KR101118691B1 - Active siRNA-Inorganic Layered Double Hydroxide Nanohybrid, a Process for Preparing the Same, and a Pharmaceutical Composition Comprising the Same for Treating Targeted Tumor - Google Patents

Abstract

The present invention is a nanocomposite of siRNA (small interfering RNA) and a target-oriented layered inorganic hydroxide, a powerful gene therapy agent, a method for preparing the same, and a pharmaceutical composition for treating a tumor containing a target-oriented siRNA-layered inorganic hydroxide nano hybrid It is about.

In the nanocomposite according to the present invention, in vivo stability of siRNA forming a nanohybrid with a layered inorganic hydroxide to which a target-directed multifunctional ligand capable of specifically binding to a tumor marker may be improved. It can be widely used in general-purpose target-directed tumor therapies since it shows the tumor therapeutic activity of siRNA at relatively low concentrations.

siRNA, Layered Inorganic Hydroxide, Targeted, Nanohybrid, Gene Therapeutics

Description

Active siRNA-Inorganic Layered Double Hydroxide Nanohybrid, a Process for Preparing the Same, and a Pharmaceutical Composition Comprising the Same for Treating Targeted Tumor}

The present invention relates to a nano hybrid of a small interfering RNA (siRNA) and a target-oriented layered inorganic hydroxide, a method for producing the same, and a pharmaceutical composition for treating a tumor containing the nano hybrid, which is a powerful gene therapy agent.

Safe and efficient gene delivery techniques for gene therapy have been studied for a long time and various gene carriers and delivery techniques have been developed. Gene transfer techniques using viruses such as adenoviruses and retroviruses, and gene transfer techniques using nonviral vectors using liposomes, cationic lipids, and cationic polymers have been developed. However, to date, the method of using the virus itself as a carrier for gene therapy is not convinced that the delivered gene enters the host chromosome and does not cause abnormal function of the host gene or activate the oncogenic gene. Continued expression, even in small amounts, may result in autoimmunity or, if a modified form of viral infection from a viral carrier, results in no effective defense immunity. Therefore, instead of using viruses, methods for fusing genes into liposomes, methods using lipids or polymers with cations, methods using inorganic nanoparticles, and the like have been studied to improve their respective disadvantages. These non-viral vectors are much lower in efficiency than viral vectors, but considering the safety and economy in vivo, there are advantages in that they have less side effects and a lower production price. In addition, the most important emerging approach in the current gene delivery system is the section on target specific delivery. When the gene is directly administered in vivo, all organs and cells in vivo are attacked by the same gene, thereby damaging normal cells and normal tissues, and thus, technology development for selective gene delivery and treatment method is important.

On the other hand, siRNA has recently been found to have an excellent effect on inhibiting the expression of specific genes in animal cells, which has attracted the spotlight as a gene therapy agent. It is expected to replace antisense oligonucleotides (ODN). siRNA is a short, double-stranded RNA strand consisting of 19 to 23 nucleotides, and targets the mRNA of the gene to be treated complementary to these sequences to suppress gene expression. However, since siRNA is degraded in a short time in vivo due to its low stability, its treatment efficiency drops drastically, the dosage of expensive siRNA must be increased, and its anionicity makes it easy to penetrate the same negatively charged cell membrane. There is a problem that it is difficult, and as a result, the intracellular delivery is poor (Celia M. & Henry, Chemical and Engineering News December, 22, 32-36, 2003). In addition, although siRNA is composed of double strands, the binding of the ribose sugar constituting the RNA is chemically very unstable compared to the binding of the deoxyribose sugar constituting the DNA, so that the half-life is rapidly degraded within 30 minutes in vivo. It becomes. Recently, attempts have been made to improve the stability by introducing various functional groups into siRNAs to protect them from degrading enzymes (see Frank Czauderna et al., Nucleic Acids Research 31, 2705-2716, 2003). Technology for securing stability and efficient cell membrane permeability can be said to be in the development stage. In addition, in order to obtain the therapeutic effect of siRNA in consideration of their instability in the blood simply using a method of continuously injecting only a high concentration of siRNA, but its efficiency is known to be low, and economically, siRNA is used as a gene therapy agent. In order to use, it is necessary to develop a new non-viral carrier manufacturing technology that is easy to intracellular delivery. According to Korean Patent Registration No. 10-0883471, by intracellular stability by improving the in vivo stability of siRNA using a hybrid conjugate covalently linked siRNA and hydrophilic polymer and a polymer electrolyte complex micelle composed of the conjugate and a cationic compound It has been reported that it is possible to efficiently deliver soluble siRNAs and to exhibit siRNA activity at relatively low concentrations.

However, there has been no report on the pharmaceutical composition for the treatment of tumors using non-viral layered inorganic hydroxides to improve the stability of siRNAs and to develop target-oriented gene therapeutics. As a result, the present inventors have tried to develop target-oriented gene therapeutics using siRNA. The present invention has been completed by confirming that siRNA can be nano-hybridized with a layered inorganic hydroxide to which a tumor-specific multifunctional ligand is bound.

Disclosure of Invention An object of the present invention is to encapsulate siRNA in a layered inorganic hydroxide and to specifically bind to a tumor marker to improve the intracellular delivery efficiency of siRNA, and to prepare a nano hybrid conjugated with a target-oriented polyfunctional ligand. To provide.

Another object of the present invention is to provide a pharmaceutical composition for tumor treatment containing a target-oriented siRNA-layered inorganic hydroxide nano hybrid and a pharmaceutically acceptable carrier.

In order to achieve the above object, the present invention provides a target-oriