JP2006028041A - Nucleic acid-containing nano particle - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an external preparation and an injection each containing a highly absorbable nucleic acid and having an effect enabling percutaneous or mucosal nucleic acid absorption hitherto sufficiently not been achieved, without a viral carrier. <P>SOLUTION: The subject nano particle is produced by non-covalently covering the periphery of a complex of a nucleic acid with a monovalent to trivalent basic salt and a divalent or trivalent metal salt or a cationic polymer, with an ionic functional group-having hydrophilic polymer, and a method for producing the same is also provided. A skin or mucosa application type external agent and an injection each containing the nano particles. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a nanoparticle containing a nucleic acid, and more particularly to a nanoparticle containing a nucleic acid and a method for producing the same, a skin and mucosa application comprising the nanoparticle, and a parenteral preparation for injection.

  It is believed that so-called gene therapy becomes possible by administering a nucleic acid in vivo. They are made possible by incorporating oligonucleic acid molecules such as foreign genes and RNA into the cell.

  By the way, the epidermis cells of the skin and mucous membrane are used as a transdermal administration site for systemic diseases such as hereditary skin diseases, immune diseases, cancer, atopic dermatitis, etc. This is a useful target site for administration of nucleic acids. In addition, the epidermis is an organ that secretes various growth factors and cytokines and affects the local and whole body. Thus, it is considered possible to supply the target protein throughout the body via epidermal cells.

  Until now, carriers for administering many nucleic acid molecules have been designed and developed. Virus-based carriers are another strong candidate, but there are still problems with their safety. There are also methods of introduction by electroporation or gene gun, but they require special equipment and lack general versatility. As non-viral carriers, liposomes, micelles, dendrimers, polymers, fine particles and the like have been developed.

  However, these carriers functioned as good carriers in an in vitro system and do not always function in the same manner in vivo. Therefore, it is necessary to design the optimum carrier for the target tissues / cells and administration routes in consideration of them.

  The skin is covered with a stratum corneum and controls the permeability of foreign molecules. The permeability of DNA to the skin is low, and even if it permeates, it is difficult to be taken into cells in the form as it is. Therefore, it is required that some carrier is used to be taken up by epidermal cells. However, in vitro, even if it is a complex that can be easily taken into cells, its function cannot be fully exhibited unless it penetrates the stratum corneum.

  Until now, attempts have been made to introduce nucleic acid molecules by the calcium phosphate method or liposomes (Non-Patent Document 1), but the introduction efficiency has not been sufficient. One reason is the physical properties of the complex of DNA and carrier. In order to improve the permeability to the skin, it is preferable to reduce the particle size of the carrier. From some studies so far, it is said that the particle size required for improving the permeability is 100 nm or less. However, in the case of a complex of calcium phosphate containing nucleic acid or a complex of nucleic acid and a cationic polymer or cationic lipid, precipitates or particles of several hundred nm are formed.

  Furthermore, a cationic polymer (Patent Document 1) in which a hydrophobic substituent is introduced by a covalent bond, a cationic block polymer (Patent Document 2) in which a hydrophilic nonionic polymer is introduced by a covalent bond, a hydrophilic ionic polymer A technique has been developed in which a complex with a nucleic acid is formed by a graft polymer introduced by covalent bonding (Non-patent Document 2) and transferred into cells. With these technologies, it was possible to form a DNA complex with a small particle size, but by covering the surface with a hydrophilic chain, the affinity for the cell was weakened, and the nucleic acid molecule was taken into the cell. Has been suppressed (Non-patent Document 3). In addition, there is a technique for producing nanocapsules by dispersing a nonionic surfactant (Patent Document 3) and a report of forming a complex of a nucleic acid and a cationic polymer with a polymer having a multivalent charge ( Patent Document 4 and Patent Document 5).

  In the present invention, a hydrophilic polymer having an ionic functional group is used, and a nucleic acid / inorganic compound complex or a nucleic acid / cationic polymer complex is made into nanoparticles, thereby enhancing the skin / mucosal absorbability of the nucleic acid, and the epidermis. We have developed a non-viral carrier that can function in nucleic acids in cells.

