KR102525360B1 - Calcium phosphate nanoparticles and methods for their synthesis - Google Patents

Abstract

The present invention relates to calcium phosphate nanoparticles synthesized by using only phosphate of phosphorylated amino acid as a source of phosphorus and a method for synthesizing the same. A method for synthesizing calcium phosphate nanoparticles according to an embodiment of the present invention includes a first step of adding and mixing an aqueous solution containing a calcium precursor to a solution containing a phosphorylated amino acid, and adding a basic substance containing a hydroxyl group to the mixed solution, A second step of mixing is included.

Description

Calcium phosphate nanoparticles and methods for their synthesis {Calcium phosphate nanoparticles and methods for their synthesis}

The present invention relates to calcium phosphate nanoparticles and methods for their synthesis. Specifically, the present invention relates to a method for synthesizing calcium phosphate nanoparticles by using only phosphate of phosphorylated amino acid as a source of phosphorus.

Calcium phosphate is a material that exists in nature, and in particular, accounts for 50 to 70 w/w.% of bone components in the human body and thus has very high biocompatibility. Therefore, studies on applying calcium phosphate to biological applications such as drug delivery, bone regeneration, and tissue regeneration are being actively conducted.

The synthesis of existing calcium phosphate nanomaterials is carried out through a co-precipitation method or a sol-gel method, using inorganic materials as main materials, such as Ca and P, as raw materials. However, calcium phosphate naturally has a mechanism for synthesizing organic materials in vivo, which is very different from conventional inorganic material-based calcium phosphate synthesis.

Due to these differences, calcium phosphate nanomaterials synthesized based on inorganic materials have a dense surface and are toxic when treated in large amounts to biological tissues.

One object of the present invention is to provide a method for synthesizing porous calcium phosphate nanoparticles by utilizing only phosphate of phosphorylated amino acid as a source of phosphorus.

One object of the present invention is to provide porous calcium phosphate nanoparticles synthesized by the above method and utilize them as a drug delivery system.

One object of the present invention is to provide a method for preparing a nanostructure for bone regeneration by sintering the calcium phosphate nanoparticles.

A method for synthesizing calcium phosphate nanoparticles according to an embodiment of the present invention includes a first step of adding and mixing an aqueous solution containing a calcium precursor to a solution containing a phosphorylated amino acid, and adding a basic substance containing a hydroxyl group to the mixed solution, A second step of mixing is included.

In one embodiment, the temperature during the mixing is preferably maintained at 55 to 65 ° C.

In one embodiment, the mixing of the first step is preferably performed for 5 to 15 minutes, and the mixing of the second step is preferably performed for 7 to 9 hours.

In one embodiment, the phosphorylated amino acid is phosphoserine, phosphotyrosine, phosphothreonine, phosphohistidine, phosphoarginine, phospholysine, It may contain at least one selected from phosphoaspartic acid, phosphoglutamic acid, and phosphocysteine.

In one embodiment, the calcium precursor is calcium chloride (CaCl ₂ ), calcium nitrate (Ca(NO ₃ ) ₂ ), calcium acetate (Ca(C ₂ H ₃ O ₂ ) ₂ ) and calcium ethoxide (Ca(OEt) ₂ ) may include any one or more selected from among.

In one embodiment, the basic material containing a hydroxyl group may include sodium hydroxide (NaOH) or ammonium hydroxide (NH ₄ (OH)).

In one embodiment, the concentration of the phosphorylated amino acid is in the range of 1 to 100 mM and the concentration of the calcium precursor is in the range of 0.9 to 100 mM, the concentration of the hydroxyl group-containing basic material is in the range of 200 to 400 mM, calcium phosphate nanoparticles can be synthesized.

In one embodiment, after the second step, the synthesized calcium phosphate nanoparticles may have a porous crystalline structure. Specifically, after the second step, the synthesized calcium phosphate nanoparticles may be porous hydroxyapatite.

Meanwhile, calcium phosphate nanoparticles according to an embodiment of the present invention are prepared according to the above method, include a plurality of pores, and have a hydroxyapatite crystal structure.

In one embodiment, the calcium phosphate nanoparticles have a grain shape, and the nanoparticles may have a size of 120 to 170 nm.

In one embodiment, the surface potential of the calcium phosphate nanoparticles is characterized in that -3 to -5 mV.

