Metal nanoparticle-containing calcium phosphate composite particles and method for producing the same

Abstract

To provide calcium phosphate composite particles containing metal-nanoparticles for high-sensitivity bioimaging, and a method of producing the same.SOLUTION: There are provided calcium phosphate composite particles containing metal-nanoparticles of good dispersibility, with the metal-nanoparticles discretely distributed across whole calcium phosphate matrix at a high filling degree and the molar ratio of the metal element relative to calcium being 0.4 or more. There is provided a method of producing calcium phosphate composite particles containing metal-nanoparticles, comprising a step of mixing / stirring metal-nanoparticles, a calcium-containing liquid and a phosphorous acid-containing liquid to prepare a supersaturated solution, and leaving the mixture stand still or stirring the same for a specified period of time, with the metal-nanoparticle being characterized by comprising a polar functional group derived from a low molecule, disposed on the surface thereof.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to metal nanoparticle-containing calcium phosphate composite particles and a method for producing the same. [Background technology]

[0002] Because metal nanoparticles possess unique physical properties and functions that differ from bulky solid metals, technologies that apply these properties and functions are used in various fields. For example, in the fields of biology and medicine, metal nanoparticles are used as bioimaging agents for the visualization or detection of specific elements in cells, tissues, and living organisms, as well as for the visualization of structures and biological processes. In particular, metal nanoparticles with a diameter of 6 nm or less are generally excreted from the body as urine by the kidneys, so there is little concern about their retention or accumulation in the body. On the other hand, it is not easy to deliver and accumulate a sufficient amount for bioimaging at the target site in the body (e.g., a tumor site). In fact, some fluorescent metal nanoparticles with low brightness have low detection sensitivity, making them difficult to detect in tissues with strong autofluorescence or in vivo.

[0003] Strategies for improving bioimaging capabilities using metal nanoparticles include increasing the brightness of the metal nanoparticles and improving the delivery efficiency of the metal nanoparticles to the target site in the body. Controlling the structure and composition of the particles is effective in increasing the brightness of metal nanoparticles, but in some cases, supporting them on a support (matrix) is also effective. For example, supporting fluorescent metal nanoparticles on a matrix restricts the Brownian motion of the particles, which reduces the probability of dynamic extinction and can improve brightness. Non-patent document 1 discloses that the fluorescence brightness of gold nanoparticles is improved by supporting them on a matrix made of mesoporous silica. On the other hand, an effective method for improving the delivery efficiency of metal nanoparticles is to support the metal nanoparticles on a larger submicron-sized matrix to create composite particles containing metal nanoparticles.

[0004] As the main component of the matrix supporting metal nanoparticles, calcium phosphate, which is an inorganic component of human teeth and bones, is considered particularly useful because it has low toxicity, excellent biocompatibility, and decomposes into serum ions under weakly acidic conditions for absorption by the body. Based on this background, metal nanoparticle-containing calcium phosphate composite particles are expected to have applications as high-performance bioimaging agents.

[0005] To efficiently deliver metal nanoparticles to target sites within the body without impairing their functions (fluorescence, excretion after release from the matrix, etc.), it is necessary to create metal nanoparticle-containing calcium phosphate composite particles that contain a large amount of metal nanoparticles, are highly packed and discretely supported throughout the matrix, and have excellent dispersibility in aqueous solutions containing bodily fluids. However, obtaining dispersible metal nanoparticle-containing calcium phosphate composite particles without using dispersants such as surfactants is not easy. Furthermore, it has been difficult to support metal nanoparticles with extremely high surface energy in a discrete state with high packing density throughout the calcium phosphate matrix without agglomerating them.

[0006] Patent Document 1 discloses a method for synthesizing dispersible metal nanoparticle-containing calcium phosphate composite particles. However, this method requires the addition of a third component that functions as a dispersant to the composite particles. Non-Patent Document 2 discloses a method for synthesizing dispersible metal nanoparticle-containing calcium phosphate composite particles without using a dispersant by using metal nanoparticles whose surfaces are modified with a certain type of polymer chain. However, in this method, the packing density of the metal nanoparticles in the calcium phosphate matrix is ​​not sufficient because the surface of the metal nanoparticles is covered with a relatively thick polymer layer (molar ratio of metal element to calcium = 0.15 or less). Non-Patent Document 3 discloses a method for synthesizing core-shell type metal nanoparticle-containing calcium phosphate composite particles by forming a core by introducing metal nanoparticles into polyacrylic acid and then covering its surface with calcium phosphate. However, in this method, the metal nanoparticles are only supported in the core portion and hardly supported in the calcium phosphate shell portion. Non-Patent Documents 4 and 5 disclose a method for synthesizing metal nanoparticle-containing calcium phosphate composite particles by irradiating a suspension of metal ion-containing calcium phosphate particles with laser light to precipitate metal nanoparticles in a calcium phosphate matrix. However, this method made it difficult to control the size and distribution of metal nanoparticles in the calcium phosphate matrix. [Prior art documents] [Patent Documents]

[0007] [Patent Document 1] Patent No. 6998008 [Non-patent literature]

[0008] [Non-Patent Document 1] L. Ma et al., Analyst, 143 (2018) 5388-5394 [Non-Patent Document 2] M. Nakamura et al., Colloids and Surfaces B: Biointerfaces, 194 (2020) 111169 [Non-Patent Document 3] L. Li et al., Small, 11 (2015) 3162-3173 [Non-Patent Document 4] M. Nakamura et al., Materials, 12 (2019) 4234 [Non-Patent Document 5] M. Nakamura et al., Acta Biomaterialia, 46 (2016) 299-307 [Overview of the project] [Problems that the invention aims to solve]

[0009] This document provides calcium phosphate composite particles containing metal nanoparticles for high-sensitivity bioimaging, and a method for producing the same. [Means for solving the problem]

[0010] In one embodiment of the present invention, a dispersible metal nanoparticle-containing calcium phosphate composite particle is provided, which contains metal nanoparticles and calcium phosphate, wherein the metal nanoparticles, having polar functional groups on their surface throughout the calcium phosphate matrix, are supported discretely and with high packing density, and the molar ratio of the metal element to calcium is 0.4 or higher.

[0011] The metal nanoparticles may also be gold nanoparticles.

[0012] The polar functional groups on the surface of metal nanoparticles may be derived from low molecular weight molecules.