Patent Publication No. 2004-510829 Patent publication 2003-528613 Patent publication 2003-524654 Patent publication 2003-503362 Patent Publication 2001-511171 Human Genetics (4) p.2279-85 (1995) S.T.P.Pharmaco Sciences (11) p.97-102 (2001) Gene Ther. (5) p.1425-33 (1998)

Therefore, an object of the present invention is to provide a technique for improving the permeability to skin and mucous membranes and transporting nucleic acids more efficiently into epidermal cells without using a virus.
In order to solve such problems, the present inventors have developed a DNA complex having a small particle size by a new method, and found that the nanoparticles can penetrate into the skin and mucous membranes and efficiently transport nucleic acids into epidermal cells. The present invention has been completed.

  Therefore, the present invention provides nanoparticles that enable excellent nucleic acid uptake by skin / mucosal administration. The present invention also provides nanoparticles that can be used as an injection.

More specifically, the present invention provides:
(1) Nucleic acid-containing nanoparticles comprising reacting a nucleic acid, a monovalent to trivalent basic salt, a divalent or trivalent metal salt, and a hydrophilic polymer having an ionic functional group,
(2) a nucleic acid-containing nanoparticle comprising a nucleic acid, a cationic polymer and a hydrophilic polymer having an ionic functional group,
(3) The monovalent or trivalent basic salt is selected from carbonate, hydrogen carbonate, phosphate, hydrogen phosphate, oxalate, lactate, and urate (1) The nucleic acid-containing nanoparticles described,
(4) The nucleic acid-containing nanoparticles according to (1), wherein the divalent or trivalent metal salt is a calcium salt, a zinc salt, an iron salt or a copper salt,
(5) The cationic polymer is selected from polylysine, polyarginine, polyhistidine, a polypeptide containing a cationic amino acid, polyethyleneimine, chitosan, a cationic dendrimer, and a DNA binding protein. Nucleic acid-containing nanoparticles of
(6) In (1) or (2), the hydrophilic polymer having an ionic functional group is selected from polyethylene glycol, lipid-polyethylene glycol, polyamino acid, polypeptide, protein, oligosaccharide and polysaccharide. The nucleic acid-containing nanoparticles described,
(7) The nucleic acid-containing nanoparticle according to (6), wherein the ionic functional group of the hydrophilic polymer having an ionic functional group is a phosphate group, a sulfate group, or a carboxyl group,
(8) The nucleic acid-containing nanoparticle according to any one of (1) to (7), wherein the particle diameter is 10 to 500 nm,
(9) Nucleic acid characterized in that a nucleic acid, a hydrophilic polymer having an ionic functional group, a divalent or trivalent metal salt and a monovalent to trivalent basic salt are allowed to act in water or a buffer solution. A method for producing the contained nanoparticles,
(10) A method for producing a nucleic acid-containing nanoparticle characterized by allowing a hydrophilic polymer having a nucleic acid, a cationic polymer and an ionic functional group to act in water or a buffer solution,
(11) The nucleic acid-containing nanoparticle according to any one of (1) to (10), wherein the nucleic acid is selected from DNA, RNA, phosphodiester derivatives thereof, 2′-O methyl RNA, peptide nucleic acids, and chimeras thereof. particle,
(12) The nucleic acid has a foreign gene expression function by plasmid DNA or an endogenous gene expression suppression or enhancement function by antigene, antisense, ribozyme, decoy or RNA interference, etc. Nucleic acid-containing nanoparticles of
(13) An external preparation for skin or mucous membrane containing the nucleic acid-containing nanoparticles according to any one of (1) to (12),
(14) The external preparation is selected from ointments, gels, sublingual tablets, oral tablets, liquids, sprays for oral and lower respiratory tracts, suction agents, suspension agents, poultices, and patches. Topical preparation,
(15) An injectable preparation containing the nucleic acid-containing nanoparticles according to any one of (1) to (12),
Is to provide.