Meanwhile, another embodiment of the present invention may include a drug delivery system in which a drug is loaded into a plurality of pores included in the calcium phosphate nanoparticles.

Another embodiment of the present invention may include a drug delivery system in which a drug is adsorbed on the surface of the calcium phosphate nanoparticles.

Meanwhile, a method for manufacturing a nanostructure for bone regeneration according to an embodiment of the present invention includes sintering the calcium phosphate nanoparticles at a temperature of 100 to 500°C.

In one embodiment, the nanostructure for bone regeneration includes nanopores and micropores, and is characterized in that it has a three-dimensional hierarchical structure.

According to the present invention, it is possible to synthesize porous calcium phosphate nanoparticles having high biocompatibility without an additional phosphorus source by using only phosphate of phosphorylated amino acid, which is an organic material, as a source of phosphorus.

In addition, by adjusting the temperature at the time of synthesizing the nanoparticles, it is possible to prevent the mechanism of precipitation of calcium hydroxide and to allow the synthesis to proceed sufficiently with calcium phosphate.

In addition, the calcium phosphate synthesized according to the present invention has high biocompatibility and can be used in biological applications such as drug delivery, bone regeneration, and tissue regeneration.

1 is a schematic diagram showing a method for synthesizing calcium phosphate nanoparticles according to an embodiment of the present invention and a TEM image of nanoparticles synthesized thereby.
2 is a SEM image of calcium phosphate nanoparticles synthesized according to an embodiment of the present invention.
3 is a SEM image of calcium phosphate nanoparticles synthesized using various phosphorylated amino acids according to an embodiment of the present invention.
Figure 4 shows TEM, SAED and EDS analysis images of calcium phosphate nanoparticles synthesized according to an embodiment of the present invention.
5 shows the hydrodynamic size and surface potential measurement results and ICP-OES analysis results of calcium phosphate nanoparticles synthesized according to an embodiment of the present invention.
6 shows infrared spectroscopy (FT-IR) spectra of calcium phosphate nanoparticles, hydroxyapatite and phosphoserine according to an embodiment of the present invention.
Figure 7 shows the results of a cytocompatibility test (MTT assay) of calcium phosphate nanoparticle-derived nanostructures and comparative examples according to an embodiment of the present invention (left), and the results of an experiment for differentiation of osteoblasts into osteocytes (ALP assay) (right) ).

Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings. Since the present invention may have various changes and various forms, specific embodiments are illustrated in the drawings and described in detail in the text. However, this is not intended to limit the present invention to a specific form disclosed, and should be understood to include all modifications, equivalents, and substitutes included in the spirit and scope of the present invention. Like reference numerals have been used for like elements throughout the description of each figure.

Terms used in this application are only used to describe specific embodiments and are not intended to limit the present invention. Singular expressions include plural expressions unless the context clearly dictates otherwise. In this application, terms such as "comprise" or "have" are intended to designate that there is a feature, step, operation, component, part, or combination thereof described in the specification, but one or more other features or steps However, it should be understood that it does not preclude the possibility of existence or addition of operations, components, parts, or combinations thereof.

1 is a schematic diagram showing a method for synthesizing calcium phosphate nanoparticles according to an embodiment of the present invention and a TEM image of nanoparticles synthesized thereby.

Referring to FIG. 1, the method for synthesizing calcium phosphate nanoparticles according to an embodiment of the present invention includes a first step of adding and mixing an aqueous solution containing a calcium precursor to a solution containing a phosphorylated amino acid, and containing a hydroxyl group in the mixed solution. A second step of adding and mixing a basic material to

The phosphorylated amino acid is used as a phosphorus source of calcium phosphate nanoparticles. Phosphorylated amino acids include, for example, phosphoserine, phosphotyrosine, phosphothreonine, phosphohistidine, phosphoarginine, phospholysine, phospho Aspartic acid, phosphoglutamic acid, phosphocysteine, and the like may be used, but are not limited thereto.

The calcium precursor is used as a calcium (Ca ²⁺ ) source of calcium phosphate nanoparticles, for example, calcium chloride (CaCl ₂ ), calcium nitrate (Ca(NO ₃ ) ₂ ), calcium acetate (Ca(C ₂ H ₃ O ₂ ) ₂ ), calcium ethoxide (Ca(OEt) ₂ ) and the like may be used, but are not limited thereto.

