organic solvent. 3) a step of concentrating the solute in one solvent using a separation membrane of water and a hydrophilic organic solvent. However, these means are for illustrative purposes and are not limited thereto. The recovery of the CaP fine particles can be achieved, for example, by centrifuging the CaP fine particle precipitate and removing the supernatant. Further, the CaP fine particles can be recovered by filtering the CaP fine particle suspension using a filter having an appropriate pore size. However, the collecting means is not limited to these. In addition, for example, methylcellulose or a surfactant can be added to keep the obtained CaP fine particles from agglomerating so as to keep the fine particles, or the pH is kept neutral or weakly alkaline so that the CaP fine particles do not dissolve. A buffering agent or the like can also be added. As described above, in order to use the CaP fine particles of the present invention as, for example, a medicine, it is possible to appropriately mix various preparation additives available to those skilled in the art into the CaP fine particles.

  Without being bound to any particular theory, CaP fine particles having a Ca / P molar ratio of 1.3 or more produced by the method of the present invention are trapped inside the CaP fine particles in which the supported molecules are not easily dissolved. Thus, the supported molecules cannot be easily detached from the CaP fine particles. Further, when the CaP fine particles are gradually dissolved, desired supported molecules are gradually released from the CaP fine particles. Therefore, the CaP microparticles of the present invention are useful as a medicament for releasing a supported molecule in a desired tissue or organ over a long period of time (this medicament is referred to as “sustained release medicament” in the present specification). It is known that CaP having a Ca / P molar ratio of 1.3 or more is safe even when inserted in a place other than bone, and the CaP microparticles of the present invention can be used as a highly safe sustained-release medicine. CaP having a Ca / P molar ratio of 1.3 or more is poorly soluble in body fluids, but for example, in weakly acidic organelles (such as lysosomes), CaP microparticles dissolve faster than neutral conditions. It can be used as an effective sustained-release drug for allowing a supported molecule to act inside a cell. CaP has the property of being taken up into cells by phagocytosis (Cheung, HS, et al., Proc. Soc. Exp. Biol. Med., 173 (2), pp.181-9, 1983). Fine particles are easily taken up into cells by phagocytosis, and phagosomes generated by phagocytosis are fused with lysosomes to become weakly acidic. As a result, the supported molecule is released inside the cell, and the molecule can directly act on the cell from inside the cell. If CaP fine particles having a Ca / P molar ratio of 1.3 or more carry two or more kinds of drugs as supported molecules, the two or more kinds of drugs can simultaneously act on cells. Further, CaP having a Ca / P molar ratio of 1.3 or more can be slowly dissolved by the action of cells or the like in a living body even under neutral conditions. The sustained release property of the supported molecule can be adjusted as appropriate depending on the type and amount of the supported molecule, the size of the CaP fine particles and the type of CaP (such as chemical composition and crystallinity), or the type of the substrate on which the film is formed, It can correspond to various uses.

  When CaP fine particles having a Ca / P molar ratio of 1.3 or more are rapidly precipitated by the method of the present invention, supported molecules dissolved in the solvent are taken into the fine particles, but rapidly precipitated CaP fine particles are Since it precipitates on the fine CaP fine particle nucleus currently disperse | distributed in a calcium-phosphate supersaturated solution instead of on large CaP like a CaP coating metal flat plate, it remains in the state of a CaP fine particle suspension as a whole. Addition of fine CaP fine particle nuclei from outside does not wait for the precipitation of small CaP fine particle nuclei that naturally occur in the calcium phosphate supersaturated solution, but the growth of CaP fine particles is promoted. Still, it remains in the state of CaP fine particle suspension.