  One aspect of the nucleic acid nanoparticles provided by the present invention is to allow a monovalent to trivalent basic salt and a divalent or trivalent metal salt to act in the presence of a hydrophilic polymer having an ionic functional group. It consists of the composite_body | complex which incorporated the nucleic acid in the conjugate | bonded_body of the basic salt and metal salt which were formed by. As the conjugate of the basic salt and the metal salt, zinc carbonate and calcium phosphate are preferable. In this case, it was found that nanoparticles having a hydrophilic polymer chain having an ionic functional group on the surface of the composite were formed.

  Another embodiment of the nucleic acid nanoparticles provided by the present invention comprises a complex of a cationic polymer and a nucleic acid, and the periphery of the complex is formed by a non-covalent bond with a hydrophilic polymer having an ionic functional group. It is coated. In this case, the cationic polymer and the nucleic acid interact with each other, and at the same time, the hydrophilic polymer having an ionic functional group is mixed to suppress the aggregation of the complex, and the hydrophilic polymer chain is formed on the surface. It was found that the arranged nanoparticles were formed.

  Furthermore, the present invention provides a skin or mucosa-applied external preparation and injection containing the nucleic acid nanoparticles described above.

The nanoparticle provided by the present invention has a small particle size by disposing a hydrophilic polymer having an ionic functional group on its surface, and the nucleic acid-containing nanoparticle that easily passes through the stratum corneum due to the small particle size. Particles.
Further, in the nanoparticles provided by the present invention, the nucleic acid carrier complex and the hydrophilic polymer having an ionic functional group interact with each other by non-covalent bonds, so that after passing through the stratum corneum, the ionic functional group can be easily obtained. The hydrophilic polymer having a group is released from the complex, and the nucleic acid is easily taken up by the cell. Further, the hydrophilic polymer having a free ionic functional group has an effect of increasing the permeability to the epidermis and acts as an absorption promoter for particles.
Therefore, by using the nanoparticles provided by the present invention, it becomes possible to easily transport nucleic acids into cells in skin and mucous membranes, supply specific proteins to the whole body by gene expression, External preparations and injections that enable control or treatment of skin diseases are provided.

  In the present invention, as described above, a nanoparticle containing a nucleic acid is converted into a nucleic acid, a hydrophilic polymer having an ionic functional group, a divalent or trivalent metal salt, a monovalent to trivalent basic salt, Alternatively, a nucleic acid-containing nanoparticle comprising acting in a buffer solution and a method for producing the same, and a nucleic acid, a hydrophilic polymer having an ionic functional group, and a cationic polymer are allowed to act in water or a buffer solution. A nucleic acid-containing nanoparticle and a method for producing the same, and a skin or mucosa-applied external preparation and injection containing the nanoparticle.

  The nanoparticles of the present invention have a diameter of 10 to 500 nm, preferably 10 to 200 nm, and more preferably 10 to 100 nm. The particle size can be adjusted by the blending ratio of the hydrophilic polymer having an ionic functional group to be used and a divalent or trivalent metal salt, monovalent to trivalent basic salt, or the concentration thereof. . Furthermore, it can be adjusted by the blending ratio of the nucleic acid, the hydrophilic polymer having an ionic functional group, or the cationic polymer, or the concentration thereof.

  Examples of the nucleic acid contained in the nanoparticles provided by the present invention include DNA, RNA, phosphodiester derivatives thereof, 2′-O methyl RNA, peptide nucleic acids, and chimeras thereof, but are not limited thereto. is not.

  The nucleic acid contained in the nanoparticles provided by the present invention has a function of expressing a foreign gene by plasmid DNA or a function of suppressing or enhancing the expression of an endogenous gene by antigene, antisense, ribozyme, decoy, RNA interference or the like.

  The monovalent to trivalent basic salts necessary for the production of the nanoparticles of the present invention are selected from carbonates, bicarbonates, phosphates, hydrogen phosphates, oxalates, lactates and urates, Of these, carbonates, hydrogen carbonates, phosphates and hydrogen phosphates are preferred.

