JP2007097884A - Formation method of dna/chitosan complex - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an easy formation method of a DNA/chitosan complex capable of using DNA for various types of applications while stably retaining the characteristics of DNA. <P>SOLUTION: Buffer solution is made to act on DNA/chitosan complex to provide it with formability and form the DNA/chitosan complex into an intended shape. It is preferable in this method that the DNA/chitosan complex is acquired as sediment by reacting DNA with chitosan in aqueous medium. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a method for forming a DNA / chitosan complex.

  In recent years, DNA has attracted attention as a novel functional material. This is due to the fact that DNA has a regular double helix (double stranded) structure that is structurally stable. DNA having a double helix structure is a naturally-derived material, and is easy to conduct electricity, and has a property of incorporating various compounds such as dyes into the double helix structure. For these reasons, DNA is expected to be used in various applications such as medical materials, conductive materials, recording elements (CD-R, DVD, etc.), organic EL elements, and the like.

  On the other hand, DNA that has been separated and purified from raw materials such as salmon shiroko without destroying the double helix structure is water-soluble and has a major problem that it lacks stability when applied to the above-mentioned uses in its original form. Have. For this reason, stabilization by conjugation of DNA as a polyanion with a synthetic lipid or the like which is a polycation that electrostatically binds to the polyanion has been attempted (Japanese Patent Laid-Open No. 2001-327591).

As one of the methods for stabilizing such DNA, JP-A-10-77235 discloses a method for stabilizing DNA as a complex with chitosan. Japanese Patent Application Laid-Open No. 2001-199903 describes a complex of a nucleic acid and chitosan which is considered suitable for the treatment in the gene field. Japanese Patent Application Laid-Open No. 2001-500109 discloses a composition comprising a granular complex of chitosan and nucleic acid as a composition suitable for gene delivery to epithelial cells.
JP 2001-327591 A JP-A-10-77235 JP 2001-199903 A JP 2001-500109 A

  By using DNA as a chitosan complex, there is an advantage that it can be used for the intended purpose with good stability without impairing the function of the DNA. Furthermore, since both DNA and chitosan are biological materials, DNA / chitosan complexes are considered to have less safety and metabolic risks than DNA / synthetic lipid complexes, and chitosan is more abundant than synthetic lipids. Since it can be procured at a low cost, it is considered commercially advantageous. However, unlike the DNA / synthetic lipid complex, the DNA / chitosan complex is insoluble in an organic solvent and thus has a drawback that it is extremely difficult to mold.

  An object of the present invention is to form a DNA / chitosan complex that maintains the double helix structure of DNA and can stably obtain its properties into a shape suitable for a desired purpose such as a medical or dental material. An object of the present invention is to provide a molding method that enables easy molding.

  As a result of various studies on the production method and characteristics of the DNA / chitosan complex, the present inventors have found that the precipitate obtained by reacting DNA and chitosan with deionized water is washed with a buffer and then frozen. By drying, a DNA / chitosan complex applicable as a medical or dental material can be obtained, and furthermore, the production conditions such as the mixing ratio of DNA and chitosan, the type and concentration of the washing buffer solution can be appropriately changed. Thus, it was found that the DNA content, properties, porosity, etc. in the DNA / chitosan complex can be adjusted. Furthermore, a novel finding was obtained that a moldable state was obtained by bringing the DNA / chitosan complex into contact with a buffer solution. The present invention has been made based on the novel findings of the present inventors.

The first aspect of the method for molding a DNA / chitosan complex of the present invention is:
Filling the DNA / chitosan complex into a mold for molding;
Supplying a buffer to the DNA / chitosan complex filled in the mold;
And lyophilizing the DNA / chitosan complex containing the buffer solution in the mold in a state of filling the mold to obtain a DNA / chitosan complex molded product molded into the shape of the mold. This is a method for forming a DNA / chitosan complex.