  When the supported molecule is a water-soluble polymer (for example, protein), a part of the supported molecule is supported so as to jump out like a bowl on the surface of the CaP fine particle having a Ca / P molar ratio of 1.3 or more, and the surface of the CaP fine particle is hydrophilic. May be sex. As a result, aggregation of the CaP fine particles is prevented, and a stable fine particle suspension can be produced. Moreover, if a substance having the same charge is disposed on the surface of the CaP fine particle having a Ca / P molar ratio of 1.3 or more, a stable CaP fine particle suspension in which the fine particles repel each other and the CaP fine particles hardly aggregate with each other can be obtained. Can do. These techniques can be appropriately used by those skilled in the art. If the supported molecule is a hydrophobic substance, the supported molecule is dissolved in a solvent having a relative dielectric constant of 80 or less so that the hydrophobic substance is contained in the CaP fine particles during rapid CaP precipitation. Be captured. A water-soluble supported molecule is dissolved in a calcium phosphate supersaturated solution, a hydrophobic substance is dissolved in a solvent having a relative dielectric constant of 80 or less, and the two solutions are mixed to form a water-soluble supported molecule in one step. A supported molecule and a hydrophobic supported molecule can be simultaneously supported on CaP fine particles having a Ca / P molar ratio of 1.3 or more.

  Furthermore, after producing a CaP fine particle suspension having a Ca / P molar ratio of 1.3 or more carrying a desired supported molecule, the CaP fine particles are recovered by a method well known to those skilled in the art such as a centrifugal separation method or a membrane separation method. If this fine particle is suspended in a solution in which another molecule (second molecule) having an affinity for the supported molecule is dissolved, the second molecule binds to the supported molecule. . By collecting the CaP fine particles bound to the second molecules thus obtained, the CaP fine particles bound to the second molecules can be produced. For example, by carrying a monoclonal antibody protein on CaP microparticles and binding an antigen protein that specifically binds to the antibody, CaP microparticles carrying the antigen protein can be produced. It can be used as a preparation. In addition, a sustained release CaP fine particle preparation containing the molecule and the supported molecule can be produced by selecting a molecule that is easily adsorbed directly to CaP and adsorbing the molecule to the CaP fine particle supporting the supported molecule.

  Further, using the same principle as an ELISA (enzyme-linked immunosorbent assay) method using an antibody, a CaP fine particle having a Ca / P molar ratio of 1.3 or more carrying the first molecule as a supported molecule is produced. A CaP fine particle capable of binding a second molecule having an affinity for the first molecule to the fine particle and further supporting a third molecule having an affinity for the second molecule and supporting three kinds of molecules. Can also be manufactured. For example, as a first molecule, a receptor protein that is abundant in the cell membrane is selected, and as a molecule having affinity for the receptor protein, for example, a cytokine, a chemokine, a peptide hormone, a macromolecule having immunoadjuvant activity, or the like is used. Can be bound as a molecule. Low molecular weight compounds (including fatty acid compounds such as prostaglandins, steroid hormones, and drugs such as anticancer drugs such as adriamycin and cisplatin) should also be bound via appropriate molecules that have affinity for the target molecule. Furthermore, by combining these low molecular weight compounds with the affinity for CaP microparticles, it is possible to easily produce a sustained-release drug carrying the target molecule.

  The CaP microparticles having a Ca / P molar ratio of 1.3 or more of the present invention in which an anticancer agent is supported as a supported molecule are useful as sustained-release pharmaceuticals for cancer treatment. As the anticancer agent, for example, mitomycin C, adriamycin, cisplatin or the like can be used. These anticancer agents are supported on CaP microparticles as supported molecules by the method of the present invention, or are bound to appropriate molecules having an affinity for these anticancer agents, and further, the affinity of these anticancer agents for CaP microparticles is utilized. Can be supported on the surface of the CaP fine particles. Thus, by administering the CaP microparticles of the present invention loaded with an anticancer agent as a sustained-release pharmaceutical to a cancer local area, a sustained local concentration of the anticancer agent can be maintained while avoiding frequent administration, and its cancer killing Cancer treatment with reduced systemic side effects by cell action becomes possible. Furthermore, CaP microparticles carrying a supported molecule useful for enhancing immunity can be used as an immune adjuvant. For example, when CaP microparticles carry proteins produced by microorganisms such as tuberculin and phagocytosed by human phagocytic cells, the cells release a large amount of cytokines. This can enhance the immunity.

  In order to easily produce CaP fine particles having a Ca / P molar ratio of 1.3 or more according to the present invention, (1) a calcium phosphate supersaturated aqueous solution and (2) separate containers in a state in which a solvent having a relative dielectric constant of 80 or less is sterilized. The kit filled in can be used. The supported molecules can be dissolved in either one or both of the above (1) and (2), and then the formation of microparticles is about 30 after mixing by simply mixing both without dispensing and weighing operations. Complete within minutes. Such a kit is suitable for use in an aseptic environment such as an operating room because it does not require dispensing or weighing and is used only once. The type, chemical composition, capacity, and the like of the calcium phosphate supersaturated aqueous solution and the solvent having a relative dielectric constant of 80 or less included in the kit can be appropriately selected by those skilled in the art depending on the type of supported molecule to be used.

Hereinafter, the present invention will be described more specifically with reference to examples. However, the scope of the present invention is not limited to the following examples.
Example 1: Phase and composition of CaP microparticles produced from a calcium phosphate supersaturated solution Sodium chloride, potassium chloride, calcium chloride, sodium acetate, potassium dihydrogen phosphate, magnesium chloride, xylitol, sodium bicarbonate Electrolyte concentration: Na ⁺ 147.15, K ⁺ 4.02, Ca ²⁺ 2.24 Cl ⁻ 155.7 mM), P solution (electrolyte concentration: Na ⁺ 45.00 K ⁺ 25.00 Mg ^{2+) _{50 Cl - 45.01 H 2 PO 4}} - 10.00 CH 3 COO - 20.00mM), carbonate solution (electrolyte _{^{concentration: Na + 166, HCO 3 -}} 166mM) was prepared. These liquids were mixed in a polystyrene container so as to have various Ca / P molar ratios and carbonic acid concentrations to obtain calcium phosphate supersaturated solutions (Table 1). The solution volume after mixing is 15 mL in all cases. None of the solvent having a relative dielectric constant of 80 or less, the calcium phosphate fine particle nucleus liquid, and the supported molecules are mixed. After mixing, when left at 37 ° C. for 2 days, the supersaturated solution becomes cloudy and fine particles are produced. The obtained fine particles were subjected to suction filtration with a membrane filter having a pore size of 0.45 μm, washed three times with ultrapure water, and freeze-dried at −80 ° C. The filtrate was analyzed by an induction plasma emission spectroscopy (ICP) method to quantify the amount of Ca reduction and P reduction in the solution due to fine particle formation. From this value, the Ca / P molar ratio of the fine particles was calculated (Table 1). The phase of the freeze-dried fine particles was identified by a powder X-ray diffraction method (CuKα ray). The X-ray diffraction pattern of the fine particles obtained in Experiment Nos. 2 and 7 in Table 1 is shown in FIG. Further, the infrared absorption spectrum of the fine particles was measured by the KBr tablet method (FIGS. 2 and 3), and the carbonic acid content of the fine particles was quantified by measuring the absorption intensity (wave number 1430, 1460 cm ⁻¹ ) of the carbonate group (Table 1). ). For determination of the carbonic acid content, carbonic acid-containing apatite (carbonic acid content: 6.3% by weight) manufactured by STK Ceramics was used as a standard, and a calibration curve prepared after mixing with KBr powder at various ratios was used.

The results in Table 1 indicate that CaP fine particles having a Ca / P molar ratio of 1.3 or more are produced from the calcium phosphate supersaturated solution of the present invention. FIG. 1 shows the powder X-ray diffraction patterns of the fine particles obtained in Experiment Nos. 2 and 7. In Experiment Nos. 1, 3-6, 8, and 9, the tendency of the powder X-ray diffraction pattern was the same. In other words, each of the powder X-ray diffraction patterns of the fine particles has one broad peak having an apex at a diffraction angle of 30 °, and small peaks appearing at positions 29.4 ° and 31.8 ° of the base of the peak. did. The large broad peak at 30 ° demonstrates the presence of amorphous calcium phosphate, and the small peaks at 29.4 ° and 31.8 ° demonstrate the presence of calcium carbonate and apatite, respectively.
A separately prepared amorphous calcium phosphate and calcium carbonate were mixed at various ratios, and powder X-ray diffraction experiments were conducted to compare peak intensities, thereby quantifying the calcium carbonate content of the fine particles obtained in Experiment Nos. 1-9. . As a result, all the fine particles were found to have a calcium carbonate content of less than 1%.