  Divalent or trivalent metal salts necessary for forming the nanoparticles provided by the present invention are zinc salts such as zinc acetate, zinc chloride and zinc sulfate, and calcium salts such as calcium carbonate, calcium chloride and calcium sulfate. , Iron salts such as iron chloride and iron sulfide, copper salts such as copper chloride and copper sulfate, and the like. Among them, zinc salts or calcium salts can be preferably used.

  Examples of the hydrophilic polymer having an ionic functional group in the present invention include polyethylene glycol, lipid-polyethylene glycol, and the like in which an ionic functional group can be introduced into a terminal, in a molecule, or in a side chain by a chemical or molecular biological technique. Hydrophilic synthetic polymers: homo- or heteropolysaccharides having ionic functional groups such as dextran, pullulan, cellulose, inulin, mannan, hyaluronic acid, or capable of introducing ionic functional groups by chemical or molecular biological techniques A polypeptide containing an amino acid having an ionic functional group, a protein, and the like. It is preferably a hydrophilic polymer having an acidic group such as a phosphoric acid group, a sulfuric acid group and a carboxyl group as the ionic functional group, and having a phosphoric acid group. Most preferred are polyethylene glycol having a phosphate group at the terminal and phosphorylethanolamine-polyethylene glycol having a phosphate ester. Furthermore, examples of the hydrophobic portion include various saturated or unsaturated alkyl chains, lipids having various saturated or unsaturated alkyl chains, steroids, polypeptides composed of hydrophobic amino acids, and the like.

  Examples of the cationic polymer necessary for forming a composite nanoparticle composed of a cationic polymer and a nucleic acid provided by the present invention include polyamino acids containing an amino acid having a cationic group such as lysine, arginine or histidine. Examples thereof include, but are not limited to, peptides or proteins, DNA-binding proteins such as histones, basic polysaccharides such as chitin and chitosan, dendrimers having a cationic group, and polyethyleneimine. Polyethyleneimine is preferred.

Below, the manufacturing method of the nanoparticle which this invention provides is demonstrated.
Nucleic acid such as DNA, hydrophilic polymer having an ionic functional group such as polyethylene glycol having a terminal phosphate group, and monovalent to trivalent basic salt are mixed in water or a buffer solution, and divalent or trivalent. Nanoparticles composed of a nucleic acid-containing complex can be prepared by adding the metal salt and allowing to stand for 1 hour or longer.

  Another method is to mix a nucleic acid such as DNA and a hydrophilic polymer having an ionic functional group such as phosphorylethanolamine-polyethylene glycol in water or a buffer solution, and a cationic polymer such as polyethyleneimine. Is added, and the mixture is allowed to stand for 1 hour or longer to produce nanoparticles comprising a nucleic acid-containing complex. A phosphate buffer (PBS) is preferably used as the buffer.

  The present invention also provides such an external preparation or injectable preparation for skin / mucosa application. Examples of such external preparations are those that can be administered in the form of application, sticking, dripping, spraying, etc., locally for the purpose of systemic and local administration / treatment, specifically, ointments, gels, sublingual Tablets, oral tablets, liquids, sprays for oral cavity and lower respiratory tract, suction agents, suspension agents, cataplasms, patches and the like can be mentioned. As a liquid, it is preferable as a nasal drop or an eye drop. In addition, application to the skin or mucous membrane, spraying to the lower respiratory tract, etc. are effective dosage forms. As an injectable agent, any of administration forms such as intravenous injection, subcutaneous injection, and intramuscular injection is possible, and it is selected according to the characteristics of each drug.

