KR101171386B1 - Core-shell structured gene-inorganic nanohybrid composite and preparation method thereof - Google Patents

Abstract

The present invention relates to a core-cell bio-inorganic nanocomposite comprising a gene which is a biomolecule and a method for preparing the same. More specifically, a two-dimensional nanosheet such as layered double hydroxide (LDH) is large. The present invention relates to a gene core-LDH cell complex having a structure in which a gene is inserted into an inorganic nanocavity formed by recombination with a gene molecule and a method of manufacturing the same. By using the gene core-LDH cell complex, a huge gene can be supported on an inorganic carrier, and excellent in stability to the external environment, and can be useful for increasing storage and delivery and its function.

Bio-inorganic nanocomposites, core-cells, exfoliation-recombination, genes, exfoliated metal hydroxides, gene-metal bilayer hydroxides, nanohybrids

Description

Core-cell structured gene-inorganic nanohybrid composite and preparation method including core-cell structure

The present invention relates to a gene-inorganic nanohybrid complex in which a large gene is encapsulated in an inorganic nanocavity and a method for preparing the same.

Hybrid technologies that fuse two elements at the nanoscale, such as inorganic-inorganic hybrids, organic-inorganic hybrids, and bio-inorganic hybrids, have recently attracted attention because they can exhibit new physicochemical properties or obtain complementary synergistic effects at the molecular level. . In particular, hybrid research with inorganic nanoparticles has been conducted from the viewpoint of increasing functionality and efficacy while overcoming the weaknesses of biomolecules or bioactive molecules ( J. Phys . Chem . Solids , 65, 373). , 2004).

Among bio-active molecules, genes, or DNAs, are ideal molecules containing various information, and they can design base sequences and structures to have new functions, and thus design synthetic genes with organic or inorganic substances, diagnosis and treatment of disease-causing gene expression. Research is being actively conducted in the fields of synthetic chemistry, biotechnology, and medicine where genes are applied, such as treatment through intracellular delivery and detection of very small amounts of macromolecules. However, when the gene itself is exposed to the external environment, it can be easily destroyed and modified by various factors (enzyme environment, chemical, physical environment, etc.), and thus the limit of effectiveness is encountered without proper stabilization measures.

Therefore, in order to overcome the unstable problem of the gene itself and to enhance the storage, storage and delivery effect, the layered double hydroxide (LDH), which is a layered inorganic nanoparticle, as an inorganic carrier capable of stabilizing the gene is noted. It has been studied, and research on this has been continued. LDH, which is one of the metal double layer hydroxides represented by the following Chemical Formula 2, is called anionic clays and is composed of a positively charged metal hydroxide layer and anion and water to offset the positive charge between the layers.

[Formula 2]

_{^{[M 2 + (1-x}} ) N 3 + x (OH) 2] [A n -] x / n yH ₂ O and

(In the LDH represented by the formula (2),

M is a divalent metal cation;

N is a trivalent metal cation;

X is a number greater than 0.1 and less than 0.4;

A is an anionic guest species;

N is the number of charges of the anion of A;

Y is a positive number greater than zero.)

Until now, intercalation chemistry of ion exchange reaction and co-precipitation reaction has been used to support bioactive molecules in the LDH. As a result, layered bio-inorganic nanocomposites have been prepared. In addition, the gene-LDH nanocomposite in which the gene is inserted into the LDH is also introduced through the ion exchange reaction and coprecipitation reaction by the strong electrostatic attraction between the LDH layers having the cationic layer charge through the ion exchange reaction and coprecipitation reaction. Bio-inorganic nanocomposites of two-dimensional layered structures have been prepared. Genes encapsulated and stabilized in inorganic nanoparticles as described above are not denatured even in the enzymatic environment, and under acidic conditions, LDH is reversibly easily dissociated and functions are gradually released as genes are gradually released (Domestic Patent Registration No. 10-0359716). J. Am . Chem . Soc . 121, 1399, 1999. Angew . Chem . Int . Ed . 39, 4041, 2000 Korean Patent Registration No. 10-0849465 Adv . Mater. , 16, 14, 2004).

