KR20140094697A - Method for manufacturing of DNA hydrogel with adhesive characteristics - Google Patents

Abstract

The present invention relates to a method for manufacturing a DNA hydrogel which has adhesive characteristics using a compound which includes two or more pyrogallol functional groups as a cross-linking agent. The present invention can be used for medicine for a gene treatment and an adhesive carrier for a local injection since the present invention is able to: synthesize the DNA hydrogel in a short time by combining a compound which includes two or more pyrogallol functional groups and a DNA solution of high concentration; control the physical properties of the DNA hydrogel according to the concentration of the compound which includes two or more pyrogallol functional groups; and emit DNA according to the decomposition degree of the compound which includes two or more pyrogallol functional groups which are a cross-linking agent when being exposed to a solution of pH7.4.

Description

TECHNICAL FIELD The present invention relates to a method for manufacturing a DNA hydrogel having an adhesive property,

The present invention relates to a method for producing a DNA hydrogel having adhesiveness. More particularly, the present invention provides a method for synthesizing a DNA hydrogel having adhesiveness by using a polyphenol compound containing two or more pyrogallol functional groups as a crosslinking agent.

DNA (deoxyribonucleic acid) is an intracellular gene transfer substance composed of phosphate group, sugar, and base, and is regulated as a nano biomaterial because it can form various types of nanostructures by controlling its base sequence. In recent years, studies are underway to synthesize DNA hydrogels or DNA nanostructures containing specific nucleotide sequences and expect the effects of gene therapy.

In particular, DNA hydrogels can be synthesized in various sizes and amounts depending on the method of synthesis. The conventional methods for synthesizing DNA hydrogels are mainly based on covalent bonding between a crosslinking agent and DNA, or self-alignment by DNA base complementary bonding. Dan luo et al. (2006) formed X, Y, and T DNA structures using complementary DNA bases and synthesized DNA hydrogels by linking these structures using DNA ligase. BjLindman et al. (2012) synthesized plasmid DNA hydrogels using covalent bonds of DNA base and EGDE (ethylene glycol diglycidyl ether) as a cross-linker. In addition, DNA hydrogels were synthesized by various methods. However, the limitations of these studies are that the process is complicated because the DNA itself is changed to induce covalent bonding, and a method of inducing a chemical crosslinking reaction mediated by the base is relatively complicated, DNA can only be applied as a carrier rather than a therapeutic drug.

Accordingly, the inventors of the present invention have succeeded in synthesizing a DNA hydrogel in a short time by mixing a DNA solution of high concentration and a compound of polyphenols containing two or more pyrogallol functional groups, and a pyrogallol functional group The degree of decomposition of a compound of a polyphenol compound having two or more pyrogallol functional groups, which is a crosslinking agent, can control the physical properties of the DNA hydrogel according to the concentration of the polyphenol compound, Releasing property, and thus the present invention has been completed.

The present invention provides an adhesive DNA hydrogel comprising a polyphenol compound containing two or more deoxyribonucleic acid (DNA) solutions and pyrogallol functional groups.

The present invention provides a method for producing an adhesive DNA hydrogel comprising a polyphenol compound containing two or more deoxyribonucleic acid (DNA) solutions and pyrogallol functional groups.

The present invention provides a drug delivery system comprising an adhesive DNA hydrogel comprising a solution of a DNA (deoxyribonucleic acid) and a compound of polyphenols containing two or more pyrogallol functional groups.

The present invention provides a method for preparing a drug delivery vehicle comprising an adhesive hydrogel prepared by mixing a solution containing a target gene and a polyphenol compound having two or more pyrogallol functional groups.

The present invention provides a gene therapeutic agent comprising a drug carrier comprising an adhesive hydrogel prepared by mixing a solution containing a target gene and a labeled polyphenol compound having two or more pyrogallol functional groups .

The present invention

There is provided an adhesive DNA hydrogel comprising a polyphenol compound represented by the following formula (1) containing at least two deoxyribonucleic acid (DNA) solutions and a pyrogallol functional group:

[Chemical Formula 1]

.

.

Wherein A is a 5- to 8-membered single ring heterocycloalkyl containing oxygen, P is pyrogallol, n is an integer from 1 to 20, and m is an integer from 1 to 5.

