KR20200072179A - Method of synthesizing calcium phosphate nanomaterials using phosphate group in nucleic acid and calcium phosphate nanomaterials using phosphate group in nucleic acid - Google Patents

Abstract

The present invention relates to a method for synthesizing a nucleic acid-based calcium phosphate nanomaterial, and a nucleic acid-based calcium phosphate nanomaterial synthesized thereby. The present invention relates to a method for synthesizing a calcium phosphate nanomaterial by using phosphorus in only a phosphate group of a nucleic acid backbone. Through the present invention, a novel nucleic acid-based porous calcium phosphate nanomaterial may be synthesized. Only the phosphate group present in the nucleic acid backbone may be used as a phosphorus source without the addition of phosphorus.

Description

Method for synthesizing nucleic acid-based calcium phosphate nanomaterials and nucleic acid-based calcium phosphate nanomaterials synthesized thereby {METHOD OF SYNTHESIZING CALCIUM PHOSPHATE NANOMATERIALS USING PHOSPHATE GROUP IN NUCLEIC ACID AND CALCIUM PHOSPHATE NANOMATERIALS USING PHOSPHATE GROUP IN NUCLEIC

The present invention relates to a method for synthesizing a nucleic acid-based calcium phosphate nanomaterial and a nucleic acid-based calcium phosphate nanomaterial synthesized thereby.

The present invention relates to a method for synthesizing calcium phosphate nanomaterials by utilizing phosphorus unique to a phosphate group of a backbone of a nucleic acid.

Synthesis of the existing calcium phosphate nanomaterials is performed through Co-precipitation (wet chemistry) or sol-gel method using inorganic materials as raw materials. Calcium phosphate is a substance that exists in nature, and in particular, it has a very high biocompatibility because it accounts for 50 to 70 w/w.% of bones in the human body. Therefore, many studies have been conducted to apply it to fields such as drug delivery, bone regeneration, and tissue regeneration.

However, calcium phosphate nanomaterials synthesized from minerals have a dense surface and show cytotoxicity when treated in large quantities in biological tissues. Originally, in vivo, calcium phosphate has a mechanism that is synthesized based on organic matter, and is very different from the synthesis of calcium phosphate based on inorganic matter. Accordingly, an attempt to make calcium phosphate based on organic materials has recently been progressed.

The present invention relates to a method and application of synthesizing calcium phosphate nanomaterials by using only the phosphate group present in the backbone of nucleic acid as a phosphorus source, which has not been progressed so far.

The present invention is to provide a method for synthesizing a calcium phosphate nanomaterial on a porous surface by using only the phosphate group present in the nucleic acid as a phosphorus source.

In addition, the present invention is to provide a biological application such as drug delivery, bone regeneration, tissue regeneration using the calcium phosphate nanomaterial synthesized by this method.

A method for synthesizing a nucleic acid-based calcium phosphate nanomaterial according to an embodiment of the present invention comprises the steps of preparing a solution containing an organic substance containing a phosphorus group obtained from a nucleic acid; Preparing a solution containing a calcium salt providing a calcium ion (Ca ^{+ 2);} Preparing a solution containing an inorganic material which provides ^- hydroxyl ion (OH); And a solution containing the organic material. A solution containing the calcium salt; And synthesizing the calcium phosphate nanomaterial by mixing the solution containing the inorganic material.

The nucleic acid is any one or more of DNA, RNA and plasmid DNA.

The calcium salt contains CaCl ₂ , and the inorganic material includes NaOH.

The content of the nucleic acid is 5 mg/mL or more, preferably 10 mg/mL or more.

The content of the calcium salt is 0.5mM or more, preferably 1mM or more.

In the step of synthesizing the calcium phosphate nanomaterial, the synthesis temperature is preferably maintained at 60°C or higher.

The nucleic acid-based calcium phosphate nanomaterial according to an embodiment of the present invention exhibits a porous characteristic and crystallinity.

In addition, the nucleic acid-based calcium phosphate nanomaterial according to an embodiment of the present invention can be used as a drug carrier carrying a drug on a porous structure or adsorbing a drug on a surface.

The nucleic acid-based calcium phosphate nanomaterial according to an embodiment of the present invention can be used as a bone regeneration nanomaterial adsorbing a protein on a porous structure. The porous nucleic acid-based calcium phosphate nanomaterial is sintered to include both a nanoporous structure and a microporous structure.

Through the present invention, a new nucleic acid-based porous calcium phosphate nanomaterial can be synthesized. Only the phosphate group present in the backbone of the nucleic acid is used as the phosphorus source, and has the characteristic of not adding additional phosphorus.