The infrared absorption spectrum of the fine particles, 550 cm ^-1, the absorption band of PO ₄ group in 1050 cm ^-1, the absorption band of CO ₃ groups in 1400-1500cm ^-1, absorption of water and OH to 1630 cm ^-1 and 3400 cm ^-1 A band appeared (Figures 2 and 3). Absorption intensity of PO ₄ group of 1050 cm ^-1 is more than three times the absorption intensity of the CO ₃ groups 1400-1500cm ^-1, the main component of fine particles indicates that it is phosphate. As described above, from the powder X-ray diffraction pattern and the infrared absorption spectrum, the main component of the fine particles is amorphous calcium phosphate.
When the carbonic acid content of the fine particles was quantified from the absorption band strength of the CO ₃ group, it was 1.9-7% by weight. Since the calcium carbonate content of the fine particles is less than 1%, 99.4% or more of the carbonate groups are incorporated into amorphous calcium phosphate and apatite.
From the above results, it is shown that a carbonic acid-containing calcium phosphate having a Ca / P molar ratio of 1.3 or more and a carbonic acid content in the range of 1% by weight to 15% by weight is produced from the calcium phosphate supersaturated solution of the present invention. It was. Furthermore, the results in Table 1 indicate that if the Ca / P molar ratio of the supersaturated solution is the same, the carbonate content of the CaP fine particles can be controlled by changing the carbonate ion concentration to be mixed.

Example 2: Effect of addition of nuclei CaP fine particles were prepared under the same conditions as in Experiment No. 7 in Table 1 of Example 1. However, dry collagen was added as a nucleus. A solvent having a relative dielectric constant of 80 or less and a supported molecule are not mixed. The same operation as in Example 1 was performed, and the powder X-ray diffraction pattern of the precipitate was measured. The result is shown in FIG. In the X-ray diffraction pattern, collagen peaks at around 20 ° and strong apatite peaks at 25.8 ° and 31.8 ° appeared. It shows that the fine particles produced can be changed from amorphous calcium phosphate to apatite by adding an appropriate nucleus to the calcium phosphate supersaturated solution.
A small peak of calcium carbonate appeared at 29.4 °. Separately prepared carbonate-containing apatite (carbonate content 6.8% by weight) and calcium carbonate were mixed in various proportions, powder X-ray diffraction was performed, and the amount of calcium carbonate was quantified by comparing peak intensities. The amount was 4% or less.

Example 3 Effect of Ethanol Addition on CaP Fine Particle Formation Conditions for supporting bovine serum albumin (hereinafter referred to as BSA) on a supersaturated calcium phosphate aqueous solution were examined. As the first step, the influence of ethanol addition on CaP fine particle production was examined. The relative dielectric constant of ethanol is 24.55.
A. Method 1. Preparation of supersaturated calcium phosphate aqueous solution Supersaturated calcium phosphate aqueous solution prepared by mixing Ringer's solution, Klinisalz B solution, and bifil sodium bicarbonate replenisher (NaHCO ₃ 13.9 mg / ml) at a ratio of 8.28: 1.24: 0.48 did. The pH was adjusted to 7.48 by slightly increasing / decreasing the sodium bicarbonate replenisher dedicated to bifill. The supersaturated calcium phosphate aqueous solution prepared in this manner is an unstable supersaturated solution, and the composition is Ca 1.85 mM, P 1.24 mM, Na 135.4 mM, Cl 134.5 mM, K 6.43 mM, Mg 0.31 mM. , CO3 7.97 mM. This solution composition corresponds to the conditions of Experiment No. 2 in Table 1.

2. Preparation and quantification of BSA solution
2.5 mg of BSA powder was gently added to the surface of 10 ml of a supersaturated calcium phosphate aqueous solution with a pH of 7.48 and dissolved at room temperature. Changes in BSA concentration in the following steps were quantified by measuring absorbance at 282 nm. There is a linear correlation between BSA concentration and absorbance at 282 nm.
3. Addition of ethanol to BSA solution and measurement of turbidity 9.5 ml of the BSA solution prepared in the previous section was taken, and 0.5 ml of 99.5% ethanol was added. This is expressed as 5% (v / v) for convenience (the ethanol concentration thereafter is also% (v / v) for convenience). Ethanol was added to 5% (v / v) to 20% (v / v), allowed to stand for 10 minutes, and turbidity was measured by absorbance at a wavelength of 450 nm.