The present invention

1) preparing DNA solution by dissolving DNA (deoxyribonucleic acid) in a solvent, and then heating; And

2) adding a compound of polyphenols represented by the following formula (1) containing two or more pyrogallol functional groups to the DNA solution heated in step 1) and stirring the mixture; and Of the present invention.

The present invention provides a drug delivery system comprising an adhesive DNA hydrogel comprising a compound of polyphenols represented by the following formula (1) containing two or more deoxyribonucleic acid (DNA) solutions and a pyrogallol functional group.

[Chemical Formula 1]

.

.

Wherein A is a 5- to 8-membered single ring heterocycloalkyl containing oxygen, P is pyrogallol, n is an integer from 1 to 20, and m is an integer from 1 to 5.

The present invention

1) heating a solution containing a target gene as a transduction material; And

2) adding a polyphenol compound represented by the following formula (1) containing at least two pyrogallol functional groups to the solution heated in step 1) and mixing to form a hydrogel; A method for producing a carrier is provided.

[Chemical Formula 1]

.

.

Wherein A is a 5- to 8-membered single ring heterocycloalkyl containing oxygen, P is pyrogallol, n is an integer from 1 to 20, and m is an integer from 1 to 5.

The present invention relates to a drug delivery system comprising an adhesive hydrogel prepared by mixing a solution containing a target gene and a polyphenol compound represented by the following formula (1) containing at least two pyrogallol functional groups Gene therapy agent.

[Chemical Formula 1]

.

.

Wherein A is a 5- to 8-membered single ring heterocycloalkyl containing oxygen, P is pyrogallol, n is an integer from 1 to 20, and m is an integer from 1 to 5.

The DNA hydrogel according to the present invention shortens the complex process of conventional DNA hydrogel synthesis and has an effect of imparting an adhesive property not possessed by a conventional DNA hydrogel. Therefore, the DNA hydrogel can be used as a medical biomaterial such as a drug delivery vehicle And can be useful as a carrier for drugs and local delivery used in gene therapy.

FIG. 1 (A) shows the dielectric properties of a compound having a molar ratio of DNA base pair to tannic acid of 2.6: 1, and (B) shows the property of a hydrogel having a molar ratio of DNA base pair to tannic acid of 1.3: 1. (C) is a graph showing the dielectric properties of a hydrogel having a molar ratio of DNA base pair to tannic acid of 0.9: 1.
FIG. 2 (A) shows the property of a hydrogel having a molar ratio of DNA base pair to tannic acid of 1.3: 1 according to the temperature, and FIG. 2 (B) shows the relationship between the DNA base pair and tannic acid molar ratio at a temperature of the hydrogel of 0.9: And the like.
Figure 3 shows FT-IR spectra of DNA and tannic acid compounds prepared by different ratios of tannic acid. (A) is tannic acid, (B) is DNA, (C), (D) And a tannic acid molar ratio of 13.1: 1, 2.6: 1, and 1.3: 1, respectively.
FIG. 4 is a graph showing the tensile properties of a hydrogel having a molar ratio of DNA base pairs to tannic acid of 1.3: 1 and 0.9: 1, respectively.
FIG. 5 is a graph showing the adhesion of a hydrogel having DNA, tannic acid of 100 w / v%, tannic acid of 150 w / v%, DNA base pair and tannic acid molar ratio of 1.3: 1 and 0.9: 1, respectively.
6 (A) is a graph showing a result of measurement of the DNA release degree of a hydrogel prepared from a DNA base pair and a tannic acid molar ratio of 1.3: 1 using 0.8% agarose gel electrophoresis. Lane 1 is the Lambda / HindIII marker, lane 2 to lane 8 are the amount of DNA release corresponding to 30 minutes, 1 hour, 3 hours, 5 hours, 8 hours, 12 hours and 24 hours, The electrophoretic band is shown by varying the concentration of DNA in the sample to 416.7 208.3, 104.2, and 52.08 / mL. 6 (B) is a graph showing the results of measurement of the tannic acid release and the degree of hydrolysis according to the time of the hydrogel prepared at a molar ratio of DNA base pair to tannic acid of 1.3: 1.