1 is a flowchart of a method for synthesizing a nucleic acid-based calcium phosphate nanomaterial according to an embodiment of the present invention.
Figure 2 shows the effect of the organic/inorganic materials used in the present invention on the formation of calcium phosphate nanomaterials.
3 shows the effect of the concentration of the DNA used in the present invention and the concentration of CaCl ₂ on the formation of nanomaterials.
4 and 5 show that the nanomaterial is synthesized at the minimum concentration of CaCl ₂ and NaOH used in the present invention.
6 shows a state in which TEM analysis of the process of synthesis of the present nanomaterial was performed.
7 to 10 show the results of EDS analysis, UV-Vis analysis, DLS (Hydrodynamic size), and Zeta potential analysis.
11 shows the results of the FT-IR analysis of the present nanomaterial.
Various embodiments are now described with reference to the drawings, and like reference numbers throughout the drawings are used to indicate similar elements. For purposes of illustration, various descriptions are presented to provide an understanding of the invention. However, it is clear that these embodiments can be practiced without these specific details. In other instances, well-known structures and devices are presented in block diagram form in order to facilitate describing the embodiments.

Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings. The present invention can be applied to various changes and may have various forms, and specific embodiments will be illustrated in the drawings and described in detail in the text. However, this is not intended to limit the present invention to a specific disclosure form, and it should be understood as including all modifications, equivalents, and substitutes included in the spirit and scope of the present invention. In describing each drawing, similar reference numerals are used for similar components.

The terms used in the present 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 indicates otherwise. In this application, the terms "include" or "have" are intended to indicate that a feature, step, operation, component, part, or combination thereof described on the specification exists, one or more other features or steps. It should be understood that it does not preclude the existence or addition possibility of the operation, components, parts or combinations thereof.

1 is a flowchart of a method for synthesizing a nucleic acid-based calcium phosphate nanomaterial according to an embodiment of the present invention.

A method for synthesizing a nucleic acid-based calcium phosphate nanomaterial according to an embodiment of the present invention includes preparing a solution containing an organic substance containing a phosphorus group obtained from a nucleic acid (S 110); Preparing a solution containing a calcium salt providing a calcium ion ^{(Ca 2 +) (S 120} ); Hydroxide ion (OH ^-) to prepare a solution containing an inorganic material providing (S 130); And a solution containing the organic material. A solution containing the calcium salt; And synthesizing the calcium phosphate nanomaterial by mixing the solution containing the inorganic material (S 140).

In step S 110, a solution containing an organic substance containing a phosphorus group obtained from a nucleic acid is prepared. The nucleic acid is preferably one or more of DNA, RNA and plasmid DNA. Such nucleic acids may be nucleic acids of various lengths or forms.

The content of such nucleic acids is preferably 5 mg/mL or more, more preferably 10 mg/mL or more. When the content of the nucleic acid is less than 5mg/mL, the amount of the nucleic acid is too small, and thus, when the calcium phosphate is synthesized, the amount of organic substances including phosphorous groups is insufficient, so that the calcium phosphate is not synthesized smoothly. This part will be further described in the embodiments described below.

In step S 120, a solution containing a calcium salt that provides calcium ions is prepared. Preferably, the calcium salt includes CaCl ₂ . Step S 120 does not necessarily have to proceed after step S 110, but can be done at the same time, and the context of the order is not related.

The ?t amount of the calcium salt is preferably 0.5 mM or more, more preferably 1 mM or more. When the content of the calcium salt is less than 0.5 mM, the amount of calcium ions is insufficient, and thus, the synthesis of calcium phosphate is not smooth. This part will be further described in the embodiments described below.

In step S 130, a solution containing an inorganic substance that provides hydroxide ions is prepared. Preferably, the inorganic material includes NaOH. The step S 130 is not necessarily performed after the step S 110 or the step S 120, but can also be performed simultaneously, and the context of the order is not related.

It plays a role of controlling the amount of OH ^- ions necessary for the formation of calcium phosphate nanomaterials, acting as a mineral that provides hydroxide ions.

In step S 140, a solution containing the organic material; A solution containing the calcium salt; And mixing the solution containing the inorganic material to synthesize a calcium phosphate nanomaterial. In the step of synthesizing the calcium phosphate nanomaterial, the synthesis temperature is preferably maintained at 60°C or higher. Because if the synthesis temperature is less than 60 ℃ Ca (OH) ₂ is likely to proceed to the mechanism that is produced. Therefore, by maintaining the temperature above 60 ℃, it is possible to induce a mechanism for the synthesis of calcium phosphate nanomaterials.