The second embodiment of the method for forming a DNA / chitosan complex of the present invention comprises:
Administering the DNA / chitosan complex in a buffer to obtain a mass of the DNA / chitosan complex;
Filling the DNA / chitosan complex mass into a mold for molding;
And a step of freeze-drying the DNA / chitosan complex in the mold in a state filled in the mold to obtain a DNA / chitosan complex molded product molded into the shape of the mold. This is a method for forming a chitosan composite.

The third aspect of the method for molding the DNA / chitosan complex of the present invention is:
In a method for forming a DNA / chitosan complex,
Administering an aqueous slurry of DNA / chitosan complex into a buffer solution to obtain a mass of a shape according to the charged state;
And a step of freeze-drying the mass of the DNA / chitosan complex to obtain a DNA / chitosan complex molded product having a shape corresponding to the charged state. .

  According to the present invention, there is provided a method capable of stably obtaining DNA characteristics and easily forming a DNA / chitosan complex useful for various applications into a shape according to a desired purpose. Can do.

  The method for molding a DNA / chitosan complex of the present invention is characterized in that moldability is imparted by allowing a molding buffer to act on the DNA / chitosan complex. Hereinafter, each aspect of the present invention will be described.

The first aspect of the method for molding a DNA / chitosan complex of the present invention is:
Filling the DNA / chitosan complex into a mold for molding;
Supplying a buffer to the DNA / chitosan complex filled in the mold;
And a step of freeze-drying the DNA / chitosan complex containing the buffer solution in the mold while filling the mold to obtain a molded DNA / chitosan complex molded into the shape of the mold.

  In this method, first, the DNA / chitosan complex is formed into a lump of an appropriate size, washed with a buffer as necessary, and filled into a mold. The mold used here has a structure that enables the DNA / chitosan complex to be formed into a desired shape. Furthermore, this mold preferably has a portion that can be opened and closed so as to enable freeze-drying described later. The size and amount of the DNA / chitosan complex mass to be filled in the mold can be appropriately selected according to the density and porosity desired in the molded product obtained after molding. When a large lump of DNA / chitosan complex is used, it may be pulverized into powder and filled in a mold. Before filling the mold, the DNA / chitosan complex may be washed with a buffer. In this case, the same washing buffer as that for molding can be preferably used. When the DNA / chitosan complex is filled in the mold, a molding buffer is supplied thereto. As a supply method, a method in which a part of the mold is opened and immersed in a buffer solution for molding while maintaining the filled state of the DNA / chitosan complex in the mold is preferable. A method of injecting a molding buffer into the mold can also be used.

  After the molding buffer is supplied to the DNA / chitosan complex in the mold, the moldability may be improved by pressing the DNA / chitosan complex in the mold as necessary. Moreover, you may remove the excess liquid in a type | mold as needed. In this manner, the DNA / chitosan complex in the mold supplied with the molding buffer is subjected to lyophilization while maintaining the filling state in the mold. When the predetermined dry state is obtained, the molded product is taken out from the mold. The molded product thus obtained has a shape given according to the structure of the mold.

The second embodiment of the method for forming a DNA / chitosan complex of the present invention comprises:
Administering the DNA / chitosan complex in a buffer to obtain a mass of the DNA / chitosan complex;
Filling the DNA / chitosan complex mass into a mold for molding;
A step of freeze-drying the DNA / chitosan complex in the mold filled in the mold to obtain a molded DNA / chitosan complex molded into the shape of the mold.

  In this method, first, the DNA / chitosan complex is put into a molding buffer solution to form a mass that can be molded in the buffer solution. For the introduction of the DNA / chitosan complex into the molding buffer, a method of preparing an aqueous slurry of the DNA / chitosan complex and introducing it into the molding buffer is suitable. For example, an aqueous slurry of DNA / chitosan complex is extruded into a molding buffer solution using an appropriate supply means such as a syringe to obtain a lump of an appropriate size and shape. The aqueous slurry can be prepared by preparing the DNA / chitosan complex as a powder having an appropriate particle size and mixing it with water. When an aqueous slurry of DNA / chitosan complex is administered into a molding buffer, a moldable mass is formed from the slurry. Next, this lump is filled into a mold for molding. In this state, if necessary, the DNA / chitosan complex in the mold may be pressurized to improve moldability, and if necessary, excess liquid in the mold may be removed.