[0013] The polar functional groups on the surface of the metal nanoparticles may be derived from glutathione or 2-mercaptopropionic acid.

[0014] The particle diameter of the metal nanoparticles may be 6 nm or less, preferably 1 nm or more and 6 nm or less, and more preferably 1 nm or more and 3 nm or less.

[0015] The diameter or major axis of the calcium phosphate composite particles containing metal nanoparticles may be 10 nm or more and 200 nm or less, preferably 30 nm or more and 100 nm or less.

[0016] The calcium phosphate in the calcium phosphate composite particles containing metal nanoparticles may be amorphous calcium phosphate.

[0017] The fluorescence intensity or X-ray absorption amount of the calcium phosphate composite particles containing metal nanoparticles may be higher than the fluorescence intensity or X-ray absorption amount of the metal nanoparticles alone.

[0018] The fluorescence intensity of the calcium phosphate composite particles containing metal nanoparticles may be 2 times or more higher than the fluorescence intensity of the metal nanoparticles alone.

[0019] The absolute value of the zeta potential of the calcium phosphate composite particles containing metal nanoparticles may be 5 mV or more.

[0020] In one embodiment of the present invention, there is provided a method for producing calcium phosphate composite particles containing metal nanoparticles, which includes a step of mixing, stirring a metal nanoparticle, a calcium-containing solution, and a phosphate-containing solution to prepare a supersaturated solution, and allowing it to stand or stir for a certain period of time, wherein the metal nanoparticles have polar functional groups derived from low molecules on their surfaces.

Effect of the Invention

[0021] It is possible to provide calcium phosphate composite particles containing metal nanoparticles for high-sensitivity bioimaging and a method for producing the same.

Brief Description of the Drawings

[0022] [Figure 1]This figure shows (a) a photograph of the dispersion of gold nanoparticle-containing calcium phosphate composite particles prepared in Example 1, (b) a transmission electron microscope image (wide area), (c) a transmission electron microscope image (magnified), and (d) the particle size distribution. [Figure 2] This figure shows the fluorescence spectrum of the gold nanoparticle-containing calcium phosphate composite particles prepared in Example 1. [Figure 3] This figure shows the fluorescence intensity per mole of gold element in the gold nanoparticle-containing calcium phosphate composite particles prepared in Example 1. [Figure 4] This is a photograph of the dispersion of gold nanoparticle-containing calcium phosphate composite particles prepared in Example 2. [Figure 5] These are transmission electron microscope images (upper panel: wide area, lower panel: magnified) of the gold nanoparticle-containing calcium phosphate composite particles prepared in Example 2. [Figure 6] This figure shows the zeta potential of the gold nanoparticle-containing calcium phosphate composite particles prepared in Example 2. [Figure 7] This figure shows the fluorescence spectrum of the gold nanoparticle-containing calcium phosphate composite particles prepared in Example 2. [Figure 8] This figure shows the fluorescence intensity per mole of gold element in the gold nanoparticle-containing calcium phosphate composite particles prepared in Example 2. [Figure 9] This figure shows the molar ratio of gold to calcium in the gold nanoparticle-containing calcium phosphate composite particles prepared in Example 2. [Figure 10] This is a photograph of the dispersion of gold nanoparticle-containing calcium phosphate composite particles prepared in Example 3. [Figure 11] This figure shows the particle size distribution of the gold-calcium phosphate composite particles prepared in Example 3. [Figure 12] This figure shows the gold concentration of the gold nanoparticle-containing calcium phosphate composite particles prepared in Example 3. [Figure 13] This figure shows the molar ratio of gold element to calcium in the gold nanoparticle-containing calcium phosphate composite particles prepared in Example 3. [Figure 14]This figure shows the fluorescence spectrum of the gold nanoparticle-containing calcium phosphate composite particles prepared in Example 3. [Figure 15] This figure shows the fluorescence intensity per mole of gold element in the gold nanoparticle-containing calcium phosphate composite particles prepared in Example 3. [Figure 16] This figure shows (a) a solution photograph, (b) a transmission electron microscope image, and (c) the particle size distribution of the gold-calcium phosphate composite particles prepared in Example 4. [Figure 17] This figure shows the fluorescence spectrum of the gold nanoparticle-containing calcium phosphate composite particles prepared in Example 4. [Modes for carrying out the invention]

[0023] The inventors discovered that by co-precipitating metal nanoparticles and calcium phosphate in a supersaturated aqueous solution of calcium phosphate to which metal nanoparticles having a certain polar functional group on its surface were added, dispersible calcium phosphate composite particles containing metal nanoparticles with high packing density and discrete arrangement could be obtained without the need for a dispersant. In the obtained metal nanoparticle-containing calcium phosphate composite particles, the metal nanoparticles were supported with high packing density and discrete arrangement throughout the calcium phosphate matrix. Furthermore, it was found that the fluorescence intensity of the metal nanoparticles was enhanced by supporting them in a calcium phosphate matrix, leading to the completion of the present invention.

[0024] The metal nanoparticle-containing calcium phosphate composite particles according to the present invention have structural characteristics suitable for administration into the body. First, the metal nanoparticle-containing calcium phosphate composite particles have a size of 10 nm to 500 nm in diameter or major axis (determined by electron microscopy analysis, the same applies hereinafter) and are dispersible in aqueous solutions such as body fluids and injection solutions, so they can be administered into the body by intravenous or arterial injection. Once administered into the body, the metal nanoparticle-containing calcium phosphate composite particles, which contain a large amount of metal nanoparticles dispersedly and with high packing density, function as a highly sensitive imaging agent. Eventually, the metal nanoparticle-containing calcium phosphate composite particles decompose under certain conditions (time, pH, etc.) (the matrix calcium phosphate dissolves), releasing the metal nanoparticles. When the calcium phosphate in the composite particles dissolves, it becomes an ion that is originally contained in body fluids. On the other hand, the released metal nanoparticles are nano-sized and excretable by the kidneys, and due to the polar functional groups on their surface, they exhibit excellent dispersibility even in high ionic strength solutions such as body fluids, and are therefore excreted from the body. In other words, the metal nanoparticle-containing calcium phosphate composite particles according to the present invention are biodegradable particles that can deliver metal nanoparticles into living organisms, and there is no concern about adverse effects on living organisms (residue, accumulation, toxicity, etc.) from the degradation products (metal nanoparticles, etc.), making them useful in applications such as medicine, biomaterials, and imaging agents. Embodiments of the present invention will be described in detail below.