The nucleic acid-based calcium phosphate nanomaterial synthesized according to the synthesis method of the present invention described above exhibits a porous characteristic. In addition, these nucleic acid-based calcium phosphate nanomaterials exhibit crystallinity.

The method of the present invention can be extended in the following way. A method of synthesizing a nanomaterial including calcium ions according to a further embodiment of the present invention includes preparing a solution containing an organic material including polyanions including a nucleic acid polymer; Preparing a solution containing a calcium salt providing a calcium ion (Ca ^{+ 2);} Preparing a solution containing an inorganic material which provides ^- hydroxyl ion (OH); And a solution containing the organic material. A solution containing the calcium salt; And mixing the solution containing the inorganic material to synthesize nanomaterials. It can be applied as a method of synthesizing nano materials including calcium ions using polyanions containing nucleic acid polymers such as DNA and RNA.

In addition, the nucleic acid-based calcium phosphate nanomaterial synthesized in this way may be hydroxyapatite.

In this case, the synthesized porous nucleic acid-based calcium phosphate nanomaterial can be used as a drug carrier, bone regeneration nanomaterial, and tissue regeneration nanomaterial.

When used as a drug delivery system, it can be used to deliver a drug by supporting the drug on a porous structure or adsorbing the drug on a surface.

When used as a bone regeneration nanomaterial, proteins are adsorbed onto a porous structure. In addition, the sintering of the calcium phosphate nanomaterial based on the porous nucleic acid can achieve a structure including both a nanoporous structure and a microporous structure (hierarchical structure), and can be used by adsorbing proteins to the structure. It is also possible to form a scaffold structure by grafting 3D printing technology.

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

The experiment proceeds with a DNA backbone with a large number of phosphate groups among bio-derived organics as a phosphorus source. Ca ^{2 +,} and is prepared by dissolving CaCl ₂ in DI water, OH ^- is prepared by dissolving NaOH in DI water.

-Deoxyribonulceic acid from fish sperm, Sigma-Aldrich, CAS #: 100403-24-5

-Calcium chloride, anhydrous, free-flowing, Redi-DriTM, ≥97%, Sigma-Aldrich, CAS #: 10043-52-4

-Sodium hydroxide, bead,>98.0%, Samchun Chemicals, CAS #: 1310-73-2

-D.I. water, Deionized water, 18.2 MΩ cm, Sartorius Arium® pro ultrapure water system

450 mg of DNA, 30 mL of D.I. After sufficiently dissolving in water, filtering is performed to extract DNA. The extracted DNA was D.I. It is quantified by measuring UV-Vis with water dilution factor: 601. At this time, DNA is quantified using Beer's law.

1) Preparation of DNA

450 mg of DNA is dissolved in 30 mL of DI water through vortexing. Remove agglomeration using Satorius stedim biotech's Minisart® 0.45 μm microfilter. Dilution factor: 601 is measured using UV-Vis quantification method. Dilute the DNA concentration to 10 mg·mL ^-1 using DI water. At this time, 20 mL is used as the final volume. Then, the synthesis is prepared at 60°C.

2) Preparation of CaCl ₂

To prepare 1 M Stock solution, measure 110.98 g and dissolve in 1 L of DI water. At this time, since the exothermic reaction proceeds, CaCl ₂ is slowly added to the shaking DI water and dissolved. Dilution to the concentration required for one experiment to prepare in a 60℃ environment. If the variable used in the experiment is 1 mM 20 mL, 20 mL of DI water is quantified and then 20 μL is removed, and then 20 μL of 1 M CaCl ₂ is added.

3) Preparation of NaOH

To prepare a 1 M Stock solution, quantify 40 g and 1 L of D.I. Dissolve in water. At this time, the exothermic reaction proceeds, so D.I. Dissolve while slowly adding NaOH in water. Dilution to the concentration required for one experiment to prepare in a 60 ℃ environment. Assuming that the variables used in the experiment are 50% and 20 mL, 10 mL of D.I. After quantifying water, 10 mL of 1 M NaOH was added.

4) Synthesis of calcium phosphate

Prepare 20 mL of DNA solution, 20 mL of CaCl ₂ solution, and 20 mL of NaOH solution at 60°C. The synthetic conditions are magnetic stirring, 200 rpm, 60℃, 8 h.

Through the above experimental conditions, the optimum value of the content of DNA and the content of CaCl ₂ was found, and the influence of the presence or absence of NaOH was confirmed.