  The DNA / chitosan complex thus filled in the mold is freeze-dried while maintaining the filled state in the mold. When the predetermined dry state is obtained, the molded product is taken out from the mold. The molded product thus obtained has a shape given according to the structure of the mold. Note that the number of lumps filled in the mold may be one, or two or more.

The third aspect of the method for molding the DNA / chitosan complex of the present invention is:
In a method for forming a DNA / chitosan complex,
Administering an aqueous slurry of DNA / chitosan complex into a buffer solution to obtain a mass of a shape according to the charged state;
A step of freeze-drying the DNA / chitosan complex mass to obtain a DNA / chitosan complex molded product having a shape corresponding to the charged state.

  In this method, an aqueous slurry of DNA / chitosan complex is administered into a molding buffer in a desired shape to obtain a mass having that shape. A molded product having a desired shape can be obtained by freeze-drying the mass while maintaining its shape. For example, a droplet-like lump can be obtained by filling a syringe with an aqueous slurry of DNA / chitosan complex and administering it as a droplet into a molding buffer. Alternatively, a thread-like or belt-like lump can be obtained in the molding buffer by appropriately selecting the shape of the syringe outlet and extruding the aqueous slurry. It is also possible to flatten the head of the droplet-shaped mass obtained in the buffer solution for molding using a spatula or the like to form a disk shape. Furthermore, a lump corresponding to the shape of the mold can be obtained by preparing the mold in the molding buffer.

  The DNA / chitosan complex used in the molding method of the present invention may be any as long as moldability can be imparted by the molding buffer. Among these, those obtained by washing a precipitate obtained by reacting DNA and chitosan in an aqueous medium for producing a complex with a buffer solution are preferable. In this DNA / chitosan complex, the content ratio, properties, porosity, etc. of DNA in the DNA / chitosan complex can be changed by appropriately changing the production conditions such as the mixing ratio of DNA and chitosan and the type and concentration of the washing buffer. Can be adjusted.

  As the DNA forming the DNA / chitosan complex, natural DNA and synthetic DNA can be used, and can be selected according to the intended use of the molded product such as medical use or dental use. Examples of natural DNA include bacterial virus λ phage DNA, Escherichia coli chromosomal DNA, calf thymus DNA, and salmon sperm DNA. Synthetic DNA can be synthesized by a synthesizer using poly (dA), poly (dT), poly (dG), poly (dC), poly (dA-dT), poly (dG-dC), etc. Synthesizing using various synthetic DNAs with different base sequences: poly (A), poly (T), poly (G), poly (U), poly (A-T), poly (G-U), etc. Possible various synthetic RNAs with different base sequences: DNA / RNA hybrids such as poly (dG), poly (U), poly (G), poly (dC) poly (dA-dT), poly (A-T), etc. And a DNA / RNA hybrid having a complementary base pair that can be synthesized by a synthesizer. These can be used alone or in combination as required.

  Such DNA has various groups (for example, phosphate-containing groups) on four types of bases [cytosine (C), guanine (G), adenine (A), thymine (T)] forming a double helix. The entire DNA has an anionic property due to, for example, the phosphate group bonded to the terminal.

  Such DNA itself is a string having a double helix structure, and since this DNA dissolves in water, this DNA itself has no moldability. A DNA / chitosan complex can be obtained by electrostatically reacting anionic DNA with cationic chitosan by utilizing the fact that DNA has anionic property. By forming a complex in this manner, DNA is substantially not dissolved in water. Moreover, the solubility with respect to an organic solvent also becomes low.