Hereinafter, the present invention will be described in detail.

The present invention provides an adhesive DNA hydrogel comprising a polyphenol compound represented by the following formula (1) containing at least two deoxyribonucleic acid (DNA) solutions and a pyrogallol functional group:

Wherein A is a 5- to 8-membered single ring heterocycloalkyl containing oxygen, P is pyrogallol, n is an integer from 1 to 20, and m is an integer from 1 to 5.

The adhesive DNA hydrogel is formed by crosslinking a polyphenol compound represented by the following formula (1) containing two or more of DNA and pyrogallol functional groups, but is not limited thereto.

The adhesive DNA hydrogel is not limited to the one obtained by crosslinking a DNA and a polyphenol compound represented by the following formula (1) containing two or more pyrogallol functional groups at a molar ratio of 0.5: 1 to 1.5: 1.

 The DNA is preferably linear, but is not limited thereto.

The compounds of polyphenols containing two or more pyrogallol functional groups include tannic acid represented by the following formula (2), ellagitannin represented by the formula (3), gallotannin represented by the formula (4) And 3,4,5-tri-O-galloylquinic acid represented by the general formula (5), but it is not limited thereto :

The adhesive DNA hydrogel is not limited to the one in which the physical properties are controlled depending on the concentration of the polyphenol compound having two or more pyrogallol functional groups.

The adhesive DNA hydrogel preferably emits DNA, but is not limited thereto.

In addition,

1) preparing DNA solution by dissolving DNA (deoxyribonucleic acid) in a solvent, and then heating; And

2) adding a compound of polyphenols represented by the following formula (1) containing two or more pyrogallol functional groups to the DNA solution heated in step 1) and stirring the mixture; and Lt; RTI ID = 0.0 > of:

[Chemical Formula 1]

.

.

Wherein A is a 5- to 8-membered single ring heterocycloalkyl containing oxygen, P is pyrogallol, n is an integer from 1 to 20, and m is an integer from 1 to 5.

Hereinafter, the above manufacturing method will be described in detail for each step.

In the production method according to the present invention, the step (1) is a step of dissolving DNA (deoxyribonucleic acid) in a solvent to prepare a DNA solution and then heating.

The heating temperature of the DNA solution is preferably 10 ° C to 60 ° C, more preferably 40 ° C to 60 ° C, and most preferably 50 ° C to 60 ° C.

In the production method according to the present invention, the step (2) is a step of adding a polyphenol compound represented by the above formula (1) containing two or more pyrogallol functional groups to a heated DNA solution and stirring .

Preferably, the adhesive DNA hydrogel is crosslinked at a molar ratio of DNA to tannic acid in a molar ratio of 0.5: 1 to 1.5: 1, and the proportion of the polyphenols represented by the above formula (1) containing two or more pyrogallol functional groups Is more preferable.

In the examples of the present invention, the storage elastic modulus (G ') and the loss elastic modulus (G ") were measured in order to analyze the characteristics of the adhesive DNA hydrogel crosslinked with tannic acid. As a result, it was found that the compound having a molar ratio of DNA base pair to tannic acid of 2.6: It was confirmed that the hydrogel was not prepared (see Fig. 1 (A)), and it was confirmed that the hydrogel was prepared in the compound having the molar ratio of DNA base pair to tannic acid of 1.3: 1, 0.9: 1 )

In an embodiment of the present invention, an adhesive DNA hydrogel crosslinked with tannic acid The storage elastic modulus (G ') and the loss elastic modulus (G ") of the adhesive DNA hydrogel were measured to analyze the physical properties according to the temperature change. As a result, the adhesive DNA hydrogel having a molar ratio of DNA base pair to tannic acid of 1.3: (See Fig. 2 (A)). It was confirmed that the adhesive DNA hydrogel having a molar ratio of DNA base pair and tannic acid of 0.9: 1 was hydrogel-formed by hydrogen bonding (Fig. 2 B).

     In the examples of the present invention, FT-IR analysis was performed with different ratios of DNA and tannic acid to confirm the interaction between DNA and tannic acid. As the ratio of tannic acid increased, (See Fig. 3).