B. Results The results are shown in FIG. One measurement value is an average value obtained from measurement values obtained by measuring the same sample five times individually. The effect of adding ethanol is that the turbidity hardly rises up to 5% (v / v) when left for 10 minutes, but rises rapidly after 10% (v / v), almost at 15% (v / v), Near equilibrium. This result indicates that CaP microparticles are spontaneously generated in a short time by mixing the hydrophilic organic solvent with the supersaturated calcium phosphate aqueous solution containing protein. When left at room temperature for 10 minutes to 18 hours, a sufficiently visible amount of precipitate was generated even at 10% (v / v).

Example 4: Influence of ethanol concentration on loading of BSA on CaP fine particles The influence of ethanol concentration on loading BSA on CaP fine particles was examined.
A. Method 1. Preparation of CaP fine particle nucleate A very small amount (about several mg visually) of existing hydroxyapatite powder was obtained in A.1. When the mixture is added to 200 ml of the supersaturated calcium phosphate aqueous solution prepared and stirred and left to stand overnight, a large amount of CaP fine particles are precipitated. The suspension in which this was stirred was used as a CaP fine particle nucleus liquid.

2. 2. Support of BSA on CaP fine particles 2-1. In the third embodiment described above. 99.5% ethanol is added to the supersaturated calcium phosphate aqueous solution (referred to as (1)), and the ethanol concentration is 10% (v / v) (referred to as (2)) or 15% (v / v) (refer to (3) And so on). After mixing, turbidity was measured by absorbance at a wavelength of 450 nm, and protein amount was measured by absorbance at a wavelength of 282 nm.
2-2. Further, these liquids were filtered through a membrane filter having a pore size of 0.22 μm, and the absorbance of the filtrates was measured in the same manner at the above-mentioned two kinds of wavelengths.
2-3. Next, 0.03 ml of the CaP fine particle nucleus solution was added to 3.97 ml of each of the above-described filtered (1) to (3) solutions, and the absorbance was measured in the same manner at the two wavelengths described above.
2-4. Next, it was allowed to stand at room temperature for 30 minutes, and the absorbance was measured in the same manner at the two wavelengths described above.
2-5. Further, these were filtered through a membrane filter having a pore size of 0.22 μm, and the absorbance of the filtrate was measured in the same manner. The rate of decrease in the amount of BSA remaining in the filtrate at this stage was calculated.
2-6. Next, the sample was allowed to stand at room temperature for 18 hours, and the absorbance was measured in the same manner at the two wavelengths described above.
2-7. Further, these were filtered through a membrane filter having a pore size of 0.22 μm, and the absorbance of the filtrate was measured in the same manner. The rate of decrease in the amount of BSA remaining in the filtrate at this stage was calculated.

B. Results Table 2 shows the effect of ethanol addition on the loading of BSA on CAP microparticles. If the ethanol concentration to be added is 10% (v / v) or less in sub-step 2-1 of step 2 (in the case of (1) and (2)), BSA hardly decreases from the solution ( 9% and 6% respectively). On the other hand, if the ethanol concentration to be added is 15% (v / v), as much as 39% of BSA immediately decreases from the solution. That is, BSA was trapped in CaP fine particles of a size that cannot pass through 0.22 μm pores or adsorbed on the surface. This result was obtained by mixing a hydrophilic organic solvent with a supersaturated calcium phosphate aqueous solution containing BSA. It shows that BSA is trapped in CaP fine particles or adsorbed on the surface over time ((3) on the right side of 2-2 in Table 2). When CAP fine particle nucleate is added to this and allowed to stand at room temperature for 30 minutes, BSA decreases from the solution as turbidity increases (CAP fine particle precipitation). This ratio is 29% when ethanol is not added, while 53% when ethanol concentration to be added is 10% (v / v) and 15% (v / v) when ethanol is added. In the case of v), it is 43%. Furthermore, when left overnight (18 hours), this percentage becomes 40%, 65%, and 55%, respectively, with further increase in turbidity. The ethanol concentration was 10% (v / v) after 30 minutes and 18 hours. Thus, if alcohol is added and the precipitation of CaP microparticles is accelerated, protein can be carried on CAP microparticles with high efficiency. When the ethanol concentration is 10% (v / v), the pH of the solution 30 minutes after ethanol addition is 7.28 when there is no BSA and 7.50 when there is BSA, and there is no significant change in the pH of the solution. It was.