  Any chitosan complexed with DNA may be used as long as it can form a complex that can be molded according to the method of the present invention. Of these, chitosan having a molecular weight of 320 (number of glucosamine residues 2) or more is preferable when it is used as a medical material or a dental material.

  Chitosan can be prepared using crab and shrimp shells, insects, plants, microorganisms and the like as raw materials. Chitosan having a molecular weight of 320 (glucosamine residue number 2) or more, preferably a molecular weight of 1600 (glucosamine residue number 10) or more can be used. This is preferably chitosan having a molecular weight of 10,000 or more in order to express insolubility of DNA in water. In addition, when chitosan having a small molecular weight and forming a salt with hydrochloric acid or the like is used, the reaction product with DNA is water-soluble, and it is difficult to separate this reaction product from the aqueous medium that is the solvent for DNA. Therefore, it is not suitable for the purpose of forming a complex.

  The reaction between DNA and chitosan can be carried out in an aqueous medium. For example, these can be reacted by adding and mixing an aqueous solution of DNA to a stirring chitosan aqueous solution. The aqueous solution of chitosan may be adjusted in pH using acid or alkali in order to ensure its dissolved state. For example, an aqueous solution under acidic conditions such as pH 5 can be suitably used.

  The compounding ratio of DNA and chitosan can be selected according to the use of the DNA / chitosan composite molded product. For example, it can be selected from the range of 1/9 to 9/1, preferably 1/1 to 1 / 1.5 (molar ratio).

  Reaction of DNA with chitosan in an aqueous medium results in precipitation of the DNA / chitosan complex. This precipitate is washed with water or various buffers, and if necessary, further washed with water, excess water is removed by centrifugation, etc., and dried to obtain a dried DNA / chitosan complex. Can do.

  Buffers for washing precipitates include Tris buffer, borate buffer, HEPES buffer, acetate buffer, citrate buffer, glycine buffer, barbiturate buffer, phthalate buffer, cacodylate buffer Solution, carbonate buffer solution, Bis-Tris buffer solution, Bis-Tris-propane buffer solution, MES buffer solution, ADA buffer solution, PIPES buffer solution, ACES buffer solution, collamine chloride buffer solution, BES buffer solution, MOPS buffer solution, It can be selected from TES buffer, HEPPS buffer, Tricine buffer, glycinamide buffer, Bicine buffer, TAPS buffer, CHES buffer, CAPS buffer, phosphate buffer and the like. The concentration and pH of the buffer solution can be freely selected so that the desired properties and physical properties of the DNA / chitosan complex can be obtained.

  The DNA / chitosan complex dried product thus obtained can be pulverized if necessary and used in the molding method according to the present invention. In this DNA / chitosan complex, the double helix structure of DNA is almost completely left without being destroyed. Therefore, by using this DNA / chitosan complex, the function of DNA can be used stably and efficiently. Is possible.

  On the other hand, as a molding buffer used in the molding method of the present invention, a phosphate buffer, a HEPES buffer, or the like can be used. The concentration of the buffer may be a concentration that does not impair the function of the DNA, and the pH can be preferably selected from the range of pH 7 to 8.

  The DNA / chitosan composite molded product molded by the molding method of the present invention described above can be used by adhering a medical or dental pharmacologically active component or the like depending on its use.

  Hereinafter, the present invention will be described in more detail with reference to examples.

Example 1 (Production of DNA / chitosan complex; Part 1)
(A) Solution / DNA solution used: Salmon sperm-derived DNA (300 base pairs, manufactured by Nichiro) is dissolved in distilled water. (5mg / 1ml)
Chitosan solution: Chitosan (molecular weight 130,000, manufactured by Nichiro) was dissolved in 50 ml of 0.2N HCl. To this solution, 0.2N NaOH and distilled water were gradually added to make 100 ml of pH 5 solution. (5mg / 1ml)
(B) Synthesis from chitosan aqueous solution and DNA aqueous solution The DNA aqueous solution was added to 100 ml of the stirring chitosan aqueous solution and allowed to react for 1 hour. The precipitate formed after the reaction was centrifuged. The resulting precipitate was washed with 60 ml of Tris-HCl buffer (10 mM, pH 7.2) (hereinafter referred to as Tris-HCl buffer) and centrifuged again. Washing with Tris-HCl buffer was performed twice. Next, after washing with 60 ml of distilled water, centrifugation was performed. Washing with distilled water was performed 3 times. The resulting white precipitate was lyophilized. The yield was 600 mg, and the molecular weight ratio (molar ratio) of the DNA / chitosan complex (sample A) was 0.95 / 1 from the quantification of phosphorus.