In an embodiment of the present invention, a universal testing machine (UTM) was used to analyze the tensile properties of the adhesive DNA hydrogels crosslinked with tannic acid. As a result, the molar ratios of DNA base pairs and tannic acid were 1.3: 1 and 0.9: Gel. As the ratio of tannic acid increased and the maximum tensile strength increased, the extension length decreased, indicating that the hydrogel having a higher ratio of tannic acid had a more rigid physical property (see FIG. 4).

In the embodiment of the present invention, the adhesive property of the adhesive DNA hydrogel crosslinked with tannic acid was analyzed using a universal testing machine (UTM). As a result, it was confirmed that the higher the tannic acid ratio, the higher the adhesive strength (see FIG. 5) .

In an embodiment of the present invention, in order to confirm DNA release and tannic acid hydrolysis of a DNA hydrogel cross-linked with tannic acid, in vitro DNA release experiments have been conducted and as a result, the amount of DNA released increases with time, (See Fig. 6 (A)). After 96 hours, it was confirmed that 80% or more of the band was released or hydrolyzed (see Fig. 6 (B)).

Accordingly, the present invention relates to the synthesis of a DNA hydrogel having an adhesive property by a very simple method using a compound of polyphenols containing two or more pyrogallol functional groups, which are adhesive polyphenolic substances, as a crosslinking agent, and It is about the properties. A DNA hydrogel can be synthesized in a short time by mixing a high concentration DNA solution and a compound of a polyphenol compound having two or more pyrogallol functional groups, and a polyglycol compound having two or more pyrogallol functional groups Since the physical properties of the DNA hydrogel can be controlled according to the concentration of the compound of the phenol, hydrogels having various physical properties can be produced according to the concentration of the polyphenol compound containing two or more pyrogallol functional groups.

In addition, the interaction between tannic acid and DNA is directly related to the phosphate group in the skeleton of the DNA, and it is confirmed that the temperature at which the hydrogel is loosened coincides with the temperature at which the hydrogen bond breaks. The pyrogallol group at the terminal of tannic acid and the backbone of the DNA interact by hydrogen bonding to form a bridge between the DNA chains to form a DNA adhesive hydrogel. Therefore, it is considered that a compound capable of cross-linking the DNA chain is possible if it is a compound having at least two pyrogallols. In addition, in order to examine the release effect of DNA, a compound linked by an ester bond, which is a hydrolyzable functional group, An adhesive DNA hydrogel can be synthesized. Most of the polyphenolic compounds contain two or more strong water-soluble functional groups such as pyrogallol and are linked by an ester bond similar to the tannic acid (formula (2)) proposed in the present invention, Hydrogels with similar properties can also be formed. Consequently, compounds of polyphenols containing two or more pyrogallol functional groups can synthesize hydrogels.

The present invention also provides a drug delivery system comprising an adhesive DNA hydrogel comprising a solution of a deoxyribonucleic acid (DNA) and a polyphenol compound represented by the following formula (1) containing at least two functional groups of pyrogallol do.

[Chemical Formula 1]

.

.

Wherein A is a 5- to 8-membered single ring heterocycloalkyl containing oxygen, P is pyrogallol, n is an integer from 1 to 20, and m is an integer from 1 to 5.

The hydrogel is a material having a characteristic of being swollen in an aqueous solution, and is structurally stable because it is generally composed of a hydrophilic organic polymer material and has a cross-linked network structure through covalent bonding or noncovalent bonding. In addition, due to the ability to adsorb and discharge large amounts of solvent, the physical properties of the hydrogel are similar to those of fluid in that they can be used in a variety of fields, such as in vivo diagnostics, drug / gene delivery, , Chemical separation, chemical and biological sensors, optical materials and the like.

In addition,

1) heating a solution containing a target gene as a transduction material; And

2) adding a polyphenol compound represented by the following formula (1) containing at least two pyrogallol functional groups to the solution heated in step 1) and mixing to form a hydrogel; A method for producing a carrier is provided.

[Chemical Formula 1]

Wherein A is a 5- to 8-membered single ring heterocycloalkyl containing oxygen, P is pyrogallol, n is an integer from 1 to 20, and m is an integer from 1 to 5.