[0025] [Calcium phosphate composite particles containing metal nanoparticles] This invention relates to metal nanoparticle-containing calcium phosphate composite particles containing metal nanoparticles and calcium phosphate. Metal nanoparticle-containing calcium phosphate composite particles refer to submicron-sized dispersible particles consisting of a matrix mainly composed of a calcium phosphate compound and containing at least metal nanoparticles as a support. Submicron-sized particles here refer to particles with a diameter or major axis of 10 nm to 1000 nm, but in this embodiment in particular, it refers to particles with a diameter or major axis of 10 nm to 500 nm that can be administered into the body by intravenous or arterial injection and are suitable for uptake by cells. In this embodiment, the metal nanoparticle-containing calcium phosphate composite particles are preferably particles with a diameter or major axis of 10 nm to 200 nm, and among them, particles with a diameter or major axis of 30 nm to 100 nm are particularly preferred, in order to enable cancer-specific accumulation in the body due to the Enhanced Permeability and Retention (EPR) effect.

[0026] In this embodiment, the metal nanoparticle-containing calcium phosphate composite particles contain a large amount of metal nanoparticles dispersedly and with high density throughout the calcium phosphate matrix. The molar ratio of the metal element to calcium in the metal nanoparticle-containing calcium phosphate composite particles is preferably 0.4 or higher, and more preferably 0.4 to 3.0. The state of discretely supported metal nanoparticles refers to a state in the calcium phosphate matrix where adjacent metal nanoparticle cores are neither in contact with nor bonded to each other. Other substances exist between the cores, and electron microscopy analysis reveals regions around each core with different compositions and crystal structures from the core itself.

[0027] In this embodiment, dispersible particles refer to particles that can maintain a monodisperse state for 30 minutes or more immediately after generation or after redispersion in an injection solution, and do not aggregate or settle. The maintenance of a monodisperse state can be confirmed by particle size distribution using dynamic light scattering (DLS). Particles that become monodisperse after 30 minutes, either immediately after generation or after redispersion in an injection solution, by applying vibration such as ultrasonic irradiation or vortexing are also acceptable. Achieving a monodisperse state allows for in vivo administration by intravenous or arterial injection. Suitable injection solutions for redispersing the particles include sterile water for injection. From the viewpoint of stability (solubility) of the calcium phosphate compound constituting the particle matrix and safety when administered in vivo, a weakly acidic to weakly alkaline aqueous solution with a pH of 5 to 9, preferably 6.5 to 8.0, is preferred for the injection solution.

[0028] In this embodiment, the support material is metal nanoparticles, which, when supported on the surface and inside the calcium phosphate particles, exhibit functions useful for biological research, diagnosis, and treatment, and contribute to maintaining the dispersibility of the composite particles. The metal nanoparticles may be a single particle or two or more types of particles. In addition to metal nanoparticles, the support material may also contain substances other than metal nanoparticles.

[0029] In this embodiment, metal nanoparticles are nanoparticles having a core made of a substance whose composition formula includes a metal element. The polar functional groups on the surface of the metal nanoparticles govern the dispersibility in a calcium phosphate supersaturated solution and the interaction with calcium phosphate and its components (ions and calcium phosphate clusters), and thus affect the structure and properties of the resulting metal nanoparticle-containing calcium phosphate composite particles. Therefore, the substance forming the core of the metal nanoparticles is not limited. Examples include gold nanoparticles with fluorescence and X-ray CT (Computed Tomography) contrast ability, quantum dots such as fluorescent CdSe / ZnS nanoparticles, and iron oxide nanoparticles with MRI (Magnetic Resonance Imaging) contrast ability. Among these, gold nanoparticles are particularly preferred because they are effective as probes for fluorescence observation of cell and tissue sections, photothermal converters for hyperthermia therapy, sensitizers (X-ray sensitizers) for radiotherapy, and contrast agents for X-ray CT.

[0030] The metal nanoparticles in this embodiment have polar functional groups on their surface. Preferably, the polar functional groups are those that have a strong interaction with calcium phosphate compounds or their constituent components (ions or calcium phosphate clusters) and are effective in maintaining the dispersibility of the metal nanoparticles. Examples include polar functional groups such as carboxyl groups, sulfo groups, and phosphate groups, which have a negative charge in near-neutral solutions, and polar functional groups such as amino groups and quaternary ammonium groups, which have a positive charge, as well as nonionic polar functional groups such as hydroxyl groups, carbonyl groups, and thiol groups. The polar functional groups on the surface of the metal nanoparticles may be one type or two or more types.

[0031] Polar functional groups may be introduced to the surface of metal nanoparticles by modifying the surface of the metal nanoparticles with a molecule containing one or more polar functional groups. Preferably, the molecule having polar functional groups is a low molecular weight of 1000 or less, which has strong interactions not only with calcium phosphate compounds or their components (ions or calcium phosphate clusters) but also with metal nanoparticles, and can maintain the dispersibility of the metal nanoparticles. If a polymer with a molecular weight exceeding 1000 is used instead of a low molecular weight, the surface of the metal nanoparticles will be coated with a thick polymer layer, increasing the distance between metal nanoparticles in the calcium phosphate matrix and reducing the density of nanoparticle packing (the elemental ratio of metal to calcium will decrease). By surface modification with a low molecular weight molecule having polar functional groups, the metal nanoparticles coated with a thin low molecular weight layer are supported in the calcium phosphate matrix discretely and with high density.

[0032] When the metal nanoparticles are gold nanoparticles or silver nanoparticles, the low molecular weight molecules having polar functional groups are preferably those having thiol groups that chemically bond to the surface of these metal nanoparticles and polar functional groups that interact with calcium phosphate or its constituent ions. Examples include glutathione, cysteine, cysteamine, mercaptoethanol, mercaptopropionic acid, ethanethiol, and their derivatives. Among these, glutathione and 2-mercaptopropionic acid are particularly preferred low molecular weight molecules.

[0033] The metal nanoparticles, possessing polar functional groups on their surface, exhibit excellent dispersibility even in high ionic strength solutions. Furthermore, in calcium phosphate supersaturated solutions, they interact with calcium phosphate compounds or their components, co-precipitating with calcium phosphate. In this way, metal nanoparticle-containing calcium phosphate composite particles can be obtained in which the metal nanoparticles are highly packed and discretely supported.