  Examples of these external preparations, bases used for preparation of injections, and other additive components include bases and various components that are pharmaceutically used for preparation of external preparations and injections. . Specifically, oily bases such as petrolatum, plastic base, paraffin, liquid paraffin, light liquid paraffin, honey beeswax, silicone oil; solvents such as water, water for injection, ethanol, methyl ethyl ketone, cottonseed oil, olive oil, peanut oil, sesame oil Nonionic surfactants such as polyoxyethylene hydrogenated castor oil, polyoxyethylene sorbitan fatty acid ester, glycerin fatty acid ester, polyoxyethylene fatty acid ester, polyoxyethylene alkyl ether, sorbitan fatty acid ester, polyoxyethylene polyoxypropylene glycol Thickeners such as polyvinylpyrrolidone, sodium carboxymethylcellulose (CMC), xanthan gum, tragacanth gum, gum arabic, gelatin, albumin; dibutylhydroxy Stabilizers such as ruene; humectants such as glycerin, 1,3-butylene glycol, propylene glycol, urea, sucrose, erythritol, sorbitol; methyl paraoxybenzoate, butyl paraoxybenzoate, sodium dehydroacetate, p-cresol, etc. It can be used by appropriately selecting depending on the dosage form. In the case of nasal drops, it is preferable to add a nasal absorption accelerator such as hydroxypropylcellulose.

  EXAMPLES The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples.

Example 1: Nanoparticle formation of DNA-cationic polymer 200 ng of plasmid DNA (β-gal-CMV), 260 ng of polyethyleneimine (PEI), and hydrophilic polymer having various ionic functional groups, or ions 2 μg, 20 μg or 200 μg of hydrophilic polymer having no functional functional group was mixed in 20 μL of PBS and allowed to stand for 1 hour. Then, it centrifuged at 8000 rpm for 5 minutes, and analyzed by 1% agarose gel electrophoresis.

  As a result of the analysis by electrophoresis, it was found that the plasmid did not move into the gel from the position of the gel top in any of the hydrophilic polymers, and was thus complexed with polyethyleneimine (PEI). Moreover, the plasmid was released from all the complexes by adding an excess of polyglutamic acid to the complex suspension. The results are shown in FIG.

In the electrophoretic diagram in the figure, the upper part shows the result for the suspension, the middle part shows the result for the suspension + polyglutamic acid, and the lower part shows the result for the supernatant + polyglutamic acid.
Further, in the electropherogram, 1 shows the result of DNA alone, 2 shows the result of polyethyleneimine (PEI), 3 is a gluconic acid phosphate as a hydrophilic polymer, 4 is Tween 80, and 5 is Oleic acid + polyethylene glycol (PEG), 6 shows the results when terminal phosphorylated PGE is used, 7 shows the result when diacylglycerol-PGE is used 1000 times the amount of DNA, and 8 shows phosphorylethanolamine-PEG. When 10 times the amount is used, 9 is when phosphorylethanolamine-PEG is used 100 times the amount of DNA, and 10 is when phosphorylethanolamine-PEG is used 1000 times the amount of DNA. Results are shown.

  As can be seen from the results shown in FIG. 1, the complex suspension was centrifuged at 8000 rpm, and the plasmid in the supernatant was analyzed by electrophoresis. As a result, oleic acid-polyethylene glycol and polyoxyethylene were used as hydrophilic polymers. (20) Sorbitan monooleate (Tween 80), diacylglycerol-polyethylene glycol, which is a neutral lipid that does not contain a phosphate ester in the molecule, polyethylene glycol or phosphate glucuronic acid with a phosphate group introduced at the end In this case, the plasmid band disappeared, and the DNA was precipitated together with the complex by centrifugation.

  On the other hand, in the case of phosphorylethanolamine-polyethylene glycol containing a phosphate ester in the molecule, it was revealed that the plasmid remained in the supernatant even after centrifugation at 8000 rpm. From the above results, it was found that a polyethyleneimine-plasmid complex having a small particle size was formed by mixing lipid-polyethylene glycol (containing a phosphate ester). Furthermore, as a result of the particle size measurement by light scattering, it was found that a nanoparticle composite having about 100 nm was formed.

Example 2: Expression of plasmid DNA in mouse skin Lipid-polyethylene glycol containing plasmid DNA (β-gal-CMV), polyethyleneimine and phosphate ester is mixed in PBS and allowed to stand for 1 hour. Thus, plasmid DNA nanoparticles were obtained. Thereafter, the obtained nanoparticles were applied to the back skin of mice for 3 consecutive days as DNA at a concentration of 10 μg / square cm. 24 hours after the end of administration, the skin was excised, the cells were solubilized with a cell lysate, and the β-galactosidase activity contained in the supernatant was measured. The result is shown in FIG.