Example 5: Precipitation A of BSA-supported CaP fine particles. Method
It was examined by sedimentation rate measurement whether CaP fine particles supporting BSA are difficult to aggregate. Example 1 Add 1/100 volume CAP fine particle nucleate to the supersaturated calcium phosphate aqueous solution prepared in step 1 and add 99.5% ethanol to 10% (v / v), and leave it for a sufficient time to leave the CaP fine particle suspension. Was made. Further, in Example 3, 2. Add 1/100 volume CAP fine particle nuclei solution to the BSA solution prepared in step 1, and then add 99.5% ethanol to 10% (v / v) and leave it for a sufficient time to suspend the BSA-supported CaP fine particles. A liquid was prepared. These BSA-unsupported CAP fine particle suspension and BSA-supported CAP fine particle suspension were stirred well again and allowed to stand, and the change in turbidity of the supernatant was measured by absorbance at a wavelength of 450 nm.

B. Results FIG. 5 shows the turbidity of CaP fine particles when BSA is supported (line with BSA in the figure) and CaP fine particles when not supported (line without BSA in the figure) and their sedimentation velocities. It is the figure which showed the result of having compared by the change measurement of turbidity. When CaP fine particles are precipitated for a sufficient time and suspended again, the fine particles carrying BSA have higher turbidity than the case where BSA is not carried. This result shows that finer CaP fine particles were produced because CaP fine particles carrying BSA hardly aggregated with each other.
When this suspension is allowed to stand, it is observed that the rate of decrease in turbidity is slower between 10 minutes and 60 minutes when BSA is supported than when BSA is not supported. Moreover, a peak is observed in the difference between the case of supporting BSA and the case of non-supporting BSA around 50 minutes when the CaP fine particles in the case of non-supporting BSA almost settled (FIG. 6). In the case of carrying BSA, it was visually observed that the CaP fine particles remained in the supernatant in a turbid state like a wrinkle. This is because the CaP fine particles are less likely to aggregate due to the hydrophilicity of BSA, and very fine CaP fine particles that are more stable and difficult to sink are generated and slowly sink.

Example 6: Preparation of CaP fine particles supporting BSA and mitomycin C dissolved in methanol Method 2. of the above-described third embodiment. Mitomycin C (Wako Pure Chemicals, catalog number 132-13201) was added to the BSA solution prepared in step 1 with a volume of 1/100 CAP microparticle nucleus solution and dissolved in methanol, a hydrophilic organic solvent, at a concentration of 50 μg / ml. A solution was added to examine whether BSA and mitomycin C were simultaneously supported on CAP microparticles. Since mitomycin C is a blue pigment having an absorption peak at 365 nm, it was quantified by measuring absorbance at 365 nm. BSA was quantified by measuring absorbance at 282 nm as in Example 4. The relative dielectric constant of methanol is 20.7.
4-1. As described in Example 3 above. The mitomycin C solution was added to the BSA solution prepared in step 1 at a concentration of 10% (v / v).
4-2. Further, these liquids were filtered through a membrane filter having a pore size of 0.22 μm.
Since the influence of turbidity can be removed, the absorbance at wavelengths of 282 nm and 365 nm at this stage was measured.
4-3. Next, 0.03 ml of CAP fine particle nucleus solution was added to 3.97 ml of each of the above-mentioned filtered (1) to (2) solutions.
4-4. Next, it was left for 30 minutes at room temperature.
4-5. Further, these were filtered through a membrane filter having a pore size of 0.22 μm, and the absorbance of the filtrate was measured in the same manner.
The 4-6.4-3 stage was left at room temperature for 18 hours.
4-7. Further, these were filtered through a membrane filter having a pore size of 0.22 μm, and the absorbance of the filtrate was measured in the same manner.