The basic material containing the hydroxyl group is used as a hydroxyl group (OH ^- ) source of calcium phosphate nanoparticles. At the same time, the basic substance containing a hydroxyl group plays a role in creating an alkaline solution state necessary for synthesis by adjusting the pH. Examples of the hydroxyl group-containing basic material include sodium hydroxide (NaOH) and ammonium hydroxide (NH ₄ (OH)), but are not limited thereto.

Hereinafter, the synthesis method of the present invention will be described in detail with reference to FIG.

First, a first step of adding and mixing an aqueous solution containing a calcium precursor to a solution containing a phosphorylated amino acid is performed.

In one embodiment, the solution containing the phosphorylated amino acid may be prepared by dissolving the phosphorylated amino acid in purified water. At this time, the concentration of the phosphorylated amino acid may be 1 to 100 mM. In addition, the prepared solution may be maintained at 55 to 65° C., preferably 60° C., for sufficient calcium phosphate synthesis.

In one embodiment, the aqueous solution containing the calcium precursor may be prepared by dissolving the calcium precursor in distilled water. At this time, the concentration of the calcium precursor may be 0.9 to 100 mM. And, the prepared aqueous solution may be maintained at 55 to 65°C, preferably 60°C, for sufficient calcium phosphate synthesis.

Meanwhile, even when the solutions prepared in the first step are mixed, the temperature is preferably maintained at 55 to 65°C, more preferably at 60°C. This is to allow the synthesis to proceed sufficiently with calcium phosphate during synthesis of the nanoparticles.

In addition, in the first step, mixing of the prepared solutions is preferably performed for 5 to 15 minutes. If it is out of the above range, the phosphorylated amino acid undergoes beta-decomposition to form a compound containing a large amount of hydroxyapatite. The compound formed under these conditions is synthesized as general hydroxyapatite, unlike a single compound in which the organic material to be produced in the present invention is included in a unit crystal.

Next, a second step of adding a basic substance containing a hydroxyl group to the mixed solution and mixing is performed.

In one embodiment, the basic substance containing a hydroxyl group may be dissolved in distilled water and then added to the mixed solution. In this case, the concentration of the hydroxyl group-containing basic substance may be 200 to 400 mM. In addition, the temperature of the basic solution may be maintained at 55 to 65° C., preferably 60° C., for sufficient synthesis of calcium phosphate.

In addition, even during the mixing in the second step, the temperature is preferably maintained at 55 to 65°C, more preferably at 60°C. This is to prevent a mechanism in which calcium is precipitated as calcium hydroxide (Ca(OH) ₂ ) during synthesis of the nanoparticles, and to allow the synthesis to proceed sufficiently with calcium phosphate.

Specifically, when the temperature during the mixing is maintained at less than 55° C., a mechanism in which calcium is precipitated as calcium hydroxide (Ca(OH) ₂ ) proceeds, so that the synthesis of calcium phosphate nanoparticles is not well performed. On the other hand, when the temperature exceeds 65° C., more energy is consumed to maintain the temperature, which is uneconomical.

In addition, in the second step, mixing is preferably performed for 7 to 9 hours. When it is performed for less than 7 hours, sufficient synthesis is not achieved, and when it exceeds 9 hours, thermal decomposition of phosphorylated amino acids occurs or a mechanism in which calcium is precipitated as calcium hydroxide (Ca(OH) ₂ ) proceeds, resulting in calcium phosphate nanoparticles. There is a problem in that the synthesis of is not performed well, and aggregation and coagulation of nanoparticles occur.

According to the present invention, calcium phosphate nanoparticles having high biocompatibility can be synthesized without an additional phosphorus source by using phosphorylated amino acid, which is an organic material, as a phosphorus source.

Specifically, looking at the TEM image of FIG. 1, the calcium phosphate nanoparticles synthesized through the second step have a porous crystalline structure. More specifically, the synthesized calcium phosphate nanoparticles may be porous hydroxyapatite.

Meanwhile, as another embodiment of the present invention, calcium phosphate nanoparticles prepared according to the above method may be mentioned.

The calcium phosphate nanoparticles may include a plurality of pores and have a hydroxyapatite crystal structure.

In one embodiment, the calcium phosphate nanoparticles may have a grain shape and may have a particle size of 120 to 170 nm.

