Channel synthesis method for carbonate apatite particles

Abstract

The purpose of the present invention is to provide a new technique for manufacturing carbonate apatite particles by a channel synthesis (flow synthesis) method.　The present invention is a method for manufacturing carbonate apatite particles using a channel synthesis device, wherein: a raw material solution A that includes water-soluble phosphate and a water-soluble calcium salt and a raw material solution B that includes a water-soluble carbonate are made to merge within the channel synthesis device; and carbonate apatite particles are produced within a reaction channel through which the merged solutions flow.

Description

Method for flow synthesis of carbonate apatite particles

[0001] The present invention relates to a method for producing carbonate apatite particles by a flow synthesis method.

[0002] Carbonate apatite is known to have an effect of efficiently delivering drugs such as nucleic acids into cells, and can be used as a drug delivery carrier. It has also been reported that carbonate apatite has an effect of enhancing the antitumor effect of anticancer agents and promoting the accumulation of contrast agents in tumors, etc. (Patent Documents 1 and 2). Furthermore, it has also been reported that composite particles in which miR-29a and / or miR-29b are supported on carbonate apatite particles are effective for treating inflammatory bowel disease (Patent Document 3).

[0003] Conventionally, carbonate apatite particles have been produced in a batch format, but in recent years, production techniques using flow synthesis methods have been reported. Specifically, Non-Patent Document 1 reports that carbonate apatite particles can be produced by merging an aqueous solution containing a water-soluble calcium salt with an aqueous solution containing a water-soluble phosphate and a water-soluble carbonate using a capillary microfluidic device.

[0004] Jung-Kyun Kim et al., Rapid flow synthesis of a biomimetic carbonate apatite as an effective drug carrier, ACS Appl Mater Interfaces. 2022 Jul 6;14(26):29626-29638. doi: 10.1021 / acsami.2c06900.

[0005] International Publication No. WO2015 / 125934, Japanese Unexamined Patent Publication No. 2015-151377, International Publication No. WO2018 / 199121

[0006] The carbonate apatite particles synthesized by the technique described in Non-Patent Document 1 have a large particle size, so although they can be used for treatment of bone defects and the like, they cannot be administered intravascularly. Accordingly, there is a demand for a flow synthesis technique capable of producing carbonate apatite particles having a particle size small enough to be administered intravascularly.

[0007] The present invention provides a novel technology for producing carbonate apatite particles by a flow synthesis method.

[0008] The inventors of the present invention conducted diligent studies to solve the aforementioned problems and discovered that by separating the source of constituent atoms of carbonate apatite into an aqueous solution containing a water-soluble phosphate and a water-soluble calcium salt (raw material solution A) and an aqueous solution containing a water-soluble carbonate (raw material solution B), and by combining these in a flow channel synthesis apparatus and generating carbonate apatite particles during the flow of the combined liquid, it is possible to produce carbonate apatite particles with a particle size small enough to be administered intravenously. The present invention was completed by further studies based on this finding.

[0009] In other words, the present invention provides inventions in the following embodiments: Item 1. A method for producing carbonate apatite particles using a flow channel synthesis apparatus, comprising: combining a raw material solution A containing a water-soluble phosphate and a water-soluble calcium salt with a raw material solution B containing a water-soluble carbonate in the flow channel synthesis apparatus, and generating carbonate apatite particles in a reaction channel through which the combined liquid flows. Item 2. The method according to Item 1, wherein the raw material solution A or the raw material solution B further contains a drug. Item 3. The method according to Item 2, wherein the drug is a nucleic acid. Item 4. The method according to any one of Items 1 to 3, wherein the raw material solution A or the raw material solution B further contains a dispersant. Item 5. The method according to Item 4, wherein the dispersant is at least one selected from the group consisting of chondroitin sulfate sodium, carmellose sodium, chitosan, sialic acid, and albumin. Item 6. The method according to any one of Items 1 to 5, wherein a diluent is combined at the downstream outlet of the reaction channel. Item 7. The method according to Item 6, wherein the diluent contains a dispersant. Item 8. The manufacturing method according to Item 7, wherein the dispersant is at least one selected from the group consisting of chondroitin sulfate sodium, carmellose sodium, chitosan, sialic acid, and albumin. Item 9. The manufacturing method according to any one of Items 1 to 8, wherein air is supplied to the reaction channel. Item 10. The manufacturing method according to any one of Items 1 to 9, wherein the inner diameter of the reaction channel is 5 to 8 mm. Item 11. The manufacturing method according to any one of Items 1 to 10, wherein a mixing promoting section is provided between a confluence section for combining raw material solution A and raw material solution B and the reaction channel for promoting the mixing of raw material solution A and raw material solution B. Item 12. The manufacturing method according to any one of Items 1 to 11, wherein the average particle size of the carbonate apatite particles produced is 700 nm or less.Item 13. The manufacturing method according to any one of items 1 to 12, wherein the flow channel synthesis apparatus is provided with: a raw material solution A supply channel for flowing raw material solution A; a raw material solution B supply channel for flowing raw material solution B; an air supply channel for flowing air; a confluence section for confluence of raw material solution A and raw material solution B; a confluence section for confluence of raw material solution B and air; a reaction channel through which the combined liquid of raw material solution A and raw material solution B flows; a diluent supply channel for flowing a diluent; a confluence section provided at the downstream outlet of the reaction channel for confluence of the confluence liquid and the diluent; and a discharge channel for discharging the reaction-finished liquid obtained by confluence of the confluence liquid and the diluent.

[0010] According to the present invention, carbonate apatite particles with an average particle diameter of approximately 700 nm or less can be produced by a flow channel synthesis method, making it possible to easily provide carbonate apatite particles suitable as drug carriers for intravascular administration.

[0011] This is a schematic diagram of the configuration of the flow channel synthesis apparatus used for producing carbonate apatite particles of the present invention. These are micrographs of carbonate apatite particles synthesized under the conditions of Examples 1-1 to 1-3. These are micrographs of carbonate apatite particles synthesized under the conditions of Examples 2-1 and 2-2. These are micrographs of carbonate apatite particles synthesized under the conditions of Examples 3-1 and 3-2. These are micrographs of carbonate apatite particles synthesized under the conditions of Examples 4-1 to 4-4, carbonate apatite particles synthesized under the conditions of Example 4-2 and subjected to ultrasonic treatment, and carbonate apatite particles synthesized under the conditions of Example 4-3 and subjected to ultrasonic treatment. These are micrographs of carbonate apatite particles synthesized under the conditions of Examples 5-1 and 5-2. These are micrographs of carbonate apatite particles synthesized under the conditions of Examples 6-1 to 6-3. These are micrographs of carbonate apatite particles synthesized under the conditions of Examples 7-1 and 7-2. These are micrographs of carbonate apatite particles synthesized under the conditions of Examples 8-2 and 8-3. These are micrographs of carbonate apatite particles synthesized under the conditions of Examples 7-1 and 7-2. This figure shows the X-ray diffraction (XRD) pattern of carbonate apatite particles obtained in Example 5-1 (n=2). This is the FT-IR spectrum obtained in Example 5-1 by total internal reflection (IRE: diamond, incident angle: 45°, vertical axis scale 555 cm). -1 This figure shows the peak intensity normalized by the peak intensity derived from nearby phosphates. This is a schematic diagram of the configuration of the flow channel synthesis apparatus for carbonate apatite particles used in Comparative Example 1. This is a micrograph of carbonate apatite particles synthesized under the conditions of Comparative Example 1.