  As shown in FIG. 2, β-galactosidase expression was not observed when DNA was administered using polyethyleneimine, but it was prepared using lipid-polyethylene glycol and polyethyleneimine containing phosphate ester. In the nanoparticles, strong expression was observed. Therefore, it was proved that DNA was transferred into the skin cells by the nanoparticles.

Example 3: Absorption of oligo DNA into mouse skin HEX end-labeled oligo DNA (14mer), lipid-polyethylene glycol containing polyethyleneimine and phosphate group were mixed and allowed to stand for 1 hour to complex oligo DNA Body particles were obtained. Thereafter, the particles were applied to the back skin of the mouse as DNA at a concentration of 100 μg / square cm. Two hours later, the skin was excised to produce a section and observed with a fluorescence microscope. The results are shown in FIGS. 3.1 to 3.4.

  Fig. 3.1 shows a phase contrast microscopic image of HE-stained mouse skin to which nothing was applied, Fig. 3.2 shows the results of the non-application group mice, and Fig. 3.3 shows the results of the complex with polyethyleneimine. FIG. 3.4 is a fluorescence micrograph showing the result of a complex of a polyethyleneimine / phosphate group-containing lipid-polyethylene glycol, and in the figure, the arrow indicates the epidermis portion. As a result, it was found that the uptake into the skin was enhanced in the DNA mixed with polyethylene-imine and a lipid-polyethylene glycol containing a phosphate group.

Example 4 Preparation of Oligo DNA-Containing Zinc Carbonate or Calcium Phosphate Nanoparticles Oligo DNA, sodium bicarbonate and terminal phosphorylated polyethylene glycol are mixed, and after adding zinc chloride, DNA-containing nanoparticles are allowed to stand for 2 hours. Got. When this nanoparticle was measured by light scattering, its particle size was 30 to 300 nm.
Similarly, oligo-DNA, sodium dihydrogen phosphate and terminal phosphorylated polyethylene glycol were mixed, and after adding calcium chloride, the mixture was allowed to stand for 2 hours to obtain DNA-containing nanoparticles. The particle size of this particle was 200 to 500 nm.
From the results of analysis by electrophoresis, DNA was complexed with nanoparticles in any particle.

  When the same operation was performed in the absence of terminal phosphorylated polyethylene glycol, large aggregates were formed, but it was found that small nanoparticles were formed by mixing terminal phosphorylated polyethylene glycol. It was. Therefore, it has been found that terminal phosphorylated polyethylene glycol is effective for producing nanoparticles in a system of zinc carbonate or calcium phosphate and nucleic acid.

Example 5: Incorporation of oligo DNA into cells FITC-labeled oligo DNA, sodium bicarbonate, zinc chloride and polyethylene glycol having a phosphate group at the end were mixed in PBS to obtain nanoparticles. The nanoparticles were added to RAW, 3T3L1 or FRSK (keratinocyte) cells, and DNA uptake was observed with a fluorescence microscope. As a control, only oligo DNA and a complex with lipofectamine were used. The results are shown in FIGS. 4.1 to 4.3.

Fig. 4.1 shows the result of oligo DNA alone, Fig. 4.2 shows the result of complex with lipofectamine, and Fig. 4.3 shows the result of nanoparticles prepared using zinc carbonate and polyethylene glycol having phosphate group. It is a fluorescence microscope which shows.
As can be seen from the results shown in FIG. 4, remarkable incorporation of DNA into RAW cells was confirmed with nanoparticles prepared using zinc carbonate and polyethylene glycol having a phosphate group. Similarly, in 3T3L1 or FRSK cells, DNA uptake was observed.

Example 6: Absorption of oligo DNA into mouse skin HEX end-labeled oligo DNA (14mer), sodium bicarbonate, zinc chloride and polyethylene glycol having a phosphate group at the end were mixed in PBS to obtain nanoparticles. It was. Thereafter, DNA was applied to the back skin of the mouse at a concentration of 100 μg / square cm. Two hours later, the skin was excised, sections were prepared and observed with a microscope. The results are shown in FIGS. 5.1 to 5.4.