[0034] The surface of the metal nanoparticle-containing calcium phosphate composite particles has polar functional groups of metal nanoparticles, which can impart a zeta potential (absolute value) of 5 mV or more, preferably 10 mV or more, to the composite particles. Because the metal nanoparticle-containing calcium phosphate composite particles have a zeta potential (absolute value) of 5 mV or more, they can maintain a monodisperse state for a long time due to mutual repulsion, even in high ionic strength solutions. Therefore, there is no need to support a third component that functions as a dispersant within the composite particles, nor is there any need to add dispersants such as surfactants to the solution.

[0035] Metal nanoparticle-containing calcium phosphate composite particles have metal nanoparticles dispersed and highly packed throughout the calcium phosphate matrix. When the metal nanoparticles are fluorescent metal nanoparticles, the calcium phosphate matrix restricts the Brownian motion of the fluorescent metal nanoparticles, reducing the probability of dynamic extinction. For this reason, the fluorescence intensity of the fluorescent metal nanoparticle-containing calcium phosphate composite particles is higher than that of the fluorescent metal nanoparticles alone. Preferably, the fluorescence intensity of the fluorescent metal nanoparticle-containing calcium phosphate composite particles is at least twice as high as that of the fluorescent metal nanoparticles alone. Here, fluorescence intensity refers to the fluorescence intensity per mole of metal element. When the fluorescent metal nanoparticles are gold nanoparticles, preferably, the fluorescence intensity of the gold nanoparticle-containing calcium phosphate composite particles is at least twice as high as that of the gold nanoparticles alone, and more preferably at least 10 times higher.

[0036] Furthermore, if the metal nanoparticles are metal nanoparticles with X-ray contrast properties, the amount of X-ray absorption due to the calcium phosphate matrix is ​​added, resulting in a higher amount of X-ray absorption than that of the metal nanoparticles alone.

[0037] The metal nanoparticles are smaller than the metal nanoparticle-containing calcium phosphate composite particles. Specifically, the diameter or major axis (core size, determined by electron microscopy) of the metal nanoparticles is preferably 6 nm or less, and more preferably between 1 nm and 6 nm. A particularly preferred diameter or major axis (core size) for metal nanoparticles is between 1 nm and 3 nm, as this allows for high renal excretion of the metal nanoparticles alone.

[0038] The composition and structure of the calcium phosphate compound contained in the metal nanoparticle-containing calcium phosphate composite particles are not limited. The calcium phosphate compound is a compound containing at least a phosphate ion and a calcium ion, and may be amorphous calcium phosphate, a crystalline calcium phosphate compound, or a mixture thereof. Examples of crystalline calcium phosphate compounds include, but are not limited to, hydroxyapatite, carbonate apatite, α-tricalcium phosphate, β-tricalcium phosphate, or octacalcium phosphate. It may be amorphous calcium phosphate immediately after production and spontaneously crystallize during subsequent washing, drying, storage, or in a dispersion, or it may be artificially crystallized by adding aging treatment, hydrothermal treatment, etc. Since the solubility of calcium phosphate changes depending on its crystal structure and crystallinity, the solubility can be adjusted by controlling the crystal structure and crystallinity depending on the application. Furthermore, some or all of the constituent ions (phosphate, calcium, or hydroxide ions, etc.) of the above calcium phosphate compound may be substituted with other ions (carbonate ions, fluoride ions, zinc ions, sodium ions, magnesium ions, or potassium ions, etc.). Particularly preferred examples of calcium phosphate compounds include amorphous calcium phosphate, octacalcium phosphate, and low-crystallinity hydroxyapatite, which exhibit excellent biodegradability and can be synthesized easily and rapidly in the liquid phase.

[0039] Metal nanoparticle-containing calcium phosphate composite particles may contain, in addition to the calcium phosphate and metal nanoparticles mentioned above, other materials such as substances useful for diagnosis or treatment as supported materials. Any substance that has a strong interaction with the calcium phosphate compound can be co-supported in the calcium phosphate matrix. Examples of such substances include molecules with polar functional groups, such as nucleic acids (DNA, RNA, miRNA, or siRNA), nucleotides, proteins, peptides, glycans, antibodies, enzymes, or coenzymes, which may be used as they are, or substances that have been compounded with polar functional groups that have a strong interaction with the calcium phosphate compound may be used.

[0040] In metal nanoparticle-containing calcium phosphate composite particles, the calcium phosphate compound serves as a matrix that supports the metal nanoparticles on the surface and within the calcium phosphate particles. By being supported in a submicron-sized calcium phosphate matrix, the metal nanoparticles are delivered efficiently to target sites in the body without being excreted by the kidneys. For example, the EPR effect of submicron-sized particles can deliver metal nanoparticles specifically to cancer cells and allow them to accumulate in cancer tissue. To further enhance the delivery efficiency of metal nanoparticles, a substance that specifically binds to target cells, organs, or tissues may be added to the surface of the metal nanoparticle-containing calcium phosphate composite particles.

[0041] In the acidic environment of the body (e.g., cancerous tissue or inflamed areas), or within endosomes after being taken up into cells in the affected area, calcium phosphate compounds dissolve into calcium ions and phosphate ions, which are naturally present in body fluids. The freed metal nanoparticles are excreted from the body due to their high renal excretion rate.

[0042] [Manufacturing method] Metal nanoparticle-containing calcium phosphate composite particles can be synthesized in a supersaturated aqueous solution of calcium phosphate (hereinafter sometimes simply referred to as a supersaturated solution). Specifically, a supersaturated solution is prepared by mixing metal nanoparticles, a calcium-containing solution containing calcium ions, and a phosphate-containing solution containing phosphate ions. pH adjusters may be added as needed during mixing. After mixing, it is preferable to stir the supersaturated solution homogenized by vortexing or shaking. By letting this supersaturated solution stand or stirring for a certain period of time, the desired composite particles can be obtained. The obtained composite particles may be used as is immediately after production, or they may be washed and then redispersed in an injection solution before use.