[0012] The present invention relates to a method for producing carbonate apatite particles using a flow channel synthesis apparatus, characterized in that a raw material solution A containing a water-soluble phosphate and a water-soluble calcium salt is combined with a raw material solution B containing a water-soluble carbonate within the flow channel synthesis apparatus, and carbonate apatite particles are generated in a reaction channel through which the combined liquid flows. The present invention's manufacturing method will be described below.

[0013] [Carbonated Apatite] The carbonated apatite produced in this invention is B-type carbonated apatite, Ca 10-a (PO4) 6-b (CO3) c It is a compound represented by [formula].

[0014] [Raw material solution A] Raw material solution A used in the present invention is an aqueous solution containing a water-soluble phosphate and a water-soluble calcium salt.

[0015] The type of water-soluble phosphate is not particularly limited, but examples include sodium dihydrogen phosphate, disodium hydrogen phosphate, potassium dihydrogen phosphate, potassium hydrogen phosphate, etc. These water-soluble phosphates may also be in hydrate form. These water-soluble phosphates may be used individually or in combination of two or more. Among these water-soluble phosphates, sodium dihydrogen phosphate is a preferred example. The concentration of the water-soluble phosphate in raw material solution A should be appropriately set so that the concentration of the water-soluble phosphate in the combined solution of raw material solution A and raw material solution B satisfies the range described later. Specifically, the concentration of the water-soluble phosphate in raw material solution A is 0.2 to 25 mM, preferably 0.8 to 18 mM, more preferably 1.7 to 8.5 mM, even more preferably 2.6 to 6.8 mM, and particularly preferably 3.5 to 6 mM.

[0016] The type of water-soluble calcium salt is not particularly limited, but examples include calcium chloride, calcium bromide, calcium iodide, calcium lactate, etc. These water-soluble calcium salts may also be in hydrate form. These water-soluble calcium salts may be used individually or in combination of two or more. Among these water-soluble calcium salts, calcium chloride is a preferred example. The concentration of calcium chloride in raw material solution A should be appropriately set so that the concentration of water-soluble calcium salt in the combined liquid of raw material solution A and raw material solution B satisfies the range described later. Specifically, the concentration of water-soluble calcium salt in raw material solution A is 2 to 170 mM, preferably 3.5 to 130 mM, more preferably 8.5 to 85 mM, even more preferably 12 to 60 mM, and particularly preferably 17 to 34 mM.

[0017] As for the ratio of water-soluble phosphate to water-soluble calcium salt in raw material solution A, for example, 0.1 to 20 moles, preferably 0.5 to 15 moles, more preferably 1 to 12 moles, even more preferably 3 to 10 moles, and particularly preferably 5 to 8 moles of water-soluble calcium salt per mole of water-soluble phosphate.

[0018] The pH of raw material solution A is not particularly limited, but for example, it can be 1 to 7, preferably 1 to 5, and more preferably 1 to 4. When a drug (such as nucleic acid) is incorporated into the carbonate apatite particles produced, from the viewpoint of increasing the amount of drug incorporated into the carbonate apatite particles, the pH of raw material solution A is more preferably 1 to 3, and particularly preferably 1.5 to 2.3. To adjust the pH of raw material solution A to the above range, an appropriate amount of acid can be added.

[0019] The type of acid used to adjust the pH of raw material solution A is not particularly limited, but hydrochloric acid is a preferred example. When adjusting the pH of raw material solution A using hydrochloric acid, the amount of hydrochloric acid added can be appropriately set to satisfy the pH range, but for example, the hydrochloric acid concentration of raw material solution A can be 0.2 to 50 mM, preferably 2 to 35 mM, and more preferably 3.5 to 25 mM.

[0020] When incorporating a drug (such as nucleic acid) into the carbonate apatite particles to be manufactured, the drug may be added to raw material solution A. The type of drug is as described in the [Raw Material Solution B] section. When raw material solution A contains a drug, the concentration of the drug in raw material solution A should be set appropriately so that the concentration of the drug in the combined solution of raw material solution A and raw material solution B satisfies the range described later. Specifically, the concentration of the drug in raw material solution A can be about 1 to 20,000 μg / ml. More specifically, when nucleic acid is used as the drug, the nucleic acid concentration in raw material solution A can be about 10 to 350 μg / ml, preferably about 35 to 180 μg / ml. However, it is preferable to add the drug to raw material solution B, and in one embodiment of the present invention, raw material solution A does not contain a drug.

[0021] Furthermore, a dispersant may be added to raw material solution A. The type of dispersant is as described in the [Raw Material Solution B] section. When a dispersant is included in raw material solution A, the concentration of the dispersant in raw material solution A should be appropriately set so that the concentration of the dispersant in the combined liquid of raw material solution A and raw material solution B satisfies the range described later. Specifically, the concentration of the dispersant in raw material solution A is 0.001 to 2% by weight, preferably 0.008 to 1% by weight, and more preferably 0.01 to 1% by weight. However, as described in the [Raw Material Solution B] section, it is preferable to add the dispersant to raw material solution B, and in one embodiment of the present invention, raw material solution A does not contain a dispersant.

[0022] [Raw material solution B] Raw material solution B used in the present invention is an aqueous solution containing a water-soluble carbonate.

[0023] The type of water-soluble carbonate is not particularly limited, but examples include sodium bicarbonate, potassium bicarbonate, sodium carbonate, potassium carbonate, etc. These water-soluble carbonates may be used individually or in combination of two or more. Among these water-soluble carbonates, sodium bicarbonate is a preferred example. The concentration of the water-soluble carbonate in raw material solution B should be appropriately set so that the concentration of the water-soluble carbonate in the combined liquid of raw material solution A and raw material solution B satisfies the range described later. Specifically, the concentration of the water-soluble carbonate in raw material solution B is 0.2 to 40 mM, preferably 0.2 to 25 mM, more preferably 2 to 15 mM, even more preferably 3.5 to 10 mM, and particularly preferably 5 to 8 mM.

[0024] When incorporating a drug into the manufactured carbonate apatite particles, it is desirable to add the drug to the raw material solution B. The type of drug is not particularly limited, but examples include antitumor agents, antibiotics, immunosuppressants, steroidal anti-inflammatory drugs, nonsteroidal anti-inflammatory drugs, α-adrenergic receptor blockers, adrenergic receptor agonists, angiotensin II receptor antagonists, angiotensin-converting enzyme inhibitors, calcium channel blockers, antifungal agents, antibacterial agents, antiviral agents, local anesthetics, anti-allergic agents, antihistamines, hemostatic agents, antipruritic and anti-inflammatory agents, peripheral vasodilators, etc. The type of drug may be nucleic acids such as DNA, RNA, antisense nucleic acids, siRNA, miRNA, etc.; proteins, small molecule compounds, etc. However, since carbonate apatite particles are highly suitable as delivery carriers for nucleic acids, nucleic acids are a preferred example of a drug to be added to the raw material solution B. These drugs may be used individually or in combination of two or more.

[0025] When a drug is included in raw material solution B, the concentration of the drug in raw material solution B should be set appropriately according to the type of drug used, the amount of drug to be incorporated into the carbonate apatite particles, etc., and should be set appropriately so that the concentration of the drug in the combined solution of raw material solution A and raw material solution B satisfies the range described later. Specifically, the concentration of the drug in raw material solution B can be said to be about 2 to 25,000 μg / ml. More specifically, when nucleic acids are used as the drug, the nucleic acid concentration in raw material solution B can be said to be about 2 to 1,300 μg / ml, preferably about 10 to 630 μg / ml. From the viewpoint of efficiently incorporating nucleic acids into carbonate apatite particles, the nucleic acid concentration in raw material solution B can be more preferably 25 to 500 μg / ml, even more preferably 25 to 250 μg / ml, and particularly preferably 75 to 140 μg / ml.

[0026] Furthermore, a dispersant may be added to the raw material solution B. By including a dispersant in the raw material solution B, the particle size and polydispersity index (PI) of the generated carbonate apatite particles can be reduced, making it possible to obtain carbonate apatite particles that are homogeneous, fine, and have a narrow particle size distribution. The type of dispersant is not particularly limited, but examples include polysaccharides such as sodium chondroitin sulfate, sodium carmellose, chitosan, and sialic acid; and proteins such as albumin. These dispersants may be used individually or in combination of two or more. Among these dispersants, polysaccharides are preferred, and sodium chondroitin sulfate is more preferred.

[0027] When a dispersant is included in raw material solution B, the concentration of the dispersant in raw material solution B can be appropriately set according to the type of dispersant used, and should be set appropriately so that the concentration of the dispersant in the combined liquid of raw material solution A and raw material solution B satisfies the range described later. Specifically, the concentration of the dispersant in raw material solution B can be 0.001 to 3% by weight, preferably 0.01 to 12% by weight. From the viewpoint of efficiently synthesizing homogeneous and minute carbonate apatite particles with a narrow particle size distribution, the concentration of the dispersant in raw material solution B can be preferably 0.01 to 0.5% by weight, more preferably 0.02 to 0.3% by weight, even more preferably 0.02 to 0.2% by weight, and particularly preferably 0.02 to 0.15% by weight.

[0028] [Diluent] The diluent is a solution that is added to the combined solution of raw material solution A and raw material solution B, and is used as needed to stop or reduce the rate of formation of carbonate apatite particles.

[0029] The diluent may be water, but it is preferably an aqueous solution containing a dispersant. The type of dispersant is not particularly limited, but examples include polysaccharides such as chondroitin sulfate sodium, carmellose sodium, chitosan, and sialic acid; and proteins such as albumin. Among these dispersants, carmellose sodium and albumin are preferred.

[0030] When a dispersant is included in the diluent, the concentration of the dispersant in the raw material solution B can be appropriately set according to the type of dispersant used, etc., but for example, the concentration of the dispersant in the diluent can be 0.001 to 5% by weight, preferably 0.005 to 2% by weight. More specifically, when a polysaccharide is used as the dispersant, the concentration of the polysaccharide in the diluent can be more preferably 0.1 to 1% by weight, even more preferably 0.2 to 0.8% by weight, particularly preferably 0.3 to 0.6% by weight, and even more preferably 0.4 to 0.6% by weight. Also, when a protein is used as the dispersant, the concentration of the protein in the diluent can be more preferably 0.01 to 1% by weight, even more preferably 0.02 to 0.5% by weight, and particularly preferably 0.05 to 0.2% by weight.

[0031] [Flow Channel Synthesis] The manufacturing method of the present invention is carried out by combining raw material solution A and raw material solution B in a flow channel synthesis apparatus and generating carbonate apatite particles in a reaction channel 4 through which the combined liquid flows. A flow channel synthesis apparatus refers to an apparatus that carries out a reaction by flowing raw materials through a fine flow channel (tube, etc.).

[0032] The raw material solution A can be supplied into the reaction channel 4 by a raw material solution A supply channel 1 provided in the flow channel synthesis apparatus. The inner diameter of the raw material solution A supply channel 1 is not particularly limited, but is limited to the extent that the raw material solution A can be supplied at the flow rate described later. For example, it can be about 1 to 10 mm, preferably about 1.5 to 6 mm, and more preferably about 2 to 4 mm. The length of the raw material solution A supply channel 1 is not particularly limited, but for example, it can be about 0.1 to 30 m, preferably about 0.5 to 20 m, and more preferably about 5 to 15 m. The material of the raw material solution A supply channel 1 is not particularly limited and may be any of the following: silicon, polyethylene, polypropylene, polytetrafluoroethylene (PTFE), metal, etc.

[0033] The temperature conditions of the raw material solution A supplied into the reaction channel 4 are not particularly limited and may be at room temperature (20°C), cooled, or heated, but heated is preferable. By preheating the raw material solution A supplied into the reaction channel 4, the temperature of the combined liquid in the reaction channel increases, and the amount of carbonate apatite particles produced can be increased. When preheating the raw material solution A supplied into the reaction channel, the temperature can be, for example, 40 to 70°C, preferably 45 to 65°C, and more preferably 50 to 55°C. To preheat the raw material solution A supplied into the reaction channel 4, for example, the raw material solution A supply channel in the flow channel synthesis apparatus can be placed in a temperature-adjustable constant temperature bath.

[0034] The raw material solution B can be supplied into the reaction channel 4 by a raw material solution B supply channel 2 provided in the flow channel synthesis apparatus. The inner diameter of the raw material solution B supply channel 2 is not particularly limited, but is limited to the extent that the raw material solution B can be supplied at the flow rate described later. For example, it is about 1 to 10 mm, preferably about 1.5 to 6 mm, and more preferably about 2 to 4 mm. The length of the raw material solution B supply channel 2 is not particularly limited, but for example, it is about 0.1 to 30 m, preferably about 0.5 to 20 m, and more preferably about 5 to 15 m. The material of the raw material solution B supply channel 2 is not particularly limited and may be any of the following: silicon, polyethylene, polypropylene, polytetrafluoroethylene (PTFE), metal, etc.

[0035] The temperature conditions of the raw material solution B supplied into the reaction channel 4 are not particularly limited and may be at room temperature, cooled, or heated, but a preferred example is at room temperature.

[0036] In one embodiment of the manufacturing method of the present invention, the flow rates of raw material solution A and raw material solution B supplied into the reaction channel 4 should be set such that, in the combined liquid of raw material solution A and raw material solution B, there are 0.5 to 20 moles, preferably 3 to 15 moles, more preferably 5 to 10 moles, and even more preferably 7 to 8 moles of water-soluble carbonate per mole of water-soluble calcium salt.

[0037] Furthermore, in one embodiment of the manufacturing method of the present invention, the flow rates of raw material solution A and raw material solution B supplied into the reaction channel 4 should be set according to the respective compositions of raw material solution A and raw material solution B, so that the concentrations of each component in the combined liquid of raw material solution A and raw material solution B satisfy the following ranges: • Concentration of water-soluble phosphate in the combined liquid: for example, 0.1 to 15 mM, preferably 0.5 to 10 mM, more preferably 1 to 5 mM, even more preferably 1.5 to 4 mM, and particularly preferably 2 to 3.5 mM. • Concentration of water-soluble calcium salt in the combined liquid: for example, 1 to 100 mM, preferably 2 to 75 mM, more preferably 5 to 50 mM, even more preferably 7 to 35 mM, and particularly preferably 10 to 25 mM. • Concentration of water-soluble carbonate in the combined liquid: for example, 0.1 to 15 mM, preferably 0.5 to 10 mM, more preferably 1 to 5 mM, even more preferably 1.5 to 4 mM, and particularly preferably 2 to 3.5 mM. - When raw material solution A and / or raw material solution B contain a drug, the concentration of the drug in the combined solution is approximately 1 to 10,000 μg / ml. If nucleic acids are used as the drug, the nucleic acid concentration is, for example, 1 to 500 μg / ml, preferably 5 to 250 μg / ml, more preferably 10 to 200 μg / ml, even more preferably 20 to 100 μg / ml, and most preferably 30 to 55 μg / ml. - When raw material solution A and / or raw material solution B contain a dispersant, the concentration of the dispersant in the combined solution is approximately 0.001 to 1% by weight, preferably 0.005 to 0.5% by weight, more preferably 0.005 to 0.2% by weight, even more preferably 0.01 to 0.1% by weight, most preferably 0.01 to 0.07% by weight, and even more preferably 0.01 to 0.05% by weight. - When adjusting the pH of raw material solution A with hydrochloric acid, the concentration of hydrochloric acid in the combined solution is, for example, 0.1 to 30 mM, preferably 1 to 20 mM, and more preferably 2 to 15 mM.