Fig. 5.1 shows a phase contrast microscopic image of HE-stained mouse skin to which nothing was applied, Fig. 5.2 shows the results of the non-application group mice, and Fig. 5.3 shows a commercially available transfection reagent. FIG. 5.4 is a fluorescence micrograph showing the result of the complex with a certain lipofectamine, and FIG. 5.4 shows the result of the complex with zinc carbonate / polyethylene glycol containing a phosphate group at the terminal. Indicates the epidermis part.
As a result, in Lipofectamine, oligo DNA is localized in the stratum corneum, whereas in nanoparticles prepared using zinc carbonate and polyethylene glycol having a phosphate group, oligo DNA is taken into the skin. I understood.

Example 7 Production of Ointment / Hydrogel Nanoparticles obtained in Example 4 (oligoDNA nanoparticles obtained using sodium bicarbonate and zinc chloride), white petrolatum, sodium carboxymethylcellulose and paraoxy An appropriate amount of methyl benzoate was taken and mixed until the total amount became homogeneous to obtain an ointment and a hydrogel.

Example 8: External patch (aqueous patch)
Formula:
Nanoparticles obtained in Example 4 0.1 parts by weight (oligoDNA nanoparticles obtained using sodium dihydrogen phosphate and calcium chloride)
Polyacrylic acid 2.0 parts by weight Sodium polyacrylate 5.0 parts by weight Sodium carboxymethylcellulose 2.0 parts by weight Gelatin 2.0 parts by weight Polyvinyl alcohol 0.5 parts by weight Glycerin 25.0 parts by weight Kaolin 1.0 part by weight Aluminum hydroxide 0.6 parts by weight Tartaric acid 0.4 parts by weight EDTA-2-sodium 0.1 parts by weight Purified water balance Based on the above ingredients, an external preparation (aqueous poultice) was obtained by a conventional method.

Example 9: Injection The plasmid DNA nanoparticle obtained in Example 2 was dissolved in distilled water for injection, contained an isotonic agent, further adjusted to pH 6.9, and then filled into a vial. High temperature and high pressure sterilization was performed to obtain an injection.

  As described above, the present invention provides a nucleic acid-containing nanoparticle having a small particle diameter by arranging a hydrophilic polymer having an ionic functional group on the surface thereof, and easily passing through the stratum corneum due to the small particle diameter. It has the effect of increasing the permeability to the epidermis and acts as a particle absorption promoter. Therefore, nucleic acid can be easily transported into cells in the skin and mucous membranes, and it can be used as an external preparation or injection for supplying specific proteins to the whole body, controlling the immune system or treating skin diseases by gene expression. Useful.

2 is a photograph showing the results of electrophoresis of a complex comprising various hydrophilic polymers, polyethyleneimine, and DNA in Example 1. FIG. 3 is a result showing expression of plasmid DNA in mouse skin in Example 2. It is the phase-contrast microscope image which carried out the HE dyeing | staining of the mouse | mouth skin section | slice which has not applied | coated nothing in Example 3. FIG. In Example 3, it shows the result of transfer of the fluorescent label oligo DNA to the mouse skin, and is a skin section fluorescence micrograph of a control mouse to which nothing is applied. FIG. 3 is a fluorescence micrograph of a mouse skin slice showing the result of transfer of fluorescently labeled oligo DNA to mouse skin in Example 3 and applied with complex particles with polyethyleneimine. In Example 3, the results of the transfer of fluorescently labeled oligo DNA to mouse skin were shown, and a mouse skin slice fluorescence micrograph applied with lipid-polyethylene glycol complex particles containing polyethyleneimine / phosphate groups. It is. In Example 5, it is a result which shows the uptake of oligo DNA to a RAW cell, and is a fluorescence micrograph of only oligo DNA. FIG. 6 is a fluorescence micrograph showing the result of the incorporation of oligo DNA into RAW cells and the result of complex with lipofectamine in Example 5. FIG. FIG. 6 is a fluorescence micrograph showing the results of nanoparticles prepared using polyethylene glycol having a zinc carbonate-phosphate group, which is a result of showing oligo DNA uptake into RAW cells in Example 5. FIG. It is the phase-contrast microscope image which carried out HE dyeing | staining of the mouse | mouth skin section | slice which has not applied | coated anything in Example 6. FIG. It is a skin section fluorescence micrograph of the control mouse | mouth which shows the result of transfer of the fluorescence label oligo DNA to the mouse skin in Example 6, and does not apply | coat anything. It is a skin microscopic fluorescence micrograph of the mouse | mouth which showed the result of transfer of the fluorescence label oligo DNA to the mouse skin in Example 6, and apply | coated the composite particle with a lipofectamine. FIG. 6 is a fluorescence micrograph of a skin slice of a mouse coated with composite particles of zinc carbonate and a polyethylene glycol having a phosphate group at the end, showing the results of transfer of fluorescently labeled oligo DNA to mouse skin in Example 6. FIG.