Hereinafter, the above manufacturing method will be described in detail for each step.

In the production method according to the present invention, the above step 1) is a step of preparing a solution containing the target gene and then heating.

The heating temperature of the DNA solution is preferably 10 ° C to 60 ° C, more preferably 40 ° C to 60 ° C, and most preferably 50 ° C to 60 ° C.

In the preparation method according to the present invention, the step 2) is a step of adding a polyphenol compound represented by the formula (1) containing two or more pyrogallol functional groups to a heated solution, To prepare a drug delivery vehicle.

Preferably, the adhesive hydrogel is crosslinked at a molar ratio of 0.5: 1 to 1.5: 1 with a polyphenol compound represented by the formula (1) containing two or more of the target gene and pyrogallol functional groups, and the ratio of tannic acid Is more preferable.

The drug delivery vehicle is capable of controlling the release of a target gene according to pH.

The present invention also relates to a drug delivery system comprising an adhesive hydrogel prepared by mixing a solution containing a target gene and a polyphenol compound represented by the following formula (1) containing at least two pyrogallol functional groups, To provide a gene therapy agent.

[Chemical Formula 1]

Wherein A is a 5- to 8-membered single ring heterocycloalkyl containing oxygen, P is pyrogallol, n is an integer from 1 to 20, and m is an integer from 1 to 5.

The gene therapy agent can synthesize a hydrogel in a short time by mixing a solution containing a target gene at a high concentration and a polyphenol compound having two or more pyrogallol functional groups, and the pyrogallol functional group The physical properties of the hydrogel can be controlled according to the concentration of the polyphenol compound containing two or more compounds, and when the compound is exposed to the acid 7.4 solution, the compound of the polyphenols containing two or more pyrogallol functional groups The hydrogel produced according to the present invention can be used as a drug for gene therapy and as an adhesive carrier for topical administration.

It should be noted, however, that the following examples and experimental examples are illustrative of the present invention and that the present invention is not limited to the following examples and experimental examples.

< Example > With tannic acid Bridged  Adhesiveness DNA Hydrogel  Produce

The adhesive DNA hydrogel crosslinked with polyphenol molecules according to the present invention is prepared by mixing a high concentration DNA solution with tannic acid. DNA and tannic acid have a hydrogen bond as indicated by the following formula (6).

150 mg of the DNA represented by the following formula (7) is dissolved in 3 mL of tertiary distilled water, and the solution is divided into 400 캜 and heated at 55 캜 for 10 minutes.

Tannic acid represented by the following formula 2 is prepared at a concentration of 0.5 g / mL, 1 g / mL, and 1.5 g / mL.

(2)

Tannic acid is added to the heated DNA solution at 40 ℃ each time, and the adhesive DNA hydrogel is synthesized according to the concentration of tannic acid, and it is kept at room temperature for 30 minutes to stabilize the crosslinking by tannic acid. The compound synthesized with 0.5 g / mL solution of tannic acid had a molar ratio of DNA base pair to tannic acid of 2.6: 1, and the compound synthesized with 1 g / mL of tannic acid and 1.5 g / mL of solution had a molar ratio of DNA base pair to tannic acid of 1.3: , 0.9: 1.

< Experimental Example  1> With tannic acid Bridged  Adhesiveness DNA Hydrogel  Character analysis

In order to confirm whether the compound prepared in the above Example had hydrogel properties, the adhesive DNA hydrogel crosslinked with tannic acid was analyzed using a Rheometer.

Specifically, the storage modulus (G ') and loss modulus (G ") of the Rheometer were measured at a frequency of 0.1-10 rad / sec.

Specifically, 440 μL of the adhesive DNA hydrogel prepared on the sample holder of the Rheometer was placed, and then a uniform plate of 100 Pa at a frequency of 0.1 to 10 rad / sec was applied using a parallel plate of 20 mm in thickness The degree of deformation of the material was confirmed by the storage elastic modulus (G ') and the loss elastic modulus (G ") while shear stress was applied. The storage elastic modulus (G ') and the loss elastic modulus (G &quot;) were measured by the following equation.