[0038] Specifically, the flow rate of the raw material solution A supplied into the reaction channel 4 is 1 to 35 ml / min, preferably 5 to 30 ml / min, more preferably 10 to 25 ml / min, and even more preferably 15 to 20 ml / min.

[0039] Specifically, the flow rate of the raw material solution B supplied into the reaction flow path 4 is 0.5 to 30 ml / min, preferably 1 to 25 ml / min, more preferably 5 to 20 ml / min, and still more preferably 10 to 15 ml / min.

[0040] The flow rate ratio of the combined raw material solution A and raw material solution B is appropriately set according to the composition of raw material solution A and raw material solution B, etc. For example, relative to a flow rate of 1 for raw material solution A, the flow rate of raw material solution B is 0.2 to 2, preferably 0.3 to 1.2, more preferably 0.4 to 0.8, and still more preferably 0.6 to 0.7.

[0041] In the flow path synthesis apparatus used in the present invention, it is preferable that a mixing promoting part M for promoting mixing of raw material solution A and raw material solution B is provided between the merging part C1 where raw material solution A and raw material solution B merge and the reaction flow path 4, to increase the mixing ratio of the merged liquid flowing into the reaction flow path 4. A mixing promotion flow path refers to a flow path provided with a means for increasing the mixing ratio of a merged liquid of two liquids. Specific examples of the mixing promotion flow path include a narrow-diameter flow path part, a static mixer, and the like.

[0042] The narrow-diameter flow path part is a member having a flow path with an inner diameter smaller than that of the raw material solution A supply flow path 1 and the raw material solution B supply flow path 2. For the inner diameter of the narrow-diameter flow path part, for example, it is about 0.8 times or less, preferably about 0.7 times or less, more preferably about 0.5 times or less, and still more preferably about 0.4 times or less of each inner diameter of the raw material solution A supply flow path 1 and the raw material solution B supply flow path 2. More specifically, the inner diameter of the narrow-diameter flow path part is 0.2 to 3 mm, preferably 0.2 to 2 mm, more preferably about 0.5 to 1.5 mm, and still more preferably about 0.8 to 1.2 mm. In addition, the length of the narrow-diameter flow path part M is not particularly limited, and for example, it is about 1 to 100 cm, preferably about 2 to 50 cm, more preferably about 3 to 30 cm, and still more preferably about 5 to 15 cm. The material of the part is not particularly limited, and may be any of silicon, polyethylene, polypropylene, metal, and the like.

[0043] A static mixer is a stationary mixer with no driving parts, and is an in-line mixer that stirs and mixes the fluid that has entered the mixer by dividing, re-merging, and reversing direction of the fluid using a stationary mixing element installed inside the mixer.

[0044] The combined liquid of raw material solution A and raw material solution B produces carbonate apatite particles while flowing through the reaction flow path 4.

[0045] The inner diameter of the reaction flow path 4 is not particularly limited, and examples thereof include 2 to 20 mm, preferably 3 to 15 mm. From the viewpoint of suppressing the formation of by-products and efficiently producing carbonate apatite particles, the inner diameter of the reaction flow path 4 is more preferably 4 to 12 mm, still more preferably 5 to 8 mm, particularly preferably 5 to 7 mm. The length of the reaction flow path 4 may be appropriately set according to the inner diameter of the reaction flow path 4 and the like, and examples thereof include 5 to 200 cm, preferably 10 to 100 cm. From the viewpoint of suppressing the formation of by-products and efficiently producing carbonate apatite particles, the length of the reaction flow path 4 is more preferably 10 to 90 cm, still more preferably 15 to 80 cm, particularly preferably 20 to 60 cm. The material of the reaction flow path 4 is not particularly limited, and may be any of silicon, polyethylene, polypropylene, metal, or the like.

[0046] The temperature conditions for the reaction flow path 4 are not particularly limited. For example, if the raw material solution A has been pre-heated as described above, the set temperature of the reaction flow path 4 may be room temperature. Further, the temperature of the reaction flow path 4 may be set to, for example, 40 to 70°C, preferably 45 to 65°C, more preferably 50 to 55°C.

[0047] The sum of the flow rate of the raw material solution A and the flow rate of the raw material solution B to be combined is the flow rate of the combined liquid in the reaction flow path 4. Specific examples of the flow rate of the combined liquid in the reaction flow path 4 include 2.5 to 65 ml / min, preferably 7 to 55 ml / min, more preferably 15 to 45 ml / min, still more preferably 25 to 35 ml / min.

[0048] The residence time (reaction time) of the combined liquid in the reaction channel 4 is determined by the flow rate of the combined liquid and the length of the reaction channel 4, but specifically, it can be 1 to 100 seconds, preferably 2 to 60 seconds, more preferably 3 to 45 seconds, and even more preferably 4 to 12 seconds.

[0049] Furthermore, air may be supplied to reaction channel 4. By supplying air to reaction channel 4, the amount of carbonate apatite particles produced can be increased. To supply air to reaction channel 4, any of the following methods may be used: directly introducing air into reaction channel 4, introducing air into raw material solution A supply channel 1, or introducing air into raw material solution B supply channel 2. A preferred example is introducing air into raw material solution B supply channel 2. To introduce air into raw material solution B supply channel 2, a confluence section C2 is provided to merge air into raw material solution B supply channel 2, and air is supplied to the confluence section C2. When supplying air to reaction channel 4, the amount of air supplied is not particularly limited, but for example, 1 to 40 ml / min, preferably 5 to 30 ml / min, and more preferably 10 to 20 ml / min can be cited. Here, air refers to the volume (ml) at room temperature and atmospheric pressure (20°C, 1 atm).

[0050] Since carbonate apatite particles are generated in the combined liquid discharged from the downstream outlet of reaction channel 4, carbonate apatite particles may be recovered from the combined liquid discharged from the downstream outlet of reaction channel 4. However, it is desirable to add a diluent to the combined liquid discharged from the downstream outlet of reaction channel 4 to stop or reduce the generation rate of carbonate apatite particles. To add a diluent to the combined liquid discharged from the downstream outlet of reaction channel 4, a diluent supply channel 5 through which the diluent flows can be connected to the downstream outlet of reaction channel 4. The ratio of combined liquid to diluent to be added is not particularly limited, but for example, the ratio of diluent to the flow rate of combined liquid is 0.1 to 2, preferably 0.2 to 1, more preferably 0.3 to 0.8, and even more preferably 0.4 to 0.6.

[0051] Since the combined liquid discharged from the downstream outlet of the reaction channel 4, or the reaction termination liquid obtained by combining the combined liquid discharged from the downstream outlet of the reaction channel 4 with the diluent, contains carbonate apatite particles, the carbonate apatite particles can be recovered from the combined liquid or the reaction termination liquid.

