TECHNICAL FIELD

The present invention relates to a targeting nanoparticle for efficient oral gene delivery, and more particularly to a nanoparticle for gene delivery, comprising: an ionic polymer conjugated with bile acid or a bile acid derivative; and a gene, and to a pharmaceutical composition comprising the same. Moreover, the present invention relates to a nanoparticle for gene delivery, which is used to deliver a gene or a therapeutic protein to targeted small intestine cells in order to minimize side effects and provide the efficiency of current therapeutic technology, and to a pharmaceutical composition comprising the same.

BACKGROUND ART

Gene therapy treats disease by delivering a therapeutic gene to a target organ to express a new protein in cells. The gene therapy is not to treat the symptoms of disease, but is a way to treat disease by eliminating the cause of the disease. The gene therapy has better selective therapeutic effects than general drug therapies, and can be applied for a long period of time by improving the cure rate of diseases difficult to control by other therapies.

The gene therapy is a next-generation therapeutic technology disclosed for the treatment of various diseases, but when a macromolecule such as DNA, RNA or the like is delivered into cells in an aqueous solution state, it is degraded rapidly by a specific enzyme in vivo and the intracellular deliver)/rate thereof is low. For this reason, in order to make the gene therapy effective, it is essential to develop a gene carrier that can safely deliver a therapeutic gene to a target cell so as to obtain high expression efficiency.

Gene carriers should have low or no toxicity and should be able to selectively and effectively deliver genes to targeted cells. Such gene carriers may be largely divided into viral and non-viral gene carriers.

Viral vectors use retro virus (RV), adeno virus (AV), adeno-associated virus (AAV) or the like as a gene carrier, and have higher gene delivery efficiency than non-viral vectors. However, when these viral vectors are introduced into the body, the vectors themselves cause problems such as immune responses or cancer induction. In addition, there is a limitation on the size of a gene that can be inserted into the vector.

Non-viral vectors such as polymers or nanoparticles have advantages over viral vectors in that they have a low biological risk, are non-immunogenic, can be modified, and can be produced in large amounts, and the size of genes that can be delivered by them is less limited. Due to these advantages, many studies thereon have recently been conducted. In particular, methods employing non-viral vectors such as cationic polymers have been much studied because of the simplicity of production methods or a relatively low risk. In a specific mechanism, the non-viral vector stabilizes a negatively charged gene by forming a complex through electrostatic interaction with the gene, and the complex binds to the cell surface by the electrical interaction between the positive charge on the complex surface and the negative charge on the cell surface, after which the gene is released from the endosome into the cytoplasm.

Specific examples of these cationic polymers include cationic homopolymers such as protamine, polyethyleneimine (PEI), polyamidoamine, poly-L-lysine (PLL), polyaminoester, polypropyleneimine and the like, and cationic block copolymers such as polyethylene glycol (PEG)-block-poly-L-lysine and PEG-PEI. These cationic polymers protect a gene such as DNA from enzymatic degradation by compressing the gene into a nanoparticle, and help enable the gene to penetrate rapidly into cells and allow the gene to escape from the endosome.

Most non-viral vectors have advantages, such as biodegradability, low toxicity, non-immunogenicity, the ease of use, and the like, over viral vectors, but have problems such as relatively lower transfection efficiency than viral vectors, limited particle sizes, and the like.

Polyethyleneimine (PEI), which is currently most extensively studied as a non-viral vector, also has problems, such as significantly in vivo low transfection efficiency, high cytotoxicity, and low gene expression efficiency due to low blood compatibility, and the like.