However, conventional ion exchange methods and coprecipitation methods for preparing two-dimensional multilayer bio-inorganic nanocomposites or gene-LDH nanocomposites with LDH, which are layered inorganic nanoparticles introduced to stabilize bioactive molecules such as conventional genes, There are the following problems. 1) Since the interlayer spacing of LDH is limited to the extent of expansion, it is possible to carry genes of three-dimensional huge or unusual structure such as plasmid gene, gene with nanoparticles showing quantum effect, gene with organic phosphor attached, etc. 2) the length or size of genes that can be stabilized depending on the particle size of LDH, even if intercalated between LDH layers ( J. Phys . Chem . Solids . 67, 1028, 2006), 3) organic and It is very difficult to support anionic genes that contain some positive charges due to the attachment of inorganic substances. 4) To produce single-phase gene-LDH nanocomposites, excess genes and long reaction times are required in the chemical reaction step. There is a problem in that it is economically disadvantageous.

Therefore, the present inventors have found that bio-inorganic nanohybrid complexes in which large bio-active molecules are encapsulated with inorganic nanoparticles, genes having three-dimensionally large or unusual structures or genes having some positive charges are encapsulated in LDH, and have excellent stability to the external environment. While studying to produce a stabilized gene-LDH nanohybrid complex regardless of particle size and gene length and size of LDH nanohybrid complex and LDH, large genes were encapsulated in LDH inorganic carriers by exfoliation-recombination. It has excellent stability, improves storage, delivery and its function, improves the weakness of multi-phase gene-LDH nanocomposites whose gene stability depends on the particle size of LDH, and can stabilize not only genes but also large biomolecules. Learn how to make core-cell bio-inorganic nanocomposites The present invention was completed.

It is an object of the present invention to provide a gene-inorganic nanohybrid complex in which a gene is encapsulated in an inorganic nanocavity.

In addition, another object of the present invention to provide a method for producing the gene-inorganic nanohybrid complex.

In order to achieve the above object, the present invention provides a gene-inorganic nanohybrid complex represented by the following formula (1):

[Formula 1]

_{^{[M 2 + (1-x}} ) N 3 + x (OH) 2] [A GEN n -] z and yH ₂ O

(In Formula 1, wherein M, N, A, n. X, y and z are as defined herein).

In addition, the present invention provides a method for preparing a gene-inorganic nanohybrid complex represented by Chemical Formula 1. Gene-inorganic nanohybrid complex represented by the formula (1) is encapsulated in the LDH inorganic carrier by the encapsulation-recombination of LDH encapsulated in the LDH inorganic carrier, excellent stability in the external environment, increase storage, delivery and its function, It is possible to improve the weakness of the multilayered gene-LDH nanocomposite whose stability depends on the particle size of LDH, and to stabilize not only genes but also large biomolecules.

According to the present invention, a large gene is encapsulated in an LDH inorganic carrier by exfoliation-recombination of LDH, thereby providing a core-cell gene-LDH nanohybrid complex that is excellent in stability to the external environment, and can increase storage, delivery, and function thereof. In addition to the limitations of conventional interlayer insertion methods that prevent encapsulation of large genes and the stability of genes, the weakness of multilayered gene-LDH nanohybrid complexes, which depend on the particle size of LDH, can be improved, as well as the genes other than genes. Inorganic encapsulation for stabilization of biomolecules can also be used to provide useful applications for providing core-cell bio-inorganic nanocomposites.

The present invention provides a large gene-inorganic nanohybrid complex having a core-cell structure.

Hereinafter, the present invention will be described in detail.