[0043] The solutions used as raw materials for calcium phosphate supersaturated solutions are not limited. Examples of calcium-containing solutions containing calcium ions include aqueous solutions of calcium chloride, calcium nitrate, calcium acetate, calcium carbonate, calcium sulfate, calcium lactate, calcium citrate, or intravenous fluid preparations containing calcium ions. Examples of phosphate-containing solutions containing phosphate ions include aqueous solutions of dipotassium hydrogen phosphate, dicalcium hydrogen phosphate, potassium dihydrogen phosphate, calcium dihydrogen phosphate, phosphate-buffered saline, or intravenous fluid preparations containing phosphate ions. As pH adjusters, pH buffers that can adjust the pH of the supersaturated solution to near neutral may be used, or alkalizing agents that can gradually increase the pH of the supersaturated solution to an alkaline pH of 7 or higher may be used. Examples of alkalizing agents include intravenous fluid preparations containing sodium bicarbonate, sodium carbonate, potassium bicarbonate, potassium carbonate, or bicarbonate ions, which can increase the pH of the solution by decarboxylation.

[0044] The temperature at which the calcium phosphate supersaturated solution is allowed to stand or be stirred should be above the freezing point and below the boiling point of the solution. However, if the temperature is too high, the precipitation of calcium phosphate in the calcium phosphate supersaturated solution will be accelerated, making it difficult to obtain submicron-sized composite particles. The ideal temperature range is 0°C to 50°C, with room temperature to 40°C being particularly suitable.

[0045] There is no limit to the time for standing or stirring the calcium phosphate supersaturated solution. Standing or stirring may take as little as 0.1 seconds, but prolonged standing or stirring may cause the composite particles to continue growing in the calcium phosphate supersaturated solution. Furthermore, an increase in the concentration of the generated composite particles may reduce dispersibility. In such cases, the standing or stirring time should be shortened, for example, from 1 minute to 180 minutes, or even from 1 minute to 60 minutes. If the generated composite particles are not to be used immediately, they should be washed and stored.

[0046] The preferred concentration range for calcium ions, phosphate ions, and metal nanoparticles in a calcium phosphate supersaturated solution varies depending on their type, as well as the temperature and time during which the supersaturated solution is allowed to stand or is stirred. However, if the concentration of calcium ions or phosphate ions is too low, the loading rate of metal nanoparticles (the amount of metal nanoparticles loaded onto the generated particles relative to the amount of metal nanoparticles in the calcium phosphate supersaturated solution) decreases. On the other hand, the loading rate of metal nanoparticles can be increased by moderately increasing the concentrations of these ions. [Examples]

[0047] (Example 1) Gold nanoparticle-containing calcium phosphate composite particles were prepared using gold nanoparticles surface-modified with calcium phosphate and glutathione.

[0048] "Sample preparation" (Fabrication of glutathione-modified gold nanoparticles) As reagents for synthesizing gold nanoparticles, tetrahydrate chloroauric acid (Fujifilm Wako Pure Chemical Industries, Ltd.), tetrakis(hydroxymethyl)phosphonium chloride solution (Sigma Aldrich), aqueous sodium hydroxide solution (Fujifilm Wako Pure Chemical Industries, Ltd.), and reduced glutathione (Fujifilm Wako Pure Chemical Industries, Ltd.) were used. A visking tube (AS ONE) was used as the dialysis membrane for the washing procedure. To an aqueous chloroauric acid solution (0.001 M, 235 mL), tetrakis(hydroxymethyl)phosphonium chloride solution (0.07 M, 7.5 mL) and aqueous sodium hydroxide solution (1 M, 7.5 mL) were added in sequence. Then, a reduced glutathione solution (0.25 M, 2 mL) was added, and the mixture was stirred at room temperature for 6 hours to produce glutathione-modified gold nanoparticles. After washing the obtained glutathione-modified gold nanoparticles using a dialysis membrane, the solvent was removed to prepare a concentrated dispersion of glutathione-modified gold nanoparticles (Au: 0.2 M). Note that this gold concentration was calculated assuming no loss due to reduction reactions or concentration processes. Hereafter, the glutathione-modified gold nanoparticles obtained here will be referred to as AuNC.

[0049] (Preparation of AuNC-containing calcium phosphate composite particles) First, three raw material solutions for calcium phosphate supersaturated solutions (calcium-containing solution, phosphate-containing solution, and pH adjuster) were prepared. The calcium-containing solution was prepared by mixing Ringer's Solution "Otsuka" (Otsuka Pharmaceutical Co., Ltd.) (49.553 mL) and Calcium Chloride Correction Solution 1 mEq / mL (Otsuka Pharmaceutical Co., Ltd.) (0.447 mL). The phosphate-containing solution was prepared by mixing Clinisalz® Infusion (Kyowa CritiCare Co., Ltd.) (9.633 mL) and Dipotassium Phosphate Injection 20 mEq Kit "Terumo" (Terumo Corporation) (0.367 mL). The pH adjuster was prepared by mixing Meiron® Injection 7% (Otsuka Pharmaceutical Co., Ltd.) (5 mL) and a mixture of Water for Injection (Fuso Pharmaceutical Industries, Ltd.) (20 mL).

[0050] A reaction solution (10 mL) was prepared by mixing AuNC concentrated dispersion (0.1 mL), water for injection (0.4 mL), calcium-containing solution (7.674 mL), phosphate-containing solution (0.917 mL), and pH adjuster (0.909 mL) (concentrations in the reaction solution: Ca: 5.1 mM, P: 2.6 mM, Au: 2 mM). The reaction solution was left to stand in a 37°C incubator for 60 minutes. After 60 minutes, the precipitate was washed and collected by repeated centrifugation (15,000 rpm, 15 minutes) and redispersion, and then redispersed in water for injection (10 mL) to obtain the sample dispersion. Hereafter, the sample obtained here will be referred to as AuNC / CaP.

[0051] "Evaluation of the structure and physical properties of a sample" The morphology and composition of the obtained AuNC / CaP were investigated by transmission electron microscopy (TEM) observation and radiofrequency inductively coupled plasma emission spectroscopy (ICP). In TEM observation, the morphology of AuNC / CaP dried on a grid was observed. In ICP, the powder obtained by freeze-drying the AuNC / CaP dispersion was dissolved in aqua regia, and the solution diluted 50-fold with ultrapure water was used for measurement to calculate the gold and calcium content in the sample. Furthermore, after sonication of the AuNC / CaP dispersion after 30 minutes or more had elapsed since preparation, particle size distribution measurements were performed by dynamic light scattering (DLS), zeta potential measurements by electrophoretic light scattering (ELS), and fluorescence spectrum measurements (excitation wavelength: 460 nm) were performed using a spectrofluorometer. Figure 1 shows (a) a photograph of the dispersion, (b) a transmission electron microscope image (wide area), (c) a transmission electron microscope image (magnified), and (d) the particle size distribution of the AuNC / CaP prepared in Example 1. Figures 2 and 3 show the fluorescence spectra of AuNC / CaP prepared in Example 1 and the fluorescence intensity per mole of gold element.