[0052] [Manufactured carbonate apatite particles] Furthermore, carbonate apatite particles recovered from the combined liquid or reaction completion liquid can be subjected to ultrasonic treatment, filtration, etc., as needed, to remove by-products and further reduce particle size and polydispersity index (PI).

[0053] The recovered carbonate apatite particles may be washed as needed and then formulated into the desired dosage form for use. Alternatively, preservatives, dispersants, etc., may be added to the recovered carbonate apatite particles before formulation.

[0054] The carbonate apatite particles obtained by the manufacturing method of the present invention have a small particle size and a narrow particle size distribution, making them suitable for medical use, particularly for intravascular administration. Furthermore, when a drug (such as nucleic acid) is incorporated into the carbonate apatite particles obtained by the manufacturing method of the present invention, these carbonate apatite particles can be used as drug-carrying carbonate apatite particles for medical purposes depending on the type of drug.

[0055] Furthermore, one embodiment of the carbonate apatite particles obtained by the manufacturing method of the present invention is amorphous particles.

[0056] The average particle size of the carbonate apatite particles obtained by the manufacturing method of the present invention is not particularly limited, but for example, it is about 700 nm or less, preferably 200 to 600 nm, more preferably 320 to 550 nm, and even more preferably 350 to 500 nm. The polydispersity index (PI) of the carbonate apatite particles obtained by the manufacturing method of the present invention is not particularly limited, but for example, it is 0.050 to 0.160, preferably 0.050 to 0.130, more preferably 0.050 to 0.110, and even more preferably 0.050 to 0.090. In the present invention, the average particle size and polydispersity index (PI) of the carbonate apatite particles refer to the volume-based average particle size and polydispersity index (PI) determined by dynamic light scattering.

[0057] [Flow channel synthesis apparatus] Figure 1 shows an example of a schematic diagram of the configuration of a flow channel synthesis apparatus used in the manufacturing method of the present invention.

[0058] The flow channel synthesis apparatus shown in Figure 1 includes: a raw material solution A supply channel 1 for circulating raw material solution A; a pump P1 for supplying raw material solution A to the raw material solution A supply channel 1; a raw material solution B supply channel 2 for circulating raw material solution B; a pump P2 for supplying raw material solution B to the raw material solution B supply channel 2; a confluence section C1 for combining raw material solution A and raw material solution B; a mixing promotion section M connected to the confluence section C1 to promote the mixing of the combined liquid (a mixture of raw material solution A and raw material solution B); and a reaction channel 4 connected to the mixing promotion section M to circulate the combined liquid and generate carbonate apatite particles. Alternatively, the confluence section C1 and the reaction channel 4 may be directly connected without the mixing promotion section M.

[0059] In the flow channel synthesis apparatus shown in Figure 1, a constant temperature bath B is provided for heating the supply channel 1 of the raw material solution A. However, the constant temperature bath B is not required, and may be provided in the reaction channel 4.

[0060] In the flow channel synthesis apparatus shown in Figure 1, an air supply channel 3 for circulating air; a pump P3 for supplying air to the air supply channel 3; and a confluence section C2 for combining the raw material solution B and air are provided, but these are not required. Furthermore, the confluence section C2 may be provided in the raw material solution A supply channel 1 and configured to combine the raw material solution B and air. Moreover, the confluence section C2 may be provided in the reaction channel 4 and configured to combine the combined liquid and air.

[0061] In the flow channel synthesis apparatus shown in Figure 1, there is a diluent supply channel 5 for circulating the diluent; a pump P4 for supplying the diluent to the diluent supply channel 5; a confluence section C3 for combining the combined liquid and the diluent; and a discharge channel 6 connected to the confluence section C3 for discharging the reaction completion liquid (a mixture of the combined liquid and the diluent) into a recovery container. However, these may be omitted, and the apparatus may be configured so that the combined liquid is directly discharged from the downstream outlet of the reaction channel 4.

[0062] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples.

[0063] 1. Measurement Method (1) Microscopic Observation of Carbonate Apatite Particles A solution containing carbonate apatite particles was observed under an optical microscope at 40x magnification. Any debris-like substances observed under the microscope (clearly different from carbonate apatite particles) are referred to as by-products.

[0064] (2) The average particle size (volume-based) and polydispersity index (PI) of carbonate apatite particles were measured using the backscatter method of dynamic light scattering. The instrument used was a Zetasizer Ultra (Model: ZSU5700, Manufacturer: Malvern Panalytical Ltd).

[0065] (3) Amount of calcium contained in carbonate apatite particles After diluting the solution containing carbonate apatite particles as needed, the solution containing carbonate apatite particles and 0.05N HCl were mixed in a 1:1 ratio to dissolve the carbonate apatite particles. The calcium concentration of the dissolved solution was measured using a metalloassay calcium analyzer LS (CPZIII, Metallogenics Co., Ltd.). From the measured values, the amount of calcium contained in the carbonate apatite particles produced in 25 ml or 400 ml of combined solution was calculated. Here, the combined solution refers to the mixture of raw material solution A and raw material solution B used in the example, and to the mixture of the first aqueous solution and the second aqueous solution in the comparative example.

[0066] (4) Nucleic Acid Incorporation Rate of Carbonate Apatite Particles After diluting the solution containing carbonate apatite particles as needed, the solution containing carbonate apatite particles was mixed with 0.05 M EDTA (product code: 349-02625, Fujifilm Wako Pure Chemical Industries) in a 5:1 ratio to dissolve the carbonate apatite particles. The nucleic acid concentration of the dissolved solution was measured using a NanoDrop micro spectrophotometer (Thermos Fisher Scientific). A 0.0083 M EDTA aqueous solution was used as a blank for the measurement with the NanoDrop micro spectrophotometer. The amount of nucleic acid incorporated into the carbonate apatite particles was determined from the measured values, and the ratio of the amount of nucleic acid incorporated into the carbonate apatite particles to the total amount of nucleic acid subjected to the reaction was calculated as the "Nucleic Acid Incorporation Rate of Carbonate Apatite Particles (%)".

[0067] (5) Nucleic acid / Ca ratio of carbonate apatite particles The amount of calcium and nucleic acid incorporated into the carbonate apatite particles was determined by the methods described in (3) and (4) above, and the weight ratio of the amount of nucleic acid to the amount of calcium was calculated as the nucleic acid / Ca ratio.

[0068] 2. Flow-channel synthesis of carbonate apatite particles (1) Flow-channel synthesis apparatus In each of the embodiments described below, carbonate apatite particles were produced using the flow-channel synthesis apparatus shown in Figure 1. The flow-channel synthesis apparatus includes: a raw material solution A supply channel 1 for circulating raw material solution A; a constant temperature bath B for heating the raw material solution A supply channel 1; a pump P1 for supplying raw material solution A to the raw material solution A supply channel 1; a raw material solution B supply channel 2 for circulating raw material solution B; a pump P2 for supplying raw material solution B to the raw material solution B supply channel 2; an air supply channel 3 for circulating air; a pump P3 for supplying air to the air supply channel 3; a diluent supply channel 5 for circulating diluent; and a pump for supplying diluent to the diluent supply channel 5. Pump P4; confluence section C1 for combining raw material solution A and raw material solution B; confluence section C2 for combining raw material solution B and air; mixing promotion section M connected to the confluence C1 to promote mixing of the combined liquid (mixture of raw material solution A and raw material solution B); reaction channel 4 connected to the mixing promotion section M to allow the combined liquid to flow and generate carbonate apatite particles; confluence section C3 for combining the combined liquid and diluent; and discharge channel 6 connected to confluence section C3 to discharge the reaction completion liquid (mixture of combined liquid and diluent) into a recovery container. However, depending on the conditions of the embodiment, the constant temperature bath B, air supply channel 3, air pump P3, and confluence section C3 were not provided.