Claims (15)

  A nucleic acid-containing nanoparticle comprising a nucleic acid, a monovalent to trivalent basic salt, a divalent or trivalent metal salt and a hydrophilic polymer having an ionic functional group.   A nucleic acid-containing nanoparticle comprising a nucleic acid, a cationic polymer, and a hydrophilic polymer having an ionic functional group.   The nucleic acid according to claim 1, wherein the monovalent or trivalent basic salt is selected from carbonates, hydrogen carbonates, phosphates, hydrogen phosphates, oxalates, lactates and urates. Contains nanoparticles.   The nucleic acid-containing nanoparticle according to claim 1, wherein the divalent or trivalent metal salt is a calcium salt, a zinc salt, an iron salt or a copper salt.   The nucleic acid-containing product according to claim 2, wherein the cationic polymer is selected from polylysine, polyarginine, polyhistidine, a polypeptide containing a cationic amino acid, polyethyleneimine, chitosan, a cationic dendrimer, and a DNA-binding protein. Nanoparticles.   The nucleic acid-containing nanoparticle according to claim 1 or 2, wherein the hydrophilic polymer having an ionic functional group is selected from polyethylene glycol, lipid-polyethylene glycol, polyamino acid, polypeptide, protein, oligosaccharide and polysaccharide. particle.   The nucleic acid-containing nanoparticle according to claim 6, wherein the ionic functional group of the hydrophilic polymer having an ionic functional group is a phosphate group, a sulfate group, or a carboxyl group.   The nucleic acid-containing nanoparticle according to any one of claims 1 to 7, wherein the particle has a diameter of 10 to 500 nm.   Nucleic acid-containing nanoparticles comprising a nucleic acid, a hydrophilic polymer having an ionic functional group, a divalent or trivalent metal salt, and a monovalent to trivalent basic salt that are allowed to act in water or a buffer solution Manufacturing method.   A method for producing a nucleic acid-containing nanoparticle, which comprises reacting a nucleic acid, a cationic polymer and a hydrophilic polymer having an ionic functional group in water or a buffer solution.   The nucleic acid-containing nanoparticle according to claim 1, wherein the nucleic acid is selected from DNA, RNA, phosphodiester derivatives thereof, 2′-0 methyl RNA, peptide nucleic acids, and chimeras thereof.   The nucleic acid-containing nanoparticle according to any one of claims 1 to 11, wherein the nucleic acid has a function of expressing a foreign gene by plasmid DNA or a function of suppressing or enhancing expression of an endogenous gene by antigene, antisense, ribozyme, decoy or RNA interference. .   A skin or mucosa applicable external preparation containing the nucleic acid-containing nanoparticles according to any one of claims 1 to 12.   The external preparation according to claim 13, wherein the external preparation is selected from an ointment, a gel, a sublingual tablet, an oral tablet, a liquid, a spray for oral cavity and lower respiratory tract, an aspirating agent, a suspension, a poultice, and a patch. . The injection for injection containing the nucleic acid containing nanoparticle in any one of Claims 1-12.

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