σ denotes the shear stress applied to the material, and γ ₀ denotes the maximum amplitude of the shear strain, G 'storage elastic modulus, and G "loss modulus, which are caused by the deformation of the material. In addition, ω denotes a frequency and t denotes time.

As a result, it was confirmed that the hydrogel was not prepared by confirming the physical properties of a compound having a molar ratio of DNA base pair to tannic acid of 2.6: 1 (FIG. 1 (A)). The loss elastic modulus (G ") was found to be larger than the storage modulus (G ') in most of the sections. The storage elastic modulus (G') increased as the frequency became closer to 10 rad / ). These physical properties are physical properties of viscoelastic materials.

In addition, the physical properties of the compound having a molar ratio of DNA base pair to tannic acid of 1.3: 1 and 0.9: 1, respectively, were confirmed. The storage elastic modulus (G ') and the loss elastic modulus (G ") were found to be higher as the storage elasticity (G') was larger than the loss elastic modulus (G" 1 (B) and (C))

< Experimental Example  2> With tannic acid Bridged  Adhesiveness DNA Hydrogel  Analysis of physical properties according to temperature change

The physical properties of the tannic acid-crosslinked DNA hydrogel prepared according to the above Example were analyzed according to the temperature change.

The storage modulus (G ') and the loss modulus (G') were measured by repeating the rheometer at 100 rad / sec at a frequency of 1 rad / sec for a total of 600 seconds, ) Were measured.

Specifically, 440 μL of the adhered DNA hydrogel prepared on the sample holder of the Rheometer was put on a 20 mm thick parallel plate at a constant force of 100 Pa at a frequency of 1 rad / sec ( Shear stress was applied and the temperature was changed to 30 degrees and 70 degrees at intervals of 100 seconds for three cycles of 600 seconds in total. The degree of deformation of the material according to the temperature change was confirmed by the storage elastic modulus (G ') and the loss elastic modulus (G ") through the same formula as in Experiment 1.

As a result, the physical properties of a hydrogel having a molar ratio of DNA base pairs to tannic acid of 1.3: 1 are shown. The storage elastic modulus (G ') is larger than the loss elastic modulus (G') and the physical properties of the hydrogel are shown at 30 ° C. However, as the temperature is increased, the loss elastic modulus (G " The physical properties of the material are lost. It was confirmed that the same phenomenon was repeated when the temperature change was repeated periodically. Thus, it was confirmed that the DNA hydrogels crosslinked with tannic acid had temperature-reversible physical properties (Fig. 2 (A)).

The physical properties of hydrogels with a molar ratio of DNA base pairs to tannic acid of 0.9: 1 are shown in the following description. It was confirmed that the DNA hydrogel crosslinked with tannic acid according to the present invention was in the form of a sol at a temperature of 54 ° C to 55 ° C or more. It was confirmed that the temperature of 54 ° C to 55 ° C or higher is generally similar to the temperature at which the hydrogen bond breaks, and the hydrogel is produced by hydrogen bonding of DNA and tannic acid (FIG. 2 (B)).

< Experimental Example  3> DNA Wow Tannic  Interaction Analysis

To analyze the interaction between DNA and tannic acid, we analyzed the ratio of DNA to tannic acid.

Specifically, a high concentration DNA solution and a tannic acid solution prepared in the above Example were mixed to prepare a solution in which the ratio of DNA base pair and tannic acid was 13.1: 1, 2.6: 1, 1.3: 1, respectively, A sample was prepared. KBr was used to fabricate a specimen disk for FT-IR analysis. Infrared light (IR) was irradiated on the specimen disk to confirm the degree of extinction in the wave number range of 4000-400 cm ^{-1. As a result} of examining the extinction wave number due to the functional groups present in DNA and tannic acid, The change of the absorption wave number in the mixture was analyzed.

As a result, the asymmetric stretching vibration of the phosphate group (PO) of the DNA appears at 1236.29 cm ^-1 , and as the ratio of tannic acid increases, it shifts to a lower frequency. In addition, the carbon-carbon vibration of the deoxyribose of DNA appears at 1095.49 cm ^-1 , and as the ratio of tannic acid increases as the ratio of tannic acid increases, It was confirmed that the shift to low frequency for vibration represents a direct interaction between DNA skeleton and tannic acid (FIG. 3).