The present invention provides a gene-inorganic nanohybrid complex represented by the following Chemical Formula 1 in which a huge gene is inserted into a nanocavity formed by exfoliation-reassembling of LDH and stabilized from an external environment:

_{^{[M 2 + (1-x}} ) N 3 + x (OH) 2] [A GEN n -] z and yH ₂ O

In Chemical Formula 1,

M ^{+ 2} is a divalent metal cation, and;

N ^{+ 3} is a trivalent metal cation, and;

A _GEN is a gene;

n is the number of charges of the gene of A _GEN ;

x is a number greater than 0.1 and less than 0.4;

z and y are positive numbers greater than zero.

Preferably,

In Chemical Formula 1,

Wherein M may be selected from magnesium (Mg ^{2 +),} nickel (Ni ^{+ 2),} copper (Cu ^{+ 2),} zinc (Zn ^{+ 2);}

The N may be selected from aluminum (Al ^{+ 3),} iron (Fe ^{+ 3),} vanadium (V ^{+ 3),} titanium (Ti ^{+ 3),} gallium (Ga ^{+ 3),} and;

The A is a large length gene or a three-dimensionally huge gene of 50 to 2500 bp or less, a single-stranded gene, a double-stranded gene, a plasmid gene, an artificial gene, PNP (Peptide nucleic acid), or an organic phosphor or an inorganic phosphor species attached thereto. And the like.

In addition,

Coprecipitating an aqueous solution of a divalent cation and a trivalent cation metal salt with a basic material and hydrothermally reacting to form LDH of Formula 2 (step 1);

Exfoliating the LDH formed in step 1 (step 2); And

Gene-inorganic nanohybrid complex of Chemical Formula 1 comprising the step of mixing the LDH exfoliated in step 2 with the macrogene or the macrogene to which the phosphor is attached to generate a core-cell gene-inorganic nanohybrid complex (step 3) It provides a method of manufacturing.

_{^{[M 2 + (1-x}} ) N 3 + x (OH) 2] [A n -] x / n yH ₂ O and

In Chemical Formula 2

M ^{+ 2} is a divalent metal cation, and;

N ^{+ 3} is a trivalent metal cation, and;

x is a number greater than 0.1 and less than 0.4;

A is an anionic guest species;

n is the number of charges of the anion of A;

y is a positive number greater than zero.

Hereinafter, the manufacturing method according to the present invention will be described in more detail step by step.

First, step 1 according to the present invention is a step of coprecipitating an aqueous solution of a divalent cation and a trivalent cation metal salt with a basic material and hydrothermally reacting to form LDH. The LDH is synthesized by making a divalent metal salt and a trivalent metal salt into a solution, and titrating with a base solution. Preferably, the divalent metal is magnesium (Mg ^{2 +),} nickel (Ni ^{2 +),} copper (Cu ^{2 +),} zinc (Zn ^{2 +)} may be selected from metal trivalent is aluminum (Al ^{3 +} ), Iron (Fe ^{3 +} ), vanadium (V ^{3 +} ), titanium (Ti ^{3 +} ), gallium (Ga ^{3 +} ), and the like, and the base solution may be sodium hydroxide (NaOH) or ammonia (NH ₃ ). Can be used. The pH of the reaction solution during the precipitation reaction is preferably 5 to 12, more preferably 9 to 11. When the pH of the reaction solution is 5 or less or 12 or more, since the solution is out of the region co-precipitated with the metal double layer hydroxide, there is a problem in that a single metal double layer hydroxide or metal oxide salt is generated. In addition, in order to prevent the formation of carbonate ions (CO ₃ ^{2 −} ) by carbon dioxide in the air, it is preferable to continuously add and react with nitrogen or an inert gas during the reaction.

Next, step 2 is a step of exfoliating the LDH formed in step 1 into a two-dimensional nanosheet.

In the method of the present invention, since the layered compound such as LDH has a cationic layer charge, the gene is encapsulated using the principle of mutually binding with a gene having an anionic phosphate group by electrostatic attraction. The encapsulation step may be carried out by a conventionally known ion exchange reaction or coprecipitation method, the conventional method of inserting genes between LDH layers is limited in the length of the gene to be inserted and protected depending on the size of the inorganic nanoparticles, It is unable to carry genes of huge or unusual structure in three dimensions, and it is very difficult to carry anionic genes containing some positive charges. In order to compensate for this drawback, a method of manufacturing a core-cell gene-inorganic nanohybrid complex stabilized from an external environment by inserting a large gene into a nanocavity formed by exfoliation-reassembling of LDH has been invented.