  When the concentration of the reaction product is changed in the above production method, that is, an aqueous DNA solution (7.5 mg / 1 ml) and an aqueous chitosan solution (2.5 mg / 1 ml) or an aqueous DNA solution (2.5 mg / 1 ml) and an aqueous chitosan solution ( 7.5 mg / 1 ml), the molecular weight ratio (molar ratio) of the DNA / chitosan complex was 1.28 / 1 and 0.88 / 1, and the yield was 700 mg and 300 mg.

  When the kind of the buffer is changed in the above production method, the porosity and the pore diameter in the DNA / chitosan complex are different.

  For example, phosphate buffer (10 mM, pH 7.2, 0.9% NaCl) (hereinafter referred to as PBS buffer), HEPES buffer (10 mM, pH 7.2), Tris-HCl buffer or borate buffer ( The DNA / chitosan complex washed with 10 mM, pH 7.2) has a large number of pores and a large morphology.

  If the molecular weight ratio (molar ratio) of DNA / chitosan is different in the above production method, the porosity and the pore diameter in the DNA / chitosan reaction product will be different even when treated with the same buffer. For example, the porosity of a DNA / chitosan complex washed with a Tris-HCl buffer is lower when the DNA content is lower.

Example 2 (Preparation of DNA / chitosan complex disk)
The DNA / chitosan complex (sample A) obtained in Example 1 was pulverized with a pulverizer or the like, washed with a phosphate buffer similar to that used in Example 1, and then the central part as shown in FIG. It was put in a hole of a mold (outer diameter 20, inner diameter 8, thickness 2 mm) made of silicone packing which was a hole cut into a circle. Note that the bottom of the mold is fixed to a stainless steel plate (20 × 20 × 1 mm) with an adhesive. The DNA / chitosan complex was flattened while sucking moisture of the DNA / chitosan complex with a filter paper or the like, then pressed several times with a nylon mesh (PE24, 526 μm), and then covered with the same mesh. This was immersed in the same phosphate buffer for 10 minutes, then the mesh was removed, and the water of the DNA / chitosan complex was removed with filter paper or the like. Then, it was covered with a polyethylene film and a stainless steel plate and immersed in liquid nitrogen. After pulling up and removing the polyethylene film and the stainless steel plate, it was covered with the aforementioned nylon mesh and immersed in liquid nitrogen again. The DNA / chitosan complex in the disk-shaped mold obtained by pulling up and removing the nylon mesh is freeze-dried, and the disk-shaped DNA / chitosan complex having properties suitable for dentistry and the like shown in the photograph of FIG. Got.

Example 3 (Method for producing DNA / chitosan complex formed into a string, ball, or freely molded)
Water was added to the DNA / chitosan complex (sample A) obtained in Example 1 to form a creamy slurry, which was placed in a syringe. When the slurry was injected from the needle at the tip of the syringe into the same phosphate buffer as used in Example 2, a DNA / chitosan complex whose shape was maintained immediately after injection was obtained. In this case, when the needle was continuously injected into the phosphate buffer from the tip of the needle, it could be formed into a thread shape, and when it was dropped into the phosphate buffer as a tear drop, it could be in the form of a ball. Furthermore, when the above-mentioned thread-like composite was put into a mold in a manner of laminating several pieces, and excess water was absorbed with a filter paper and freeze-dried, it could be freely formed into a form corresponding to the mold.