[0052] "result" (Solution photograph, TEM observation) The AuNC / CaP dispersion was yellowish-white (Figure 1(a)). This suggests that the white calcium phosphate contains yellow AuNC. TEM observation (Figures 1(b), (c)) revealed that the AuNC / CaP particles were nearly spherical in shape and submicron in size with a particle diameter of 65.2 nm ± 1.6 nm (mean ± standard error). Magnified images (Figure 1(c)) showed that numerous AuNC particles (black dots) (particle diameter: 2.2 ± 0.7 nm (mean particle size ± standard error)) were discretely present inside and on the surface of the calcium phosphate matrix while maintaining their particle shape.

[0053] (ICP measurement) Furthermore, when the molar ratio of gold element to calcium (Au / Ca molar ratio) of AuNC / CaP was calculated to be 0.7, it was found that we successfully created composite particles with a high density of AuNC. It is thought that in a reaction solution with a high degree of supersaturation, calcium phosphate formed a uniform nucleation, and grew as a matrix by adsorbing and incorporating the dispersible AuNC, thereby forming AuNC-containing calcium phosphate composite particles with discrete and high density of AuNC.

[0054] (DLS / ELS measurement) In the particle size distribution obtained by DLS measurement, the AuNC / CaP dispersion showed a single peak, and its average hydrodynamic diameter (number-average diameter) was 126 nm (Figure 1(d)). That is, AuNC / CaP were submicron-sized monodisperse particles. Furthermore, ELS measurement results showed that the zeta potential of AuNC / CaP was -14.5 mV. AuNC has a negative surface charge because its surface is modified with glutathione containing a carboxyl group. It is thought that the presence of AuNC on the surface caused AuNC / CaP to exhibit a relatively large negative zeta potential. Generally, particles with small absolute values ​​of zeta potential tend to aggregate, while particles with larger absolute values ​​of zeta potential exhibit higher dispersibility due to inter-particle repulsion. The threshold zeta potential for dispersible particles is considered to be 5-10 mV (absolute value), and since AuNC / CaP has a zeta potential (absolute value) that exceeds this, it is thought to have exhibited monodispersibility.

[0055] (Fluorescence spectroscopy and ICP measurement) The fluorescence spectrum measurements (Figure 2) showed that the maximum fluorescence wavelength for both AuNC and AuNC / CaP was around 570 nm. Based on the amount of gold element calculated by ICP, the fluorescence intensity per mole of gold element (at 570 nm) for both AuNC and AuNC / CaP was calculated to be 1.3 × 10⁻⁶, respectively. 7 , 4.0×10 8 This was the case (Figure 3). Comparing the two, it was found that the fluorescence intensity of AuNC / CaP was 31 times that of AuNC. From the above, we found that the fluorescence intensity of AuNC is enhanced by complexation with calcium phosphate. This is thought to be because the Brownian motion of AuNC is restricted when it is supported and immobilized in the matrix (calcium phosphate), and the probability of dynamic quenching decreases.

[0056] (Example 2: Change in AuNC concentration) In this example, samples prepared by varying the AuNC concentration were evaluated.

[0057] "Sample preparation" Samples were prepared in the same manner as in Example 1, except that the amounts of AuNC concentrated dispersion and sterile water for injection used were varied to set the gold concentration in the reaction solution to 0, 0.6, 1, 2, 4, and 10 mM (concentrations in the reaction solution: Ca: 5.1 mM, P: 2.6 mM, Au: 0-10 mM). Samples obtained from the reaction solutions with gold concentrations of 0, 0.6, 1, 2, 4, and 10 mM were designated as 1, 2, 3, 4, 5, and 6, respectively. Sample 4 is the same as the sample prepared in Example 1.

[0058] "Evaluation of the structure and physical properties of a sample" TEM observation, DLS-ELS measurement, ICP measurement, and fluorescence spectral measurement were performed on the obtained samples (1-6) using the same method as in Example 1. Figure 4 shows a photograph of the dispersion of AuNC / CaP prepared in Example 2. Figure 5 shows transmission electron microscope images of AuNC / CaP prepared in Example 2 (upper row: wide area, lower row: magnified). Figure 6 shows the zeta potential of AuNC / CaP prepared in Example 2. Figure 7 shows the fluorescence spectrum of AuNC / CaP prepared in Example 2. Figure 8 shows the fluorescence intensity per mole of gold element of AuNC / CaP prepared in Example 2. Figure 9 shows the molar ratio of gold element to calcium element of AuNC / CaP prepared in Example 2.

[0059] "result" (Solution photograph, TEM observation) The CaP dispersion of sample 1 was white, while the AuNC / CaP dispersions of samples 2-6 were yellowish-white (Figure 4). TEM observation revealed that the CaP in sample 1 contained a mixture of particles of various shapes and sizes (Figure 5). On the other hand, the AuNC / CaP dispersions of samples 2-6 all consisted of relatively uniform, nearly spherical, submicron-sized particles (Figure 5). The particle diameters of the AuNC / CaP dispersions of samples 2-6 were 91.0±1.9 nm, 90.8±1.6 nm, 65.2±1.6 nm, 51.7±1.3 nm, and 34.8±0.7 nm (mean ± standard error), respectively, and the size of the generated particles decreased with increasing gold concentration in the reaction solution. In reaction solutions with high gold concentration, it is thought that the AuNC suppressed the growth of composite particles by covering the surface of the growing calcium phosphate matrix more quickly and extensively.

[0060] (DLS / ELS measurement) In DLS measurements, CaP 1 exhibited polydispersity, making it impossible to obtain reliable results. This is thought to be due to the generation of particles of various shapes and sizes, as shown in TEM observations. On the other hand, AuNC / CaP 2-6 showed a single peak in the particle size distribution obtained by DLS measurements, and their average hydrodynamic diameters (number-average diameters) were 251, 205, 126, 83, and 62 nm, respectively, confirming that they were all submicron-sized monodisperse particles.