B. Results Table 3 shows the results. Although CaP microparticles are rapidly formed even when methanol in which mitomycin C is dissolved is added, the mitomycin C concentration immediately after the addition is unknown because of the large influence of turbidity that changes immediately after the addition. . However, if the clear filtrate after filtration through a membrane filter having a pore size of 0.22 μm is used as the starting point, 3% after 30 minutes and 8% mitomycin C after 18 hours are further supported on the CAP microparticles. Can be estimated. It is considered that 17% of BSA was loaded with mitomycin C after 30 minutes, and 29% was further loaded after 18 hours. These are the rate of increase in loading from the time when the original mixture was filtered through a 0.22 μm pore size membrane filter.

Example 7: Preparation of CaP fine particles carrying BSA and mitomycin C dissolved in dimethyl sulfoxide (DMSO) Mitomycin C was dissolved in DMSO, which is a hydrophilic organic solvent, and it was examined whether it was carried together with BSA. At this time, the influence of ethanol was also examined.
A. Method 5-1. A mixed solution was prepared with the combinations shown in Table 4. The blank is 1. Of a supersaturated calcium phosphate aqueous solution.
5-2. Further, these liquids were filtered through a membrane filter having a pore size of 0.22 μm.
Since the influence of turbidity can be removed, the absorbance at wavelengths of 282 nm and 365 nm at this stage was measured.
5-3. Next, 0.03 ml of the CAP fine particle nucleus solution was added to 3.97 ml of the filtered solution.
5-4. Next, it was left at room temperature for 30 minutes.
5-5. Further, these were filtered through a membrane filter having a pore size of 0.22 μm, and the absorbance of some filtrates was measured in the same manner.
The 5-6.5-3 stage was left at room temperature for 18 hours.
5-7. Further, these were filtered through a membrane filter having a pore size of 0.22 μm, and the absorbance of the filtrate was measured in the same manner.

In Table 4, A is 1. The supersaturated calcium phosphate aqueous solution, B, of Example 3-2. BSA solution, C is DMSO solution of mitomycin C (concentration is 0.5 mg / ml), D is DMSO, and E is 99.5% ethanol. The unit of the numerical value was ml, and each mixed solution was prepared.

B. Results Table 5 shows the rate of increase in loading from the stage when the initial mixture was filtered through a 0.22 μm pore size membrane filter. As for BSA, 10% (v / v) DMSO ((3)) is added to the case where only the calcium phosphate supersaturated aqueous solution is used ((1)), while the increase rate was 8.1% after 30 minutes. As a result, the increase rate was 23.2%, and by adding 10% (v / v) ethanol ((8)), the increase rate was 19.8%. Therefore, the effect of accelerating the precipitation of CaP fine particles by these hydrophilic organic solvents is clear. After a sufficient precipitation time (18 hours), this difference in rate of increase narrowed. The loading increase rate after 18 hours was 28.7% to 59.3% for BSA and 0.5% to 4.2% for mitomycin C after addition of various hydrophilic organic solvents (including mitomycin C solution). Accordingly, by dissolving water-soluble substances in a calcium phosphate supersaturated aqueous solution, dissolving hydrophobic substances in a hydrophilic organic solvent, and mixing the respective solutions, the CaP fine particles can be supported together in one step. it can.

In Table 5, the solution numbers correspond to the solution numbers in Table 4. Measurement was performed using A in Table 4 as a blank. Absorbance (282 nm and 365 nm) after standing at room temperature for 18 hours was measured. The CaP fine particles have already begun to precipitate in the mixed liquid of 5-1 stage, but the calculation starting point of the following loading rate is the 5-2 stage. Therefore, the loading increase rate is set.

Example 8: Capture of albumin into CaP fine particles (part 1)
It is said that albumin cannot be supported on hydroxyapatite by conventional methods. By the method of the present invention, it was proved by the following method that albumin was not only adsorbed on the surface of the CaP fine particles but also captured inside the CaP fine particles.
A. Method 6-1. 27 ml of a supersaturated calcium phosphate aqueous solution prepared in Example 3 was prepared. To this, 1/100 volume of CaP fine particle nuclei described in Example 4 and 3 ml of 99.5% ethanol (10% (v / v) for convenience) were added to form CaP fine particles, and left at room temperature for 18 hours. This is the control group.
6-2. 6.75 mg of BSA powder was added to the control group tube in which precipitation occurred, and after complete dissolution and stirring, the mixture was allowed to stand at room temperature for 18 hours.
6-3. To 54 ml of the supersaturated calcium phosphate aqueous solution prepared in Example 3 described above, 13.5 mg of BSA was added and completely dissolved, stirred and the absorbance at 282 nm was measured. To this, 1/100 volume of CaP fine particle nucleate described in Example 4 and 6 ml of 99.5% ethanol were added to produce CaP fine particles, and left at room temperature for 18 hours. This is a BSA group.
6-4. 0.22 μm filter filtration after 18 hours. The absorbance at 282 nm of both groups after filtration was measured.