In one embodiment, the surface potential of the calcium phosphate nanoparticles may be -3 to -5 mV. That is, the calcium phosphate nanoparticles of the present invention have a neutral surface potential compared to inorganic-based nanoparticles and thus have excellent biocompatibility.

The calcium phosphate nanoparticles synthesized as above have high biocompatibility and can be applied in the fields of drug delivery, bone regeneration, and tissue regeneration.

Specifically, the calcium phosphate nanoparticles of the present invention can be used as a drug delivery system.

In one embodiment, a large amount of drug may be loaded into a plurality of pores included in the calcium phosphate nanoparticle and used as a drug delivery system.

In another embodiment, a drug may be adsorbed on the surface of the calcium phosphate nanoparticle to be used as a drug delivery system. Drugs used at this time include RNA and DNA-based gene carriers; Poly-L-lactide, poly(lactic-co-glycolic acid), and collagen-type high-molecular drugs used to improve cell adhesiveness, proliferation, and differentiation; in vivo growth factors such as BMP-2 and PDGF; Fungicides or antibiotics such as gatifloxacine, ciproflaxin, tigecyclin, cefuroxine, amoxicillin, indomethacin, doxycyclin; vitamins and antioxidants of coenzyme Q10, but are not limited thereto.

In addition, the calcium phosphate nanoparticles of the present invention can be used as nanomaterials for bone regeneration. In one embodiment, the calcium phosphate nanoparticles can be used as a nanomaterial for bone regeneration by adsorbing proteins in multiple pores.

Meanwhile, in another embodiment of the present invention, a method for manufacturing a nanostructure for bone regeneration may be mentioned.

The method for preparing the nanostructure for bone regeneration includes sintering the calcium phosphate nanoparticles at a temperature of 100 to 500°C.

When the sintering is performed at a temperature of 100 to 500° C., the nanostructure for bone regeneration may maintain a hydroxyapatite structure. However, if the temperature is less than 100 ℃ sintering of the nanoparticles is not made, and if it exceeds 500 ℃ phase transition to tricalcium phosphate occurs. Therefore, it is preferable to sinter at a temperature of 100°C to 500°C.

The nanostructure for bone regeneration obtained by the above method includes nanopores and micropores and has a three-dimensional hierarchical structure. Therefore, it can be used for protein adsorption or nutrient diffusion using a three-dimensional hierarchical structure.

In addition, the nanostructure for bone regeneration may form a scaffold structure using 3D printing.

Meanwhile, the present invention may provide a method for surface modification of calcium phosphate nanoparticles in another embodiment. The method of modifying the surface of the calcium phosphate nanoparticles is to modify the surface of the nanoparticles by reacting the calcium phosphate nanoparticles with a polymer containing a thiol group (-SH) in a 1-ethyl-3-dimethylaminopropyl carbodiimide (EDC) solution. It includes steps to

The calcium phosphate nanoparticles of the present invention contain a phosphorylated amino acid functional group, and due to the presence of such a functional group, the surface of the nanoparticle can be modified by reacting with a polymer containing a thiol group (-SH). The functional group is different depending on the type of phosphorylated amino acid used in the synthesis, and may be, for example, serine, tyrosine, threonine, histidine, arginine, lysine, and the like.

In addition, the polymer containing the thiol group (-SH) is preferably 3-mercaptopropionic acid (HSCH ₂ CH ₂ CO ₂ H), but is not limited thereto.

Hereinafter, the content of the present invention will be further described with specific examples.

Nanoparticles were synthesized using a phosphoserine solution and an aqueous calcium chloride solution at various concentrations (1 mM, 10 mM, and 100 mM, respectively).

Specifically, phosphoserine solutions in which different concentrations of phosphoserine were dissolved in purified water were prepared, respectively, and maintained at 60°C. Meanwhile, aqueous solutions of calcium chloride having different concentrations were prepared, respectively, and maintained at 60°C. In addition, an aqueous sodium hydroxide solution having a concentration of 300 mM was prepared and maintained at 60°C.

Thereafter, an aqueous calcium chloride solution was added to the phosphoserine solution and mixed for 10 minutes. Next, an aqueous sodium hydroxide solution was added to the mixed solution and mixed for 8 hours to synthesize calcium phosphate nanoparticles. At this time, the temperature during mixing was maintained at 60°C.

The synthesized calcium phosphate nanoparticles were obtained by separating using centrifugation and washing three times with distilled water.