[0061] ELS measurements revealed that the zeta potentials of CaP or AuNC / CaP samples 1-6 were -0.5, -15.7, -15.9, -14.5, -15.5, and -16.4 mV, respectively (Figure 6). In the case of CaP sample 1, the repulsive force between calcium phosphate particles produced in the reaction solution without AuNC was insufficient (absolute value of zeta potential less than 1), which is thought to have resulted in particles of various shapes and sizes due to particle bonding and aggregation. On the other hand, the absolute values ​​of the zeta potentials of AuNC / CaP samples 2-6, produced from reaction solutions with added AuNC, were all around 15 mV. This indicates that the repulsive force between particles was sufficiently large, resulting in monodisperse composite particles with a relatively narrow particle size distribution, as each particle grew uniformly while maintaining a monodisperse state.

[0062] (Fluorescence spectroscopy and ICP measurement) No peaks were observed in the fluorescence spectrum of CaP 1. On the other hand, the maximum fluorescence wavelength of AuNC / CaP 2-6 was around 570 nm (Figure 7). The fluorescence intensity per mole of gold element (570 nm) for AuNC / CaP 2-6 was 5.8 × 10⁻⁶, respectively. 8 , 4.8×10 8 , 4.0×10 8 , 3.1×10 8 , and 1.9 × 10 8 As the gold concentration in the reaction solution increased, the fluorescence intensity per mole of gold (570 nm) decreased (Figure 8).

[0063] Furthermore, the molar ratio of gold to calcium (Au / Ca molar ratio) for CaP or AuNC / CaP for samples 1-6 was calculated to be 0.0, 0.4, 0.6, 0.7, 0.8, and 0.9, respectively (Figure 9). It was found that the Au / Ca molar ratio increased as the gold concentration in the reaction solution increased. This result indicates that the packing density of AuNC in the composite particles increased as the gold concentration in the reaction solution increased.

[0064] Based on the above, it is possible to adjust the particle size of the composite particles, the packing density of the metal nanoparticles, and the fluorescence intensity per mole of gold element by adjusting the gold concentration in the reaction solution.

[0065] (Example 3) In this example, samples prepared by varying the concentration of calcium ions in the calcium-containing solution and the concentration of phosphate ions in the phosphate-containing solution were evaluated.

[0066] "Sample preparation" Samples were prepared in the same manner as in Example 2, step 6, except that the concentration of calcium ions in the calcium-containing solution and the concentration of phosphate ions in the phosphate-containing solution were changed. The concentrations of calcium ions and phosphate ions in the reaction solution were increased from the conditions of Example 2, step 6, and sample prepared under conditions of Ca: 7.7 mM, P: 3.9 mM was designated as sample 7, and sample prepared under conditions of Ca: 10.3 mM, P: 5.1 mM was designated as sample 8 (gold concentration in reaction solutions 6-8: 10 mM).

[0067] "Evaluation of the structure and physical properties of a sample" ICP, DLS, and fluorescence spectral measurements were performed on the obtained samples (6-8) using the same method as in Example 1. Figure 10 shows a photograph of the AuNC / CaP dispersion prepared in Example 3. Figure 11 shows the particle size distribution of the AuNC / CaP prepared in Example 3. Figure 12 shows the gold concentration of the AuNC / CaP prepared in Example 3. Figure 13 shows the molar ratio of gold element to calcium element in the AuNC / CaP prepared in Example 3. Figure 14 shows the fluorescence spectrum of the gold nanoparticle-containing calcium phosphate composite particles prepared in Example 3. Figure 15 shows the fluorescence intensity per mole of gold element element prepared in Example 3.

[0068] "result" (Solution photo) All AuNC / CaP dispersions from 6 to 8 exhibited a yellowish-white color (Figure 10). Macroscopic examination revealed no aggregate formation, suggesting good dispersibility. Furthermore, the solution color deepened with increasing concentrations of calcium and phosphate ions in the reaction solution.

[0069] (DLS measurement) In the particle size distribution by DLS measurement (Figure 11), AuNC / CaP of 6 - 8 showed a single peak, and the average value of the hydrodynamic diameter (number average diameter) was 62, 70, and 119 nm respectively. Thus, it was confirmed that all the generated particles were sub - micron - sized monodisperse particles.

[0070] (ICP measurement) The gold concentrations in the dispersions of AuNC / CaP of 6 - 8 were 1.3, 2.9, and 4.8 mM in order (Figure 12). It was found that with the increase in the concentrations of calcium ions and phosphate ions in the reaction solution, more AuNCs were carried in the generated particles. That is, the loading rate of AuNC could be increased by increasing the concentrations of calcium ions and phosphate ions in the reaction solution. Also, when calculating the molar ratio of gold element to calcium (Au / Ca molar ratio) of AuNC / CaP of 6 - 8, they were 0.9, 0.6, and 0.6 respectively (Figure 13). It is considered that the reason is that the increase in the concentrations of calcium ions and phosphate ions further increased the supersaturation of the reaction solution, increased the nucleation frequency and growth rate of calcium phosphate, and as a result, the amount of calcium phosphate matrix holding AuNC increased.

[0071] (Fluorescence spectrum measurement) The maximum fluorescence wavelength of AuNC / CaP of 6 - 8 was around 570 nm, and the maximum fluorescence intensity increased with the increase in the concentrations of calcium ions and phosphate ions in the reaction solution (Figure 14). On the other hand, when calculating the fluorescence intensity (570 nm) per mole of gold element of AuNC and AuNC / CaP of 6 - 8, they were 1.1×10 7 、1.9×10 8 、1.7×10 8 、1.6×10 8The AuNC / CaP values ​​for groups 6-8 were 17, 15, and 15 times higher than those for AuNC, respectively, but no significant difference was observed between the comparison groups (6-8) (Figure 15). Therefore, by increasing the concentrations of calcium ions and phosphate ions in the reaction solution, we succeeded in recovering more AuNC as composite particles and improving their loading rate within the same reaction scale, while maintaining the effect of the calcium phosphate matrix in enhancing the fluorescence intensity of AuNC.

[0072] (Example 4) In this example, gold nanoparticle-containing calcium phosphate composite particles were prepared using gold nanoparticles surface-modified with 2-mercaptopropionic acid.