Example 4 (Production of DNA / chitosan complex; Part 2)
After dissolving 1 g of low molecular weight double-stranded DNA (LdsDNA) having a main peak at around 300 bp with deionized water, the final volume was 200 ml (5 mg / ml). On the other hand, chitosan (molecular weight 130,000, manufactured by Nichiro) was measured so that the molar ratio was 1.5 times that of deoxynucleotide (dNMP), dissolved in 0.2N HCl, and then 0.2 NNaOH was used. Finally, the solution was adjusted to pH 5.0 and 5 mg / ml. Thereafter, the LdsDNA solution and the chitosan solution were mixed and stirred for 1 hour at room temperature, and then the precipitate was collected by centrifugation (10000 rpm, 5 minutes). 100 ml of deionized water was added to the precipitate and centrifuged (8000 rpm, 5 minutes). This washing step was performed twice. The resulting precipitate was lyophilized and recovered as a DNA / chitosan complex.

Example 5 (DNA / chitosan complex pellet molding method)
To 200 mg of the DNA / chitosan complex recovered by the method of Example 4, 1 ml of deionized water was added and ground with a mortar to obtain a creamy slurry. This slurry is added to 50 ml of 10 mM phosphate buffer (10 mM Na ₂ HPO ₄ · 12H ₂ O, 10 mM KH ₂ PO ₄ ; pH 7.4, containing 100 mM NaCl and 3 mM KCl) and left to stand for 10 minutes. Lumps were obtained in the buffer. The obtained lump was filled in a circular frame having a bottom surface, molded while removing excess moisture with Kimwipe, and freeze-dried in a state filled in the frame to obtain pellets.

  Furthermore, when the concentration of the phosphate buffer was increased from 10 mM to 100 mM, the pellet strength improved.

Example 6
The slurry obtained in Example 5 was filled in a syringe and extruded into a similar phosphate buffer as used in Example 5 in a thread form. The obtained thread-like lump was freeze-dried while maintaining its shape to obtain a thread-like molded product.

It is a figure of the type | mold which shape | molds the pellet of a DNA / chitosan complex. It is a figure which shows the pellet of the shape | molded DNA / chitosan complex.

Explanation of symbols

1 Stainless steel plate 2 Silicone mold 3 Nylon mesh 4 DNA / chitosan complex filled in mold

Claims (6)

In a method for forming a DNA / chitosan complex,
Filling the DNA / chitosan complex into a mold for molding;
Supplying a buffer to the DNA / chitosan complex filled in the mold;
And lyophilizing the DNA / chitosan complex containing the buffer solution in the mold in a state of filling the mold to obtain a DNA / chitosan complex molded product molded into the shape of the mold. A method for forming a DNA / chitosan complex.   The molding method according to claim 1, wherein the DNA / chitosan complex is obtained as a precipitate by reacting DNA and chitosan in an aqueous medium. In a method for forming a DNA / chitosan complex,
Administering the DNA / chitosan complex in a buffer to obtain a mass of the DNA / chitosan complex;
Filling the DNA / chitosan complex mass into a mold for molding;
And a step of freeze-drying the DNA / chitosan complex in the mold in a state filled in the mold to obtain a DNA / chitosan complex molded product molded into the shape of the mold. A method for forming a chitosan composite.   4. The molding method according to claim 3, wherein the DNA / chitosan complex is obtained as a precipitate by reacting DNA and chitosan in an aqueous medium. In a method for forming a DNA / chitosan complex,
Administering an aqueous slurry of DNA / chitosan complex into a buffer solution to obtain a mass of a shape according to the charged state;
And a step of freeze-drying the mass of the DNA / chitosan complex to obtain a DNA / chitosan complex molded product having a shape corresponding to the charged state.   The molding method according to claim 5, wherein the DNA / chitosan complex is obtained as a precipitate by reacting DNA and chitosan in an aqueous medium.

Images