[0069] (2) Basic Conditions Using the flow channel synthesis apparatus described above, raw material solution A (calcium and phosphate supply solution) and raw material solution B (carbonate supply solution) were introduced into separate flow channels and merged to generate carbonate apatite in the reaction channel. A diluent was introduced into the downstream outlet of the reaction channel and the reaction completion solution was collected. The basic conditions for each example are as follows: <Basic Conditions> Raw material solution A: Raw material solution of composition A1 shown in Table 1 Raw material solution B: Raw material solution of composition B1 shown in Table 1 Diluent: Aqueous aqueous solution containing 0.45% by weight of carmellose sodium Raw material solution A supply channel 1: Silicone tube with a length of 10m, outer diameter of 5mm, and inner diameter of 3mm Raw material solution B supply channel 2: Silicone tube with a length of 0.5m, outer diameter of 5mm, and inner diameter of 3mm Air supply channel 3: None Reaction channel 4: Silicone tube with a length of 30cm, outer diameter of 8mm, and inner diameter of 6mm Diluent supply channel 5: Silicone tube with an outer diameter of 5mm and inner diameter of 3mm Discharge channel 6: Silicone tube with an outer diameter of 5mm and inner diameter of 3mm Tube junction C1: Connecting member that combines two flow paths into one flow path. Junction C2: None. Junction C3: Connecting member that combines two flow paths into one flow path. Mixing promotion section M: 10cm long, 1mm inner diameter thin silicone tube. Pumps P1, P2 and P4: Liquid transfer pumps. Pump P3: None. Supply rate of raw material solution A to supply channel 1: 15.5 ml / min. Supply rate of raw material solution B to supply channel 2: 15.5 ml / min. Supply rate of air to supply channel 2: 20 ml / min. Supply rate of diluent to supply channel 5: 15 ml / min. Constant temperature bath B: None. Temperature: All at room temperature.

[0070]

[0071] Under the above basic conditions, when raw material solution A1 and raw material solution B1 are combined, the combined solution will contain 2.7 mM NaH2PO4, 17.4 mM CaCl2, 6.75 mM HCl, 132.13 mM NaHCO3, and 24 μg / ml nucleic acids, and the pH of the combined solution will be approximately 7.6.

[0072] Note that some of the examples 1 to 9 shown below were conducted on different days, so even under the same conditions, the measured values ​​may not be exactly the same due to systematic errors, random errors, etc.

[0073] (3) Example 1: Examination of reaction channel length and reaction time As shown in Table 2, the above basic conditions were adopted except for (1) changing the reaction channel to 4 and (2) changing the mixing accelerator M, and a flow channel synthesis of carbonate apatite was carried out.

[0074]

[0075] Figure 2 shows a micrograph of the carbonate apatite particles contained in the recovered reaction solution. Table 3 shows the measurement results of the amount of calcium, average particle size, and polydispersity index (PI) of the carbonate apatite particles produced from 25 ml of the combined solution. In all three examples (1-3), carbonate apatite particles with an average particle size of 550 nm or less were produced. Furthermore, it was observed that the longer the reaction channel 4 and the longer the reaction time, the larger the particle size of the carbonate apatite particles became, and the amount of carbonate apatite particles produced (the amount of calcium contained in the carbonate apatite particles produced from 25 ml of the combined solution) tended to increase. However, in examples 1-3, where the reaction channel 4 had the longest channel, a small amount of by-products (a dust-like substance visible in the micrograph) were observed (Figure 2).

[0076]

[0077] (4) Example 2: Investigation of the concentration of carboxymethylcellulose sodium in the diluent (1) As shown in Table 4, the above basic conditions were adopted except for (1) changing the diluent, (2) changing the reaction channel 4, and (3) changing the mixing accelerator M, and the flow channel synthesis of carbonate apatite was carried out.

[0078]

[0079] A micrograph of the carbonate apatite particles contained in the recovered reaction solution is shown in Figure 3. Table 5 shows the calcium content, average particle size, and polydispersity index (PI) measurements of the carbonate apatite particles produced from 25 ml of the combined solution. From the micrograph (Figure 3), it was confirmed that a concentration of 0.45 w / v% carboxymethylcellulose sodium in the diluent (Example 2-1) resulted in carbonate apatite particles with a cleaner appearance.

[0080]

[0081] (5) Example 3: Examination of the material of the reaction channel 4 As shown in Table 6, the above basic conditions were adopted except for (1) changing the reaction channel 4 and (2) changing the mixing accelerator M, and a flow channel synthesis of carbonate apatite was carried out.

[0082]

[0083] Figure 4 shows a microscopic image of the carbonate apatite particles contained in the recovered reaction solution. Table 7 shows the measurement results of the average particle size and polydispersity index (PI) of the carbonate apatite particles. As a result, it was found that the material of reaction channel 4 had little effect on the particle size distribution of the generated carbonate apatite particles.

[0084]

[0085] (6) Example 4: Examination of temperature and air supply of raw material solution A supply channel 1 As shown in Tables 8-1 and 8-2, the above basic conditions were adopted to perform the flow channel synthesis of carbonate apatite, except that (1) raw material solution A and raw material solution B were changed to compositions shown in Table 9, (2) the supply rate of raw material solution A2 to raw material solution A supply channel 1 and the supply rate of raw material solution B2 to raw material solution B supply channel 2 were changed, (3) a constant temperature bath B was installed to heat raw material solution A supply channel 1, (4) the length of reaction channel 4 was changed, and (5) an air supply channel 3, an air pump P3 and a confluence C3 were installed to supply air to raw material solution B supply channel 2. Furthermore, when raw material solution A2 and raw material solution B2 are combined under the above conditions, the combined solution will contain 2.7 mM NaH2PO4, 17.4 mM CaCl2, 6.75 mM HCl, 132.13 mM NaHCO3, and 24 μg / ml nucleic acid, and the pH of the combined solution will be approximately 7.6.

[0086] Furthermore, the carbonate apatite particles of Example 4-2 were subjected to ultrasonic treatment ("Dispersion Nano Taro" (Thinky Co., Ltd.), 5000 rpm for 1 minute). In addition, the carbonate apatite particles of Example 4-3 were subjected to filtration of the reaction completion solution (filtration using two sheets of ADVANTEC 131 filter paper) and the filtrate was recovered.

[0087]

[0088]

[0089]

[0090] Micrographs of carbonate apatite particles contained in the recovered reaction solution (Examples 4-1 to 4-4), carbonate apatite particles from Example 4-2 after sonication, and carbonate apatite particles from Example 4-3 contained in the filtrate after filtration are shown in Figure 5. Table 10 shows the measurement results of the amount of calcium contained in carbonate apatite particles generated from 25 ml of combined solution, the nucleic acid uptake rate of the carbonate apatite particles, and the average particle size and polydispersity index (PI) of the carbonate apatite particles. When the liquid temperature in the reaction channel was increased by heating the raw material solution A supply channel 1, an increase in the amount of carbonate apatite particles generated (amount of calcium contained in carbonate apatite particles generated from 400 ml of combined solution) was observed (Examples 4-2 to 4-4). Furthermore, when air was introduced into the raw material solution B supply channel 2 and supplied to the reaction channel, an increase in the amount of carbonate apatite particles generated was observed. In addition, supplying air to the reaction channel did not have any effect on the particle size of the carbonate apatite particles generated.