< Experimental Example  4> With tannic acid Bridged  Adhesiveness DNA Hydrogel  Tensile characterization

A universal testing machine (UTM) was used to analyze the tensile properties of the tannic acid-crosslinked DNA hydrogel prepared in the above Example.

The tensile properties of the DNA hydrogels measured in the UTM are analyzed by the following equation (2).

The force (F, Load) applied to the hydrogel is represented by shear stress (σ) divided by the cross-sectional area (A) in which the hydrogel exists, and the extent (L) The strain is divided by 2 mm (L ₀ ) and expressed as strain (ε, strain).

Specifically, a sample for measurement was prepared by adhering a hydrogel having a thickness of 2 mm to the end of two OHP films having a width of 1 cm and a length of 5 cm. The prepared specimen was fixed on the grip of the UTM, and the load applied to the hydrogel and the length of the stretched hydrogel were measured by pulling up a 50 N load cell at a rate of 15 mm / min.

As a result, the tensile properties of hydrogels prepared from DNA base pairs and tannic acid molar ratios were 1.3: 1 and 0.9: 1, respectively. When the ratio of tannic acid was higher and the maximum tensile strength was increased, It was confirmed that the hydrogel having a higher ratio had a property of becoming more hardened (Fig. 4).

< Experimental Example  5> With tannic acid Bridged  Adhesiveness DNA Hydrogel  Adhesion Analysis

A universal testing machine (UTM) was used to confirm the adhesiveness of the tannic acid-crosslinked DNA hydrogel prepared in the above Example.

The adhesion of the hydrogels measured in the UTM was defined as the maximum load acting on the prepared sample. In Experimental Example 5, since the sample was prepared by pressing the hydrogel between the OHP films very thinly, the adhesion force between the OHP film and the hydrogel can be confirmed.

Specifically, the sample for measurement was prepared by slightly modifying the method of measuring the tensile properties. Width 1 cm, was prepared a hydrogel in chapter 2 of vertically 5 cm OHP film end, thereby extremely thin squeezed by the cross-sectional area 1 cm ^2. The prepared specimen was fixed on the grip of the UTM, and then the 500 N load cell was pulled up at a rate of 10 mm / min to measure the maximum force applied to the OHP film, that is, the force when the OHP film 2 terminal was cut off .

As a result, the adhesion of hydrogels prepared from DNA, tannic acid, DNA base pairs and tannic acid molar ratios of 1.3: 1 and 0.9: 1, respectively, was shown. Comparing the adhesion between DNA and tannic acid, the adhesion of tannic acid was 10 times higher than the adhesion of DNA. The adhesion of 100w / v% of tannic acid was 75.4kPa, while that of 150w / v% of tannic acid was 98.1kPa. In the case of the DNA hydrogel prepared using tannic acid having different concentrations, the adhesive strength was higher than that of DNA, and the higher the tannic acid ratio, the higher the adhesion (FIG. 5).

< Experimental Example  6> With tannic acid Bridged DNA Hydrogel DNA  Emissions and Tannic acid  Hydrolysis analysis

In order to confirm the degree of DNA release from the tannic acid-crosslinked DNA hydrogel prepared in the above <Example>, an in vitro DNA release experiment was conducted.

Tannic acid as shown in Formula 2 contains a large amount of ester bond in the molecule and is known to be hydrolyzed when exposed to an aqueous solution. Therefore, when the crosslinking of the prepared DNA hydrogel is broken due to the hydrolysis of tannic acid used as a crosslinking agent, the DNA release effect can be confirmed. In this experiment, an in vitro experiment was carried out using a pH 7.4 buffer having an acidity similar to that of the in vivo environment.

Specifically, 246.8 mg of a hydrogel having a molar ratio of DNA base pair and tannic acid of 1.3: 1 was used, and 5 mL of TRIS buffer having an acidity of 7.4 was used as a solvent for 30 minutes, 1 hour, 3 hours, 5 hours, 8 hours, At 12 and 24 hours, 100 μL of the sample from which the DNA was released was extracted. The degree of DNA release was confirmed by 0.8% agarose gel electrophoresis. The sample from which the DNA was released was then diluted to determine the absorbance of the peak at 275 nm in the UV / vis spectrum. To measure the concentration of released or hydrolyzed tannic acid, the absorbance was measured at 279 nm of the UV / vis spectrum for tannic acid 0.045, 0.028, 0.023, 0.014, 0.011 and 0.006 mg / mL solutions and then the resulting linear relationship Was used

As a result, it was confirmed that the amount of DNA to be released was increased with time, and the region of the band appeared in electrophoresis was widened (Fig. 6 (A)).