Meanwhile, Korean Patent Application Publication No. 10-2010-0122405 discloses a nanoparticle-type amphiphilic polymer nanoparticle which is an siRNA carrier obtained by bonding an siRNA to an amphiphilic polymer nanoparticle, a complex comprising a hydrophilic polymer bonded to a hydrophobic polymer, and which is produced by bonding each of chitosan and polyethyleneimine as hydrophilic polymers to the bile acid 5-β-cholanic acid, thereby producing complexes, and then mixing the complexes with each other. In particular, the above patent document discloses that spherical self-aggregates are formed in an aqueous system due to amphiphilicity caused by the hydrophobic groups of bile acid and the hydrophilic group of chitosan and polyethyleneimine (PEI), and for this reason, strongly amphiphilic polyethyleneimine and chitosan are located on the surface of chitosan-bile acid/polyethyleneimine (PEI)-bile acid complex nanoparticles, and hydrophobic bile acid is located in the core. However, it is not accessible for clinical trials due to the serious toxicity of PEI.

Accordingly, there is an urgent need to develop a gene carrier for safe and efficient gene delivery, which has enhanced transfection efficiency while retaining the advantages of conventional non-viral vectors.

Meanwhile, diabetes mellitus is a metabolic disease in which the blood glucose concentration is increased due to insufficient insulin secretion or functional abnormalities. Type 2 diabetes refers to the case in which in vivo blood glucose levels are increased without being controlled, because insulin is not properly secreted or the insulin secreted does not properly act in vivo, and type 1 diabetes refers to the case in which blood glucose levels are increased because the pancreas fails to secrete insulin. In the case of type 1 diabetes, insulin treatment is necessary. In the case of type 2 diabetes, lifestyle modification is essential, and administration of oral hypoglycemic agents may additionally be required. An eating drug is administered one to three times a day, and the administration time or side effects of the drug slightly differ depending on the time of action of the drug.

Diabetes therapies which are currently used include diet therapy, exercise therapy, sulphonylureas, biguanide-based drugs, α-glucosidase inhibitors, insulin, and the like, and various insulins and the like have been developed through many studies on the development of new drugs and have been applied to clinical practice.

Meanwhile, glucagon-like peptide-1 (GLP-1), a kind of insulin secretion peptide, is an incretin hormone which is secreted from the L-cells of the ileum and large intestine. The major action of glucagon-like peptide-1 is to increase insulin secretion and to inhibit hypoglycemia by glucose-dependent insulin secretion. Due to such characteristics, glucagon-like peptide-1 is applied for the treatment of type 2 diabetes. However, it has a very short blood half-life of about 2 minutes, and for this reason, the development of drugs using glucagon-like peptide-1 is greatly limited. Accordingly, developed and commercially available glucagon-like peptide-1 agonists include exendin-4, a glucagon-like peptide-1 analogue isolated from the salivary glands of the Gila monster. Exendin-4 has higher physiological activity than glucagon-like peptide-1 together with resistance to DPP-IV (dipeptidyl peptidase-4), and thus has an in vivo half-life of 2 to 4 hours, which is longer than that of glucagon-like peptide-1 (U.S. Pat. No. 5,424,286). However, it is impossible to expect the sufficient duration of physiological activity only by a method that increases resistance to DPP-IV. For example, currently commercially available exendin-4 (exenatide) should be administered twice a day to a patient through injection, and still has a disadvantage in that vomiting and nausea due to administration of exenatide impose a great burden on patients.

However, these therapeutic agents have low efficacy or the problem of causing many side effects such as liver dysfunction, hypoglycemia, lactic acidosis and the like.

Accordingly, there is a need for a diabetes therapeutic agent that reduces the adverse effects of conventional diabetes therapeutic agents, improves metabolic abnormalities, and is safe even when administered over a long period of time.

In addition, in order to solve these problems, a technology employing viral carriers has been developed. This technology is exposed to risks such as nonspecific immune responses, etc., and the production process is complicated, resulting in many problems in commercialization. Therefore, recent studies have been directed toward the use of non-viral carriers, that is, carriers based on cationic lipids or polymers. Although the efficiency of these non-viral carriers is lower than that of viral carriers, these non-viral carriers have advantages in that they are stable in vivo, are produced by an easy and simple process, and are low-priced. However, when a mixture is to be made using a polymer and a gene, formation of the mixture encounters great difficulty due to the characteristic hardness and weakly anionic nature of the gene.