[0091] Furthermore, when the reaction solution containing carbonate apatite particles was subjected to sonication, the particle size of the carbonate apatite particles decreased, and the polydispersity index (PI) also decreased significantly (Example 4-2). In addition, when the reaction solution containing carbonate apatite particles was filtered, the disappearance of large by-products and a significant decrease in the polydispersity index (PI) were observed (Example 4-3). In other words, it was found that homogeneous carbonate apatite particles with a narrow particle size distribution can be obtained by further sonication or filtration of carbonate apatite particles produced by flow channel synthesis.

[0092]

[0093] (7) Example 5: Examination of adding chondroitin sulfate sodium to raw material solution B As shown in Tables 11-1 and 11-2, the above basic conditions were adopted to perform the flow channel synthesis of carbonate apatite, except that (1) raw material solution A was changed to composition A2 shown in Table 9, (2) raw material solution B was changed to the composition shown in Table 12, (3) the supply rate of raw material solution A2 to raw material solution A supply channel 1 and the supply rate of raw material solution B2 to raw material solution B supply channel 2 were changed, (4) a constant temperature bath B was provided to heat raw material solution A supply channel 1, (5) the reaction channel 4 was changed, and (6) an air supply channel 3, an air pump P3 and a confluence C3 were provided to supply air to raw material solution B supply channel 2.

[0094]

[0095]

[0096]

[0097] Microscopic images of the carbonate apatite particles contained in the recovered reaction solution are shown in Figure 6. Table 13 shows the measurement results of the amount of calcium contained in the carbonate apatite particles produced from 25 ml of the combined solution, the nucleic acid uptake rate of the carbonate apatite particles, the nucleic acid / Ca ratio of the carbonate apatite particles, and the average particle size and polydispersity index (PI) of the carbonate apatite particles. As a result, although the amount of carbonate apatite particles produced (amount of calcium contained in carbonate apatite particles produced from 25 ml of the combined solution) and the nucleic acid uptake rate decreased when sodium chondroitin sulfate was included in the raw material solution B, a decrease in particle size and polydispersity index (PI) was observed, indicating that smaller and more homogeneous carbonate apatite particles could be produced. Furthermore, when sodium chondroitin sulfate was included in the raw material solution B, the generation of by-products was also suppressed (Figure 6).

[0098]

[0099] (8) Example 6: Examination of the amount of chondroitin sulfate sodium added to raw material solution B As shown in Tables 14-1 and 14-2, the above basic conditions were adopted to perform the flow channel synthesis of carbonate apatite, except that (1) raw material solution A was changed to composition A2 shown in Table 9, (2) raw material solution B was changed to the composition shown in Table 15, (3) the supply rate of raw material solution A2 to raw material solution A supply channel 1 and the supply rate of raw material solution B2 to raw material solution B supply channel 2 were changed, (4) a constant temperature bath B was provided to heat raw material solution A supply channel 1, (5) the reaction channel 4 was changed, and (6) an air supply channel 3, an air pump P3 and a confluence C3 were provided to supply air to raw material solution B supply channel 2. Furthermore, when raw material solution B with composition B3 is used, the concentration of chondroitin sulfate sodium in the combined solution is 1% by weight; when raw material solution B with composition B4 is used, the concentration of chondroitin sulfate sodium in the combined solution is 0.5% by weight; and when raw material solution B with composition B5 is used, the concentration of chondroitin sulfate sodium in the combined solution is 0.1% by weight.

[0100]

[0101]

[0102]

[0103] Microscopic images of the carbonate apatite particles contained in the recovered reaction solution are shown in Figure 7. Table 16 shows the measurement results of the calcium content, nucleic acid uptake rate, nucleic acid / Ca ratio, average particle size, and polydispersity index (PI) of the carbonate apatite particles generated from 25 ml of the combined solution. As a result, when the concentration of chondroitin sulfate sodium in the raw material solution B was reduced, an improvement in nucleic acid uptake rate was observed without causing a significant difference in the polydispersity index (PI). Furthermore, when the concentration of chondroitin sulfate sodium in the raw material solution B was 0.05 w / v% and 0.01 w / v%, no by-products were observed at all (Figure 7).

[0104]

[0105] (9) Example 7: Examination of pH of raw material solution A As shown in Tables 17-1 and 17-2, the above basic conditions were adopted to perform the flow channel synthesis of carbonate apatite, except that (1) raw material solution A was changed to the composition shown in Table 18, (2) raw material solution B was changed to composition B5 shown in Table 15, (3) the supply rate of raw material solution A2 to raw material solution A supply channel 1 and the supply rate of raw material solution B2 to raw material solution B supply channel 2 were changed, (4) a constant temperature bath B was installed to heat raw material solution A supply channel 1, (5) the reaction channel 4 was changed, and (6) an air supply channel 3, an air pump P3 and a confluence C3 were installed to supply air to raw material solution B supply channel 2.

[0106]

[0107]

[0108]

[0109] A micrograph of the carbonate apatite particles contained in the recovered reaction solution is shown in Figure 8. Table 19 shows the measurement results of the amount of calcium contained in the carbonate apatite particles produced from 25 ml of the combined solution, the nucleic acid uptake rate of the carbonate apatite particles, the nucleic acid / Ca ratio of the carbonate apatite particles, and the average particle size and polydispersity index (PI) of the carbonate apatite particles. It is known that increasing the nucleic acid / Ca ratio of the carbonate apatite particles to around 200 improves the uptake rate of the carbonate apatite particles by tumors. When the hydrochloric acid concentration in raw material solution A was increased and the pH was lowered, the nucleic acid / Ca ratio of the carbonate apatite particles increased to around 170. That is, it was confirmed that carbonate apatite particles with a high uptake rate by tumors can be obtained by controlling the pH of raw material solution A. In addition, no by-products were observed in Examples 7-1 and 7-2 (Figure 8).

[0110]

[0111] (10) Example 8: Examination of the length of the reaction channel 4 As shown in Tables 20-1 and 20-2, the above basic conditions were adopted to perform the flow channel synthesis of carbonate apatite, except that (1) raw material solution A was changed to composition A2 shown in Table 9, (2) raw material solution B was changed to composition B5 shown in Table 15, (3) the supply rate of raw material solution A2 to raw material solution A supply channel 1 and the supply rate of raw material solution B2 to raw material solution B supply channel 2 were changed, (4) a constant temperature bath B was provided to heat the raw material solution A supply channel 1, (5) the reaction channel 4 was changed, and (6) an air supply channel 3, an air pump P3 and a confluence C3 were provided to supply air to the raw material solution B supply channel 2.

[0112]

[0113]

[0114] A micrograph of the carbonate apatite particles contained in the recovered reaction solution is shown in Figure 9. Table 21 shows the measurement results of the amount of calcium in the carbonate apatite particles produced from 25 ml of the combined solution, the nucleic acid uptake rate of the carbonate apatite particles, the nucleic acid / Ca ratio of the carbonate apatite particles, and the average particle size and polydispersity index (PI) of the carbonate apatite particles. As a result, it was confirmed that shortening the length of the reaction channel 4 and thus shortening the reaction time in the reaction channel 4 improved the nucleic acid / Ca ratio of the carbonate apatite particles. Furthermore, no by-products were observed in any of Examples 8-1 to 8-3 (Figure 9).