2 is a SEM analysis image of calcium phosphate nanoparticles synthesized according to Example 1.

Referring to FIG. 2, it can be seen that calcium phosphate nanoparticles were successfully synthesized in a concentration range of 1 to 100 mM phosphoserine, 1 to 100 mM calcium chloride, and 300 mM sodium hydroxide.

In order to confirm the nanoparticle synthesis result according to the phosphorylated amino acid type, nanoparticles were synthesized using various concentrations of phosphoserine, phosphotyrosine, and phosphothreonine.

Phosphoserine (Example 2-1)

Phosphoserine solutions of different concentrations (30, 45, 60, 75, 90, 105, 150 mM) in distilled water were prepared, respectively, and maintained at 60°C. Meanwhile, aqueous solutions of calcium chloride having different concentrations (0.9, 1.2, and 1.5 mM) were prepared, respectively, and maintained at 60°C. In addition, an aqueous sodium hydroxide solution having a concentration of 300 mM was prepared and maintained at 60°C.

Thereafter, an aqueous calcium chloride solution was added to the phosphoserine solution and mixed for 10 minutes. Next, an aqueous sodium hydroxide solution was added to the mixed solution and mixed for 8 hours to synthesize calcium phosphate nanoparticles. At this time, the temperature during mixing was maintained at 60°C.

The synthesized calcium phosphate nanoparticles were obtained by separating using centrifugation and washing three times with distilled water.

Phosphotyrosine (Example 2-2)

Phosphotyrosine solutions of different concentrations (60, 75, 90 mM) dissolved in distilled water were prepared, respectively, and maintained at 60°C. Other experimental conditions were performed in the same manner as in Example 2-1 to synthesize calcium phosphate nanoparticles.

Phosphoserine (Examples 2-3)

Calcium phosphate nanoparticles were synthesized in the same manner as in Example 2-2, except that phosphothreonine was used instead of phosphotyrosine.

3 is a SEM analysis image of calcium phosphate nanoparticles synthesized according to Example 2.

Referring to FIG. 3 , it can be seen that calcium phosphate nanoparticles were successfully synthesized in a concentration range of 30 to 105 mM phosphoserine, 0.9 to 1.5 mM calcium chloride, and 300 mM sodium hydroxide.

In the case of phosphotyrosine and phosphothreonine, calcium phosphate nanoparticles were successfully synthesized in the concentration range of 60 to 90 mM.

In Example 2-1, calcium phosphate nanoparticles synthesized under conditions of phosphoserine 90 mM, calcium chloride 1.2 mM, sodium hydroxide 300 mM (hereinafter referred to as Example) were analyzed by TEM, SAED and EDS, and the results are shown in FIG. 4 shown in

Referring to (a) of FIG. 4 , it can be seen that the synthesized nanoparticles have a porous form, are calcium phosphate having a grain shape, and have a hydroxyapatite crystal structure.

In addition, in (b) of FIG. 4 , it can be seen that Ca, P, and O elements were detected as components of the nanoparticles.

Calcium phosphate nanoparticle properties

In order to evaluate the characteristics of the synthesized nanoparticles, the hydrodynamic size and surface potential of the calcium phosphate nanoparticles synthesized according to Examples 2-1 and 2-2 were measured, and ICP-OES analysis was performed. After that, the results are shown in FIG. 5 . As a comparative example, conventional hydroxyapatite nanoparticles (HAp) and DNApatite were used.

Referring to Figure 5 (a), in terms of the concentration of calcium, the size of the nanoparticles using phosphoserine (Example 2-1) is about 160 nm, and the nanoparticles using phosphotyrosine (Example 2-2) The size of was measured to be about 130 nm, showing no significant difference.

On the other hand, looking at the results of measuring the surface potential, the surface potential of the nanoparticles synthesized according to Examples 2-1 and 2-2 was -3 to -5 mV, and the conventional hydroxyapatite nanoparticles (HAp) as comparative examples It was found to have a potential close to neutral compared to that of DNApatite and DNApatite.

In addition, as shown in FIG. 5(b), when the ratio of Ca and P of the nanoparticles of the present invention was confirmed through ICP-OES, they all showed the same value of 1.6. These results indicate that the calcium phosphate nanoparticles synthesized according to the examples of the present invention have a hydroxyapatite crystal structure.