In addition, it was confirmed that 48.4% of the tannic acid contained in the tannic acid-crosslinked DNA hydrogel was released or hydrolyzed 24 hours after exposure to the TRIS buffer, and 80% or more was released or hydrolyzed after 96 hours ( 6 (B)).

Claims (14)

An adhesive DNA hydrogel comprising a polyphenol compound represented by the following formula (1) containing at least two deoxyribonucleic acid (DNA) solutions and a pyrogallol functional group:

[Chemical Formula 1]

.
Wherein A is a 5- to 8-membered single ring heterocycloalkyl containing oxygen, P is pyrogallol, n is an integer from 1 to 20, and m is an integer from 1 to 5.
The adhesive DNA hydrogel according to claim 1, wherein the adhesive DNA hydrogel is formed by crosslinking a compound of polyphenols containing at least two functional groups of DNA and pyrogallol.
The adhesive DNA hydrogel according to claim 1, wherein the adhesive DNA hydrogel is crosslinked at a molar ratio of 0.5: 1 to 1.5: 1 with a compound of polyphenols containing at least two functional groups of DNA and pyrogallol DNA hydrogel.
2. The adhesive DNA hydrogel according to claim 1, wherein the DNA is linear.
The polyphenol compound according to claim 1, wherein the polyphenol compound having two or more pyrogallol functional groups is selected from the group consisting of tannic acid represented by the following formula (2), ellagitannin represented by the formula (3) Gallotannin, and 3,4,5-tri-O-galloylquinic acid represented by the general formula (5). Characterized by an adhesive DNA hydrogel:

(2)

(3)

[Chemical Formula 4]

[Chemical Formula 5]

.
6. The adhesive DNA hydrogel according to claim 5, wherein the polyphenol compound having two or more pyrogallol functional groups is a tannic acid represented by the following formula (2): &lt; EMI ID =

(2)

.
2. The adhesive DNA hydrogel according to claim 1, wherein the adhesive DNA hydrogel is characterized in that its physical properties are controlled by the concentration of a polyphenol compound having two or more pyrogallol functional groups.
2. The adhesive DNA hydrogel according to claim 1, wherein the adhesive DNA hydrogel releases DNA.
1) preparing DNA solution by dissolving DNA (deoxyribonucleic acid) in a solvent, and then heating; And
2) adding a compound of polyphenols represented by the following formula (1) containing two or more pyrogallol functional groups to the DNA solution heated in step 1) and stirring the mixture; and : &Lt;

[Chemical Formula 1]

.
Wherein A is a 5- to 8-membered single ring heterocycloalkyl containing oxygen, P is pyrogallol, n is an integer from 1 to 20, and m is an integer from 1 to 5.
The method according to claim 9, wherein the temperature of the DNA solution in step 1) is 10 ° C to 60 ° C.
The adhesive DNA hydrogel according to claim 9, wherein the adhesive DNA hydrogel is crosslinked at a molar ratio of 0.5: 1 to 1.5: 1 with a compound of polyphenols containing at least two functional groups of DNA and pyrogallol A method for producing a DNA hydrogel.
A drug delivery system comprising the adhesive DNA hydrogel of claim 1.
1) heating a solution containing a target gene as a transduction material; And
2) adding a polyphenol compound represented by the following formula (1) containing at least two pyrogallol functional groups to the solution heated in step 1) and mixing to form a hydrogel; Method of preparing the carrier:

[Chemical Formula 1]

.
Wherein A is a 5- to 8-membered single ring heterocycloalkyl containing oxygen, P is pyrogallol, n is an integer from 1 to 20, and m is an integer from 1 to 5.
14. A gene therapy agent comprising the drug delivery system of claim 13.

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