* Confirmation of the interaction of organic/inorganic substances required for synthetic reactions

Figure 2 shows the effect of the organic/inorganic materials used in the present invention on the formation of calcium phosphate nanomaterials. According to the concentration of DNA used for synthesis (1, 5, 10 mg mL-1), the concentration of CaCl ₂ (0, 1, 5 mM) and the presence or absence of NaOH (1 M) Confirmed. When the amount of DNA as a phosphorus source is small, Ca ^{2 +} and OH ⁻ react to confirm the progress of Ca(OH) ₂ precipitation (co-precipitation), and synthesis was not progressed with the nanomaterial according to the present invention. . Amorphous precipitation was observed, and formation of nanomaterials could not be confirmed. This can be clearly seen by comparing the formation of crystalline nanomaterials as the amount of DNA increases, and it can be inferred that this is due to the influence of the phosphorus source. On the other hand, if the amount of Ca ^{2 +} is small, it was confirmed that the formation of precipitates and nanomaterials is impossible. Also, in the absence of NaOH, precipitates and nanomaterials were not formed.

* Confirmation of variables affecting nanomaterial synthesis (concentration of DNA and CaCl ₂ )

3 shows the effect of the concentration of the DNA used in the present invention and the concentration of CaCl ₂ on the formation of nanomaterials. It was confirmed that the same effects as described above were exhibited. In particular, the synthesis proceeds with nanomaterials under most experimental conditions using DNA as 10 mg mL-1. Through this experiment, it can be confirmed that the surface shape of the nanomaterial is porous, and this is because it was synthesized using an organic material such as a DNA phosphate chain. That is, when the nanomaterial is synthesized by minimizing the influence of CaCl ₂ and NaOH, the shape may be different according to different organic phosphorus sources. The next experiment proceeds with the goal of using minimal CaCl ₂ and NaOH.

* Confirmation of variables affecting nanomaterial synthesis (concentration of CaCl ₂ and NaOH)

4 and 5 show that the nanomaterial is synthesized at the minimum concentration of CaCl ₂ and NaOH used in the present invention. Check the shape of the nanomaterial that maintains the chain shape of the DNA, which is an organic-based phosphorus source, as much as possible. At this time, when using CaCl ₂ 0.5 mM, it can be confirmed that the minimum concentration is synthesized. In addition, the minimum NaOH conditions that can be synthesized in FIG. 5 were confirmed to be 30% and 1 M conditions.

* Additional analysis of TEM, EDS, and FT-IR of synthesized nanomaterials

6 shows a state in which TEM analysis of the process of synthesis of the present nanomaterial was performed. During the process of synthesizing nanomaterials, sampling was performed every hour for analysis. Through this, it was confirmed that the nanomaterial has a crystal structure and a porous surface. Image Fourier transform (bottom right of each TEM photo) analysis demonstrates that this nanomaterial has a crystal structure. In addition, it can be seen that the nanomaterials seen from the upper right of each TEM photograph are porous surfaces because the layered layers are stacked and overlapped.

7 to 10 show the results of EDS analysis, UV-Vis analysis, DLS (Hydrodynamic size), and Zeta potential analysis. While performing the EDS analysis, it shows a controllable result so that the Ca/P atomic ratio is 1.64, which is the same as that of hydroxy apatite. The conditions are DNA 10 mg mL ^-1 , CaCl ₂ 5mM, NaOH 60%. The results of the EDS analysis can also be confirmed in FIGS. 4 and 5, which are typically synthesized as 1.0 and 1.25. It can be seen that the higher the concentration of CaCl ₂ is, the higher the concentration, and indicates that it is synthesized into a calcium phosphate structure under various conditions.

UV-Vis, DLS and Zeta potential analysis were performed by sampling every hour during the synthesis process. UV-Vis analysis shows that the presence of DNA persists during the synthesis process. It indicates that the synthesized nanomaterial contains DNA.

DLS has a small initial shape and shows a larger shape over time. Zeta potential analysis shows that the synthesis process continues to have a negative charge, which is the same as that seen with normal calcium phosphate.

11 shows the results of the FT-IR analysis of the present nanomaterial. DNA, CaCl ₂ , NaOH, and synthesized nanomaterials used for synthesis were measured. The compounds used in the synthesis show the form of chemical bonds that should be possessed. However, the synthesized nano-material is chemically bonded to have a calcium phosphate _{v s (OH), v as} CH 3, v as CH 2, v 3 (CO 3 2 -) show, v _3a (PO ₄ ^3-), etc. Given, it shows the same form as the FT-IR analysis, which shows a typical calcium phosphate nanomaterial. That is, it is proved that this nanomaterial is calcium phosphate using DNA as a phosphorus source.