[0073] "Sample preparation" (Preparation of 2-mercaptopropionic acid-modified gold nanoparticles) As reagents for synthesizing gold nanoparticles, tetrahydrate chlorauric acid, reduced glutathione, and 2-mercaptopropionic acid (Fujifilm Wako Pure Chemical Industries, Ltd.) were used. To 177.5 mL of ultrapure water at 80°C, aqueous solution of chlorauric acid (0.1 M, 8 mL) and a solution of reduced glutathione (0.25 M, 4.8 mL) were added and stirred for 3 hours, then cooled in ice water (4°C) for 10 minutes. Subsequently, aqueous solution of sodium hydroxide (1 M, 5.5 mL) and 2-mercaptopropionic acid (0.25 M, 4.8 mL) were added in sequence, and the mixture was stirred at room temperature for 3 hours to produce 2-mercaptopropionic acid-modified gold nanoparticles. After washing the dispersion of 2-mercaptopropionic acid-modified gold nanoparticles using a dialysis membrane, the solvent was removed to prepare a concentrated dispersion of 2-mercaptopropionic acid-modified gold nanoparticles (Au: 0.2 M). Note that this gold concentration was calculated assuming no loss due to reduction reactions or concentration operations. Hereafter, the 2-mercaptopropionic acid-modified gold nanoparticles obtained here will be referred to as AuNC-M.

[0074] (Preparation of calcium phosphate composite particles containing AuNC-M) The sample was prepared in the same manner as in Example 3, step 8, except that AuNC-M concentrated dispersion was used instead of AuNC concentrated dispersion (concentrations in the reaction solution: Ca: 10.3 mM, P: 5.1 mM, Au: 10 mM). The obtained sample is denoted as AuNC-M / CaP.

[0075] "Evaluation of the structure and physical properties of a sample" DLS measurements and fluorescence spectral measurements were performed on AuNC-M / CaP using the same method as in Example 1. The gold concentration of the AuNC-M dispersion used for fluorescence spectral measurement was the same as that of the reaction solution used to prepare AuNC-M / CaP (Au: 10 mM). Figure 16 shows (a) a photograph of the solution, (b) a transmission electron microscope image, and (c) the particle size distribution of the AuNC-M / CaP prepared in Example 4. Figure 17 shows the fluorescence spectrum of the AuNC-M / CaP prepared in Example 4.

[0076] "result" (Solution photographs, TEM observation, ICP measurement, DLS measurement) The AuNC-M / CaP dispersion was yellowish-white (Figure 16(a)). This suggests that the white calcium phosphate contains yellow AuNC-M. TEM observation revealed that the AuNC-M / CaP particles were nearly spherical in shape, with a particle size of 74.5 nm ± 3.9 nm (mean ± standard error) (Figure 16(b)). Numerous AuNC particles (black dots) were observed to be discretely present within and on the surface of the calcium phosphate matrix, maintaining their particle shape. ICP measurement calculated the molar ratio of gold element to calcium (Au / Ca molar ratio) of AuNC-M / CaP to be 2.4, indicating successful fabrication of composite particles with high AuNC density. DLS measurement of the particle size distribution showed that AuNC-M / CaP exhibited a single peak, and its average hydrodynamic diameter (number-average diameter) was 192 nm, confirming that it was a submicron-sized monodisperse particle (Figure 16(c)).

[0077] From the above, it has become clear that discrete and highly packed metal nanoparticle-containing calcium phosphate composite particles can be produced even when using gold nanoparticles surface-modified with 2-mercaptopropionic acid.

[0078] (Fluorescence spectrum) In the fluorescence spectra of dispersions of AuNC-M and AuNC-M / CaP, it was confirmed that the maximum fluorescence wavelength was almost the same for both, around 570 nm, and that the fluorescence intensity of AuNC-M was enhanced by compounding with calcium phosphate (Figure 17). From the amount of gold element calculated by ICP, the fluorescence intensity per mole of gold element (570 nm) for AuNC-M and AuNC-M / CaP was calculated to be 4.5 × 10⁻⁶, respectively. 4 , 1.1 × 10 7 The results showed that the fluorescence intensity of AuNC-M / CaP was 242 times greater than that of AuNC-M.

[0079] From the above, it has become clear that even when using gold nanoparticles surface-modified with 2-mercaptopropionic acid, it is possible to produce composite particles containing gold nanoparticles with enhanced fluorescence intensity.

Claims

1. Dispersible calcium phosphate composite particles containing metal nanoparticles, wherein metal nanoparticles having polar functional groups derived from low molecular weights of 1000 or less on their surface are highly packed and discretely supported throughout the calcium phosphate matrix, and the molar ratio of the metal element to the calcium is 0.4 or higher.

2. The metal nanoparticle-containing calcium phosphate composite particle according to claim 1, wherein the metal nanoparticles are gold nanoparticles.

3. The metal nanoparticle-containing calcium phosphate composite particle according to claim 1, wherein the polar functional group is derived from glutathione or 2-mercaptopropionic acid.

4. The metal nanoparticle-containing calcium phosphate composite particle according to claim 1, wherein the particle size of the metal nanoparticles is 6 nm or less.

5. The metal nanoparticle-containing calcium phosphate composite particle according to claim 1, wherein the diameter or major axis of the metal nanoparticle-containing calcium phosphate composite particle is 10 nm or more and 200 nm or less.

6. The metal nanoparticle-containing calcium phosphate composite particle according to claim 1, wherein the calcium phosphate is amorphous calcium phosphate.

7. The metal nanoparticle-containing calcium phosphate composite particle according to claim 1, wherein the fluorescence intensity or X-ray absorption of the metal nanoparticle-containing calcium phosphate composite particle is higher than that of the metal nanoparticle alone.

8. The metal nanoparticle-containing calcium phosphate composite particle according to claim 1, wherein the fluorescence intensity of the metal nanoparticle-containing calcium phosphate composite particle is at least twice as high as the fluorescence intensity of the metal nanoparticle alone.

9. The metal nanoparticle-containing calcium phosphate composite particle according to claim 1, wherein the absolute value of the zeta potential of the metal nanoparticle-containing calcium phosphate composite particle is 5 mV or more.

10. The process includes mixing metal nanoparticles, a calcium-containing solution, and a phosphoric acid-containing solution, stirring to prepare a supersaturated solution, and then allowing it to stand or stir for a certain period of time. The metal nanoparticles have polar functional groups derived from low molecular weights of 1000 or less on their surface. A method for producing composite particles of calcium phosphate containing metal nanoparticles.

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