LDH formed in step 1 may be exfoliated using an ion exchange method or a specific solvent. LDH is nitric acid present in the interlayer (NO ₃ ^-), chloride (Cl ^-), carbonate (CO ₃ ^{2 -)} Substituting the ions such as the anionic ion of a long carbon chain are separated by a thin slice layer, formamide ( Formamide, HCONH ₂ ) is used as a solvent, formamide is inserted between layers with strong hydrogen bonding force between layers of metal bilayer hydroxides and expands between layers to break the lattice into sheets (JP4019138 Chem . Mater . 2005, 17, 4386).

In the present invention, since it is recombined with a gene that is a bioactive molecule, a method of exfoliation using formamide that maintains exfoliation even without applying temperature is preferable, and the concentration of exfoliated two-dimensional nanosheet is 0.01 to 0.10 wt%. desirable. If the concentration of the two-dimensional nanosheets is 0.10% by weight or more, there is a problem in that a metal double layer hydroxide which is not exfoliated into a single layer is present.

Next, step 3 is a step of generating a core-cell gene-inorganic nanohybrid complex by mixing the LDH exfoliated in step 2 with the macrogene.

The gene is a large length gene or a three-dimensionally huge gene of 50 to 2500 bp or less, and is a single-stranded gene, a double-stranded gene, a plasmid gene, an artificial gene (Peptide nucleic acid), or an organic phosphor or an inorganic phosphor species attached thereto. It may include, etc., the amount of the gene is preferably used by using a weight ratio of about 0.1 to 10 times the total weight of the peeled two-dimensional nanosheets, if the amount of the gene is more than 10 times by weight ratio perfect gene There is a problem that some of the genes are exposed to the external environment because it is not encapsulated.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are merely to illustrate the present invention, but the content of the present invention is not limited by the following examples.

< Example  1> Core-cell genes with encapsulated genes of great length LDH  Nanocomposite Synthesis

Step 1: LDH Formation of

100 nm size LDH was formed through coprecipitation and hydrothermal synthesis. _{0.3 M Mg (NO 3) 2} 6H 2 O, 0.15 M Al (NO 3) 3 to 9H ₂ O carbonate ions (CO ₃ ^{2 -)??} Dissolved in the removal of deionized water, pH is to be 10 to 11 After titration with 0.5 M NaOH until, and stirred vigorously at room temperature for 30 minutes to obtain LDH crystals. The co-precipitated LDH was placed in a hydrothermal synthesis vessel, stirred at 100 ° C. for 12 hours, separated by a centrifuge, and the unreacted salt was removed by washing to obtain LDH having a uniform size of 100 nm. Entire process has carbonate ion (CO ₃ ^{2 -)} according to the carbon dioxide in the air was carried out in a nitrogen atmosphere in order to prevent the generation.

Step 2: LDH of Exfoliation

The LDH synthesized in Step 1 was dispersed in formamide at 0.05% by weight, and maintained in a nitrogen atmosphere at room temperature with vigorous stirring for 2 days to obtain exfoliated LDH nanosheets.

Step 3: Core-Cell Genes LDH  Nanocomposite Synthesis

50 to 2500 bp double-stranded herring gene (Herring testis DNA) was mixed in a weight ratio of 1: 1 to the LDH nanosheet colloid solution separated in step 2, and stirred at room temperature for 1 hour. After completion of the reaction, the centrifuge was separated and the excess gene was completely removed by washing to obtain a core-cell gene-LDH nanocomposite. Entire process has carbonate ion (CO ₃ ^{2 -)} according to the carbon dioxide in the air was carried out in a nitrogen atmosphere in order to prevent the generation.