[0115]

[0116] (11) Example 9: Examination of nucleic acid concentration of raw material solution B As shown in Tables 22-1 and 22-2, the above basic conditions were adopted to perform the flow channel synthesis of carbonate apatite, except that (1) raw material solution A was changed to composition A2 shown in Table 9, (2) raw material solution B was changed to the composition shown in Table 23, (3) the supply rate of raw material solution A2 to raw material solution A supply channel 1 and the supply rate of raw material solution B2 to raw material solution B supply channel 2 were changed, (4) a constant temperature bath B was installed to heat raw material solution A supply channel 1, (5) the reaction channel 4 was changed, and (6) an air supply channel 3, an air pump P3 and a confluence C3 were installed to supply air to raw material solution B supply channel 2.

[0117]

[0118]

[0119]

[0120] A micrograph of the carbonate apatite particles contained in the recovered reaction solution is shown in Figure 10. Table 24 shows the measurement results of the calcium content, nucleic acid uptake rate, nucleic acid / Ca ratio, average particle size, and polydispersity index (PI) of the carbonate apatite particles produced from 25 ml of the combined solution. As a result, it was confirmed that increasing the nucleic acid concentration in raw material solution B improved the nucleic acid / Ca ratio of the carbonate apatite particles to 200 or more. Furthermore, no by-products were observed in either Example 9-1 or 9-2 (Figure 10).

[0121]

[0122] (12) Crystal and compositional analysis of carbonate apatite particles The carbonate apatite particles obtained in Example 5-1 were diluted in water and subjected to X-ray diffraction (XRD). In addition, the carbonate apatite particles obtained in Example 5-1 were diluted in water and analyzed by Fourier transform infrared spectroscopy (FT-IR).

[0123] XRD results confirmed that the carbonate apatite particles obtained in the above example were amorphous and did not contain any crystals (Figure 11). Furthermore, FT-IR results confirmed that the carbonate apatite particles obtained in the above example were mainly composed of carbonate ions and phosphate ions, and that carbonate apatite particles were successfully produced (Figure 12).

[0124] 3. Flow channel synthesis of carbonate apatite particles by the method described in Non-Patent Literature 1 (1) Flow channel synthesis of carbonate apatite particles was performed by the method described in Non-Patent Literature 1. A schematic diagram of the configuration of the flow channel synthesis apparatus used is shown in Figure 13. The flow channel synthesis apparatus includes a pump P12 for circulating the first aqueous solution and supplying it to the second aqueous solution supply channel 12; a confluence section C11 for combining the first aqueous solution and the second aqueous solution; and a reaction channel 13 connected to the confluence section C11, through which the combined liquid (a mixture of the first aqueous solution and the second aqueous solution) is circulated to generate carbonate apatite particles, and which discharges the combined liquid into a recovery container. The specific setting conditions are as follows. <Conditions> First aqueous solution: Aqueous solution with the composition shown in Table 25 Second aqueous solution: Aqueous solution with the composition shown in Table 25 First aqueous solution supply channel 11: Silicone tube with an outer diameter of 5 mm and an inner diameter of 3 mm Second aqueous solution supply channel 12: Silicone tube with an outer diameter of 5 mm and an inner diameter of 3 mm Reaction channel 13: PTFE tube with a length of 2.2 m, an outer diameter of 1.6 mm and an inner diameter of 0.5 mm Confluence C11: Connecting member that combines the two channels into one channel Pumps P11 and P12: Liquid transfer pumps Supply rate of the first aqueous solution to the first aqueous solution supply channel: 2.5 ml / min Supply rate of the second aqueous solution to the second aqueous solution supply channel: 2.5 ml / min Flow rate of the mixed solution in the reaction channel 13: 5 ml / min Residence time of the mixed solution in the reaction channel 13: 5.18 seconds

[0125]

[0126] Under the aforementioned basic conditions, when the first aqueous solution and the second aqueous solution are combined, the combined solution will contain 10 mM Ca(NO3)2・4H2O, 1 mM (NH4)2CO3, 6.7 mM (NH4)2HPO4, and 36 μg / ml nucleic acid.

[0127] (2) As a result, micrographs of the carbonate apatite particles contained in the combined solution are shown in Figure 14. The amount of calcium contained in the carbonate apatite particles generated from 25 ml of the combined solution, the nucleic acid uptake rate of the carbonate apatite particles, and the measurement results of the average particle size of the carbonate apatite particles are shown in Table 26.

[0128] In the combined solution obtained by the method described in Non-Patent Document 1, the carbonate apatite particles formed gel-like aggregates. Furthermore, the carbonate apatite particles produced in Examples 1 to 9 had an average particle diameter of approximately 300 to 500 nm and were suitable for use as a carrier for nucleic acid introduction. However, the carbonate apatite particles produced in Comparative Example 1 (method described in Non-Patent Document 1) had a large average particle diameter of approximately 5 μm and were unsuitable as a carrier for nucleic acid introduction.

[0129]

[0130] 1 Raw material solution A supply channel 2 Raw material solution B supply channel 3 Air supply channel 4 Reaction channel 5 Diluent supply channel 6 Discharge channel P1, P2, P3, P4 Pumps C1, C2, C3 Confluence B Constant temperature bath M Mixing promotion section 11 First aqueous solution supply channel 12 Second aqueous solution supply channel 12 13 Reaction channel C11 Confluence P11, P12 Pumps

Claims

1. A method for producing carbonate apatite particles using a flow channel synthesis apparatus, comprising: combining a raw material solution A containing a water-soluble phosphate and a water-soluble calcium salt with a raw material solution B containing a water-soluble carbonate in the flow channel synthesis apparatus; and generating carbonate apatite particles in a reaction channel through which the combined liquid flows.

2. The manufacturing method according to claim 1, wherein the raw material solution A or the raw material solution B further contains a drug.

3. The manufacturing method according to claim 2, wherein the drug is a nucleic acid.

4. The manufacturing method according to claim 1 or 2, wherein the raw material solution A or the raw material solution B further contains a dispersant.

5. The manufacturing method according to claim 4, wherein the dispersant is at least one selected from the group consisting of chondroitin sulfate sodium, carmellose sodium, chitosan, sialic acid, and albumin.

6. The manufacturing method according to claim 1 or 2, wherein the diluent is added at the downstream outlet of the reaction channel.

7. The manufacturing method according to claim 6, wherein the diluent contains a dispersant.

8. The manufacturing method according to claim 7, wherein the dispersant is at least one selected from the group consisting of chondroitin sulfate sodium, carmellose sodium, chitosan, sialic acid, and albumin.

9. The manufacturing method according to claim 1 or 6, wherein air is supplied to the reaction channel.

10. The manufacturing method according to claim 1 or 2, wherein the inner diameter of the reaction channel is 5 to 8 mm.

11. The manufacturing method according to claim 1 or 2, wherein a mixing promoting section is provided between a confluence section for combining raw material solution A and raw material solution B and the reaction channel for promoting the mixing of raw material solution A and raw material solution B.

12. The manufacturing method according to claim 1 or 2, wherein the average particle size of the carbonate apatite particles produced is 700 nm or less.

13. The manufacturing method according to claim 9, wherein the flow channel synthesis apparatus is provided with: a raw material solution A supply channel for flowing raw material solution A; a raw material solution B supply channel for flowing raw material solution B; an air supply channel for flowing air; a confluence section for confluence of raw material solution A and raw material solution B; a confluence section for confluence of raw material solution B and air; a reaction channel through which the combined liquid of raw material solution A and raw material solution B flows; a diluent supply channel for flowing a diluent; a confluence section provided at the downstream outlet of the reaction channel for confluence of the confluence liquid and the diluent; and a discharge channel for discharging the reaction-finished liquid obtained by confluence of the confluence liquid and the diluent.

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