Through this example, a new nucleic acid-based porous calcium phosphate nanomaterial could be synthesized. Only the phosphate group present in the backbone of the nucleic acid is used as the phosphorus source, and no additional phosphorus is added. CaCl ₂ and NaOH were used for Ca ^{2 +} source and material formation, respectively. Then, the temperature was raised to 60°C to prevent the mechanism of precipitation with Ca(OH) ₂ , so that synthesis was sufficiently performed with calcium phosphate. Thereafter, the nanomaterial synthesized through centrifuge is washed three times with DI water to supply only the synthesized nanomaterial. The completed nanomaterial shows a porous shape, and is confirmed to have a grain shape of average width: 47.85 nm and length: 202.15 nm. In addition, it was confirmed that Ca2+ and phosphorus ions maintain a specific atomic ratio, showing that calcium phosphate nanomaterials are synthesized.

Since the nanostructure of the present invention is porous and has a large specific surface area, it has strengths in protein adsorption and drug delivery. It is a component that makes up 50-70 w/w.% of bone in the body, which helps in adsorbing and regenerating bone cells. In addition, it is expected that if calcium is escaped from the calcium phosphate nanomaterial derived from the nucleic acid, it can be used for drug delivery using the remaining nucleic acid.

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

Claims (16)

Preparing a solution containing an organic substance containing a phosphorus group obtained from a nucleic acid;
Preparing a solution containing a calcium salt providing a calcium ion (Ca ^{+ 2);}
Preparing a solution containing an inorganic material which provides ^- hydroxyl ion (OH); And
A solution containing the organic material; A solution containing the calcium salt; And comprising the step of synthesizing the calcium phosphate nano-material by mixing the solution containing the inorganic material,
Method for synthesizing a nucleic acid-based calcium phosphate nanomaterial.
According to claim 1,
The nucleic acid is any one or more of DNA, RNA and plasmid DNA,
Method for synthesizing a nucleic acid-based calcium phosphate nanomaterial.
According to claim 1,
The calcium salt includes CaCl ₂ ,
Method for synthesizing a nucleic acid-based calcium phosphate nanomaterial.
According to claim 1,
The inorganic material includes NaOH,
Method for synthesizing a nucleic acid-based calcium phosphate nanomaterial.
According to claim 1,
The content of the nucleic acid is 5mg / mL or more,
Method for synthesizing a nucleic acid-based calcium phosphate nanomaterial.
According to claim 1,
The content of the nucleic acid is 10mg / mL or more,
Method for synthesizing a nucleic acid-based calcium phosphate nanomaterial.
According to claim 1,
The content of the calcium salt is 0.5mM or more,
Method for synthesizing a nucleic acid-based calcium phosphate nanomaterial.
According to claim 1,
The content of the calcium salt is 1mM or more,
Method for synthesizing a nucleic acid-based calcium phosphate nanomaterial.
According to claim 1,
In the step of synthesizing the calcium phosphate nanomaterial, the synthesis temperature is maintained at 60°C or higher,
Method for synthesizing a nucleic acid-based calcium phosphate nanomaterial.
It is synthesized according to any one of claims 1 to 9,
Which exhibits a porous characteristic,
Nucleic acid based calcium phosphate nano material.
The method of claim 10,
The nucleic acid-based calcium phosphate nanomaterial exhibits crystallinity,
Nucleic acid based calcium phosphate nano material.
In a calcium phosphate nanomaterial based on a porous nucleic acid, synthesized according to any one of claims 1 to 9,
The drug is supported on the porous structure or adsorbed on the surface.
Drug carriers.
In a calcium phosphate nanomaterial based on a porous nucleic acid, synthesized according to any one of claims 1 to 9,
Protein adsorbed on the porous structure,
Bone regeneration nanomaterial.
The method of claim 13,
Sintering the porous nucleic acid-based calcium phosphate nanomaterial to include both a nanoporous structure and a microporous structure,
Bone regeneration nanomaterial.
An organic-inorganic composite material synthesized according to any one of claims 1 to 9,
The organic-inorganic composite material is characterized in that the strength is improved,
Organic-inorganic composite material.
Preparing a solution containing an organic material including polyanions containing a nucleic acid polymer;
Preparing a solution containing a calcium salt providing a calcium ion (Ca ^{+ 2);}
Preparing a solution containing an inorganic material which provides ^- hydroxyl ion (OH); And
A solution containing the organic material; A solution containing the calcium salt; And comprising the step of synthesizing the nano-material by mixing the solution containing the inorganic material,
Method for synthesizing nanomaterials containing calcium ions.

Images