< Example  2> Core-Cell Gene with Encapsulated Gene Encapsulated LDH  Nanocomposite Synthesis

The 35 bp double-stranded gene having the organic phosphors of cyanine 3 (green) and cyanine 5 (red) attached to the LDH nanosheet colloid solution separated in step 2 of Example 1 was mixed at a weight ratio of 1: 0.75. Stirred at room temperature for 1 hour. After completion of the reaction, the centrifuge was separated and the excess gene was completely removed by washing to obtain a core-cell gene-LDH nanocomposite. Entire process has carbonate ion (CO ₃ ^{2 -)} according to the carbon dioxide in the air was carried out in a nitrogen atmosphere in order to prevent the generation.

< Experimental Example  1> Gene LDH  X-ray of Nanocomposite diffraction  analysis

X-ray diffraction analysis was performed to confirm the crystal structure of the LDH prepared in step 1 of Example 1 and the gene-LDH nanocomposite prepared in step 3 of Example 1 and Example 2.

The results are shown in Fig.

As can be seen in the 2 (a), the interlayer spacing of the LDH prepared in Step 1 of Example 1, nitric acid ion to 0.79 ㎚ ^- was identified as typical LDH that is inserted (NO _3). As can be seen in (b) and (c) of Figure 2, the gene-LDH nanocomposites prepared in step 3 of Example 1 and Example 2 do not have diffraction at a low angle of 2θ <15 °, It can be seen that the gene-LDH nanocomposite has an amorphous structure instead of the nanocomposite of the multi-layered structure.

< Experimental Example  2> gene LDH  Transmission Electron Microscopy Analysis of Nanocomposites

Transmission electron microscopy was performed to finely check the shape and crystal structure of the LDH prepared in step 1 of Example 1 and the gene-LDH nanocomposite prepared in step 3 of Example 1 and Example 2. . Each specimen was fixed in epoxy resin, cut to 50 nm thickness and observed without specific staining process.

The results are shown in Fig.

As can be seen in Figure 3 (a), the LDH prepared in Step 1 of Example 1 is a hexagonal particles of uniform size of about 100 nm, the interlayer spacing is about 0.8 nm to the nitrate ion (NO ^₃₎ it was confirmed that the LDH is inserted. As can be seen in Figure 3 (b), the gene-LDH nanocomposite prepared in step 3 of Example 1 is a very thin LDH nanosheets disorderly encapsulated the gene of a large length of several micrometers size of the card It can be seen that a core-cell nanocomposite having an amorphous structure close to the shape of a house of card was formed. As can be seen in Figure 3 (c), the gene-LDH nanocomposite prepared in Example 2 formed an inorganic cavity about 10 nm thick as the exfoliated LDH is recombined, the drug encapsulated therein It can be seen that the gene-LDH nanocomposite has a perfect core-cell structure of 150 nm size.

< Experimental Example  3> Stability Evaluation Experiment of Encapsulated Gene

In order to determine the stability of the genes encapsulated in LDH in Examples 1 and 2 were carried out the following experiment. 10 μg of the gene-LDH nanocomposite was dispersed in 10 μl of TE (Tris-EDTA) buffer solution, 20 μl of DNAase, DNase I (1000 unit) and reaction buffer (200 mM Tris-HCl, pH 8.3, 20 μl of 20 mM MgCl ₂ ) was added and incubated at 37 ° C. for 1 hour. 20 μl of stop buffer solution (50 mM EDTA) was added, incubated at 70 ° C. for 10 minutes, and then centrifuged to remove enzymes and buffer solution. After the enzyme reaction was dispersed in hydrochloric acid buffer solution of pH 4, and dissolved for 5 hours at room temperature to extract the gene, it was electrophoresed in 1% or 5% agarose gel.

The results are shown in FIG.