B. Results Table 6 shows the results. When calculated from the A282 measurement value before addition of the CaP fine particle nucleate and alcohol, it is 0.222 after the addition of the CaP fine particle nucleate and alcohol (assuming that the total capacity does not change due to the addition of alcohol). In the control group, BSA was sufficiently adsorbed 18 hours after the addition of BSA, and the amount adsorbed on the surface of the CaP fine particles can be seen from the difference from the measured A282 value of the control group after 0.22 μm filter filtration. On the other hand, the BSA content directly captured in the CaP microparticles can be calculated from the final difference between the control group and the BSA group. The combined loading of both was 30.3%. Of these, the ratio of direct capture was calculated to be about 1/5 (17.9%).

Example 9: Capture of albumin into CaP microparticles (part 2)
This experiment was an experiment that saw the reproducibility of the experiment of Example 8. The results are shown in Table 7. The BSA loading rate, which combines the adsorbed and directly captured components, was 55.4%, and the proportion of directly captured components was calculated to be about 1/5 (20.6%). Reproducibility is considered good.

Powder X-ray diffraction pattern of CaP fine particles prepared without using a solvent with a relative dielectric constant of 80 or less and supported molecules Infrared absorption spectrum of fine particles prepared under the conditions of Experiment Nos. 1-4 in Table 1 without using a solvent having a relative dielectric constant of 80 or less, a supported molecule, or a CaP nuclear liquid. Infrared absorption spectrum of fine particles prepared under the conditions of Experiment Nos. 5-9 in Table 1 without using a solvent having a relative dielectric constant of 80 or less, a supported molecule, or a CaP nuclear liquid. It is the figure which showed the influence of ethanol addition with respect to CaP fine particle production | generation. It is the figure which showed the influence of BSA regarding the sedimentation rate of CaP microparticles | fine-particles. It is the figure which showed the influence of BSA regarding the sedimentation rate of CaP microparticles | fine-particles.

Claims (8)

A method for producing calcium phosphate fine particles having a Ca / P molar ratio of 1.3 or more carrying one or more supported molecules, selected from the group consisting of methanol, ethanol , acetone, pyridine, dimethyl sulfoxide, and polyethylene glycol Including the step of precipitating calcium phosphate fine particles from the calcium phosphate aqueous solution by mixing one or two or more hydrophilic organic solvents with the calcium phosphate supersaturated aqueous solution, and either or both of the above calcium phosphate supersaturated aqueous solution and the above hydrophilic organic solvent Includes a supported molecule, the Ca / P molar ratio in the calcium phosphate supersaturated aqueous solution is 0.5 or more and 2.5 or less, the HCO ₃ ²⁻ concentration is 60 mM or less, and the particle diameter of the calcium phosphate fine particles described above The manufacturing method whose is 10 nm-10 micrometers. 2. The production method according to claim 1, wherein the calcium phosphate fine particles are carbonic acid-containing calcium phosphate fine particles containing 1 wt% to 15 wt% of carbonate group CO ₃ ^2- . The production method according to claim 1, wherein the calcium phosphate fine particles are carbonate-containing apatite containing 1 to 15% by weight of carbonate group CO ₃ ^2- . The production method according to any one of claims 1 to 3, wherein the calcium phosphate supersaturated aqueous solution contains a hydrophilic supported molecule. The method according to claim 4, wherein the hydrophilic supported molecule is albumin. 6. The production method according to claim 1, wherein the hydrophilic organic solvent contains a hydrophobic supported molecule. The method according to claim 6, wherein the hydrophobic supported molecule is an anticancer agent. The manufacturing method of any one of Claim 1 thru | or 7 including the process of mixing a calcium-phosphate fine particle nucleus liquid in precipitation of said calcium-phosphate fine particle.