DISCLOSURE

Technical Problem

Accordingly, the present inventors have conducted studies and made efforts to solve the above-described problems, and as a result, have found that when a nanoparticle for gene deliver)/comprises an ionic polymer, surface-modified with bile acid or a bile acid derivative, and a gene, and when the core of the nanoparticle for gene deliver)/comprises an ionic complex of the ionic polymer and the gene and when the bile acid or bile acid derivative having an hydrophilic ionic group is located on the surface of the nanoparticle, the in vivo stability of the nanoparticle can be improved, the above-described side effects can be reduced by minimizing the in vivo toxicity of the nanoparticle, and disorders associated with oral administration of the nanoparticle can be overcome, thereby completing the present invention.

Therefore, it is an object of the present invention to provide a nanoparticle for gene delivery, which is capable of effectively delivering a therapeutic gene to an intracellular target site and comprises: at least one ionic polymer conjugated with bile acid or a bile acid derivative; and a gene, wherein the conjugated bile acid or bile acid derivative has an ionic group.

Another object of the present invention is to provide a pharmaceutical composition for treating diabetes, which comprises the nanoparticle for gene delivery as an active ingredient.

Technical Solution

To achieve the above objects, the present invention provides a nanoparticle for gene delivery, which comprises: at least one ionic polymer conjugated with bile acid or a bile acid derivative; and a gene, wherein the conjugated bile acid or bile acid derivative has an ionic group.

In one embodiment of the present invention, the ionic polymer may be a cationic polymer.

In one embodiment of the present invention, the cationic polymer may be selected from the group consisting of chitosan, protamine, poly-L-lysine (PLL), and polyamidoamine (PAMAM).

In one embodiment of the present invention, the ionic polymer may be an anionic polymer, and in this case, the ionic polymer further comprises a cationic polymer.

In one embodiment of the present invention, the anionic polymer may be selected from among heparin, hyaluronic acid, and chondroitin sulfate, and the cationic polymer may be selected from the group consisting of chitosan, glycol chitosan, protamine, poly-L-lysine (PLL), and polyamidoamine (PAMAM).

In one embodiment of the present invention, the ionic polymer conjugated with the bile acid or bile acid derivative may be one conjugated via a disulfide bond.

In one embodiment of the present invention, the bile acid or its derivative may be selected from the group consisting of deoxycholic acid, taurodeoxycholic acid, taurocholic acid (TCA), glycocholic acid, and glycochenodeoxycholic acid. More preferably, it is taurocholic acid (TCA).

In one embodiment of the present invention, the gene may be one or more selected from among single-stranded or double-stranded DNA (deoxyribonucleic acid), single-stranded or double-stranded RNA (ribonucleic acid), plasmid DNA (pDNA), antisense oligonucleotides, ribozymes, catalytic RNA, and nucleotides. Preferably, the gene may be one or more selected from among plasmid DNA (pDNA) and antisense oligonucleotides. More preferably, the gene may be a glucagon-like peptide-1 (GLP-1) gene, a GLP-1 peptide, a GLP-1 variant, a GLP-1 derivative, a GLP-1 agonist, liraglutide, or exendin-4.

In one embodiment of the present invention, the degree of conjugation of the bile acid or its derivative may vary depending on the number of functional groups of the ionic polymer, but the bile acid or its derivative and the ionic polymer may preferably be conjugated to each other at a molar ratio of 1:1 to 1:300.

In one embodiment of the present invention, the nanoparticle for gene delivery may be absorbed regardless of the particle size, but may preferably have a particle size of 500 nm or less.

In one embodiment of the present invention, the ionic polymer conjugated with the bile acid or bile acid derivative and the gene may be bonded to each other at a molar ratio of 1:1 to 1:200.

In one embodiment of the present invention, the N/P ratio (the number of nitrogen atoms of the ionic polymer conjugated with the bile acid or bile acid derivative/the number of phosphate atoms of the gene) of the nanoparticle for gene delivery may be 1 to 200.