As can be seen in Figure 4 (a), it was confirmed that the gene of large length of 50 to 2500 bp in the core-cell gene-LDH nanocomposite prepared in Example 1 was stabilized in the enzymatic environment. As can be seen in Figure 4 (b), it was confirmed that the three-dimensional fluorescent substance attached gene in the core-cell gene-LDH nanocomposite prepared in Example 2 was stabilized in the enzymatic environment. Accordingly, the core-cell gene-LDH nanocomposite according to the present invention can encapsulate a gene of a large size regardless of length and structure and obtain stability to the external environment, thereby increasing storage, delivery and function of the gene. It can be seen that.

1 is a schematic diagram predicting the formation structure of the gene-LDH nanocomposite according to the present invention ((a) Example 1, (b) Example 2);

2 is an X-ray diffraction diagram of the gene-LDH nanocomposite according to the present invention ((a) Step 1 of Example 1, (b) Step 3 of Example 1, (c) Example 2);

3 is a transmission electron microscopy image of the gene-LDH nanocomposite according to the present invention ((a) Step 1 of Example 1, (b) Step 3 of Example 1, (c) Example 2);

Figure 4 is an electrophoretic image showing the results of the stability evaluation of the gene encapsulated in the gene-LDH nanocomposite according to the present invention ((a) step 3 of Example 1; lane 1: 100 bp marker, lane 2: 50- 2500 bp gene comparison group, lane 3: Experimental Example 3, (b) step 3 of Example 2; lane 1: 5 bp marker, lane 2: 35 bp gene comparison group, lane 3: Experimental Example 3).

Claims (12)

delete delete delete delete delete delete Coprecipitating an aqueous solution of a divalent cation and a trivalent cation metal salt with a basic material and hydrothermally reacting to form LDH of Formula 2 (step 1); Exfoliating the LDH formed in step 1 (step 2); And The LDH exfoliated in step 2 is mixed with the macrogene or the macrogene to which the phosphor is attached, but the weight ratio of the exfoliated LDH and the gene is mixed so that the weight ratio is in the range of 1: 0.1 to 1:10, and the core-cell gene-inorganic nanohybrid Method for producing a gene-inorganic nanohybrid complex of the core-cell structure comprising the step of generating a complex: [Formula 2] [M ²⁺ _(1-x) N ³⁺ _x (OH) ₂ ] [A ^n- ] _{x / n} yH ₂ O (In Formula 2, M ²⁺ is a divalent metal cation, N ³⁺ is a trivalent metal cation, x is a number greater than 0.1 and less than 0.4, A is an anionic guest species, n is the number of charges of the anion of A, y is a positive number greater than zero). Gene-inorganic nanohybrid complex represented by the following formula (1) in which a gene is encapsulated in a core-cell structure in an inorganic nanocavity prepared by the method of claim 7: [Formula 1] [M ²⁺ _(1-x) N ³⁺ _x (OH) ₂ ] [A _GEN ^n- ] _z ㆍ yH ₂ O (In the formula 1, M ²⁺ is a divalent metal cation, N ³⁺ is a trivalent metal cation, A _GEN is a gene n is the charge number of the gene of A _GEN , x is a number greater than 0.1 and less than 0.4, z and y are positive numbers greater than zero). The gene of claim 8, wherein M is any one selected from the group consisting of magnesium (Mg ²⁺ ), nickel (Ni ²⁺ ), copper (Cu ²⁺ ), and zinc (Zn ²⁺ ). Inorganic Nanohybrid Complex. 9. The N- ^amine compound according to claim 8, wherein N is any one selected from the group consisting of aluminum (Al ³⁺ ), iron (Fe ³⁺ ), vanadium (V ³⁺ ), titanium (Ti ³⁺ ), and gallium (Ga ³⁺ ). Gene-inorganic nanohybrid complex, characterized in that the. The gene-inorganic nanohybrid complex according to claim 8, wherein the gene is 50 bp or more and 2500 bp or less. The method of claim 8, wherein the gene is any one selected from the group consisting of a single-stranded gene, a double-stranded gene, a plasmid gene, an artificial gene (Peptide nucleic acid) or a gene to which an organic phosphor or an inorganic phosphor species is attached. Characterized in the gene-inorganic nanohybrid complex.

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