Calcium phosphate nanoparticles, hydroxyapatite and phosphoserine synthesized according to Example 2-1 were analyzed using infrared spectroscopy (FT-IR), and the results are shown in FIG. 6 .

As shown in FIG. 6, it can be confirmed that the calcium phosphate single compound (pS-HAp) according to the present invention has a chemical bond in common with phosphoserine used in the synthesis and existing calcium phosphate.

The above results indicate that the calcium phosphate nanoparticles according to the present invention are calcium phosphate nanoparticles using phosphoserine as a phosphorus source.

A nanostructure for bone regeneration was prepared by freeze-drying the calcium phosphate nanoparticles synthesized according to Example 2-1.

Thereafter, using osteoblasts (MC3T3-E1), a biocompatibility test (MTT assay) of the nanostructure for bone regeneration was performed, and the results are shown in FIG. 7 .

As shown in the cell compatibility test (MTT assay) result on the left side of FIG. 7, it can be seen that there is no toxicity (cell viability 80% or more) even when a very high concentration (200 μg/mL) of the nanostructure for bone regeneration is treated. .

In addition, the results of the ALP assay, which is an indicator of differentiation of osteoblasts into osteocytes, are shown on the right side of FIG. 7 .

When comparing the existing dense hydroxyapatite (HAp) and tricalcium phosphate (TCP) together, it can be seen that the nanostructure derived from a single compound (pS-HAp) greatly promotes ALP differentiation of osteoblasts over time. there is. This shows that the ability of the single compound (pS-HAp)-derived nanostructure to differentiate osteoblasts into osteocytes is superior to existing hydroxyapatite nanoparticles.

The description of the presented embodiments is provided to enable any person skilled in the art to use or practice the present invention. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein may be applied to other embodiments without departing from the scope of the present invention. Thus, the present invention is not to be limited to the embodiments presented herein, but is to be construed in the widest scope consistent with the principles and novel features presented herein.

Claims (16)

A first step of adding and mixing an aqueous solution containing a calcium precursor to a solution containing phosphorylated amino acids; and
A second step of adding a basic substance containing a hydroxyl group to the mixed solution and mixing;
The phosphorylated amino acid includes phosphoserine, phosphotyrosine or phosphothreonine,
The calcium phosphate nanoparticles synthesized after the second step include a plurality of pores, have a hydroxyapatite crystal structure, and exhibit a grain shape,
Characterized in that the size of the nanoparticles is 120 to 170 nm, and the surface potential of the calcium phosphate nanoparticles is -3 to -5 mV,
A method for synthesizing calcium phosphate nanoparticles.
According to claim 1,
Characterized in that the temperature during the mixing is maintained at 55 to 65 ° C.
A method for synthesizing calcium phosphate nanoparticles.
According to claim 1,
Mixing in the first step is performed for 5 to 15 minutes,
Characterized in that the mixing of the second step is performed for 7 to 9 hours,
A method for synthesizing calcium phosphate nanoparticles.
delete According to claim 1,
The calcium precursor is any one selected from calcium chloride (CaCl ₂ ), calcium nitrate (Ca(NO ₃ ) ₂ ), calcium acetate (Ca(C ₂ H ₃ O ₂ ) ₂ ) and calcium ethoxide (Ca(OEt) ₂ ) Characterized by including the above,
A method for synthesizing calcium phosphate nanoparticles.
According to claim 1,
Characterized in that the basic substance containing a hydroxyl group includes sodium hydroxide (NaOH) or ammonium hydroxide (NH ₄ (OH)),
A method for synthesizing calcium phosphate nanoparticles.
According to claim 1,
Characterized in that the concentration of the phosphorylated amino acid is in the range of 1 to 100 mM and the concentration of the calcium precursor is in the range of 0.9 to 100 mM, and the concentration of the basic substance containing the hydroxyl group is in the range of 200 to 400 mM, calcium phosphate nanoparticles are synthesized doing,
A method for synthesizing calcium phosphate nanoparticles.
delete delete A calcium phosphate nanoparticle prepared according to the method of any one of claims 1 to 3 and 5 to 7,
Contains a large number of pores, has a hydroxyapatite crystal structure, and exhibits a grain shape,
Characterized in that the size of the nanoparticles is 120 to 170 nm, and the surface potential of the calcium phosphate nanoparticles is -3 to -5 mV,
Calcium phosphate nanoparticles. delete delete delete delete delete delete

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