The present invention also provides a pharmaceutical composition for treating diabetes, which comprises a nanoparticle for gene delivery as an active ingredient, wherein the nanoparticle comprises: at least one ionic polymer conjugated with bile acid or a bile acid derivative; and a glucagon-like peptide-1 (GLP-1) gene, a GLP-1 peptide, a GLP-1 variant, a GLP-1 derivative, a GLP-1 agonist, liraglutide, or exendin-4, wherein the conjugated bile acid or bile acid derivative has an ionic group.

In one embodiment of the present invention, the composition may be for oral administration.

Advantageous Effects

The nanoparticle for gene delivery according to the present invention is a novel oral gene delivery system capable of regulating blood glucose levels in biological systems and insulin secretion in response to ingested meals, and comprises: an ionic polymer conjugated with bile acid or a bile acid derivative; and a gene.

The nanoparticle for gene delivery according to the present invention is a nanoparticle surface-modified with bile acid or its derivative, wherein an ionic complex comprising a negatively charged gene encapsulated in a cationic polymer due to the electrostatic interaction between the negatively charged gene and the cationic polymer is present in the core of the nanoparticle and wherein the bile acid or bile acid derivative conjugated to the cationic polymer is located on the surface of the nanoparticle while having a hydrophilic anionic group.

Furthermore, the nanoparticle for gene delivery according to the present invention is a nanoparticle surface-modified with bile acid or its derivative, wherein an ionic complex comprising a negatively charged gene encapsulated in a cationic polymer due to the electrostatic interaction between the negatively charged gene and the cationic polymer is present in the core of the nanoparticle and wherein the cationic polymer of the ionic complex, and the anionic polymer conjugated with the bile acid or bile acid derivative having a hydrophilic anionic group, are conjugated to each other by electrostatic interaction, and thus the bile acid or bile acid derivative conjugated to the anionic polymer is located on the surface of the nanoparticle while having a hydrophilic ionic group.

Accordingly, the nanoparticle for gene delivery according to the present invention protects against gastric acid through direct physical bonding between the cationic polymer and the gene, may be safely delivered to small intestine cells, and may also be used as a targeting nanoparticle that enables the GLP-1 gene to be expressed only in L-cells in the small intestine.

When the nanoparticle surface-modified with bile acid or its derivative according to the present invention was used to deliver a therapeutic gene to a target cell, it easily delivered the gene through intestinal mucosa and, at the same time, was very stable in the gastrointestinal tract due to the ionic complex present in the core of the nanoparticle. This suggests that the nanoparticle for gene delivery according to the present invention is very suitable for oral administration.

The bile acid located on the surface of the nanoparticle according to the present invention can inhibit gastrointestinal (GI) degradation of the nanoparticle, and absorb the bile acid receptor ASBT (apical sodium bile acid transporter) receptor of intestinal cell membrane in the small intestine ileum, thereby delivering the therapeutic gene into cells, and thus the nanoparticle can exhibit increased gene expression.

In particular, when the therapeutic gene used is GLP-1, a GLP-1 peptide, a GLP-1 variant, a GLP-1 derivative, a GLP-1 agonist, liraglutide or exendin-4, the bile acid located on the surface of the nanoparticle according to the present invention and the cationic polymer are separated by the enzyme glutathione of intestinal cells and pH and enter portal vein, and the separated therapeutic gene is delivered only to the nucleus of intestinal cells and expresses a large amount of the GLP-1, GLP-1 peptide, GLP-1 variant, GLP-1 derivative, GLP-1 agonist, liraglutide or exendin-4, thereby efficiently exhibiting diabetic therapeutic efficacy.

Accordingly, the nanoparticle for gene delivery according to the present invention, when administered orally to diabetic mouse models, can exhibit blood glucose-lowering effects by secreting insulin while successfully expressing GLP-1, a GLP-1 peptide, a GLP-1 variant, a GLP-1 derivative, a GLP-1 agonist, liraglutide or exendin-4, thereby effectively preventing or treating diabetes, particularly type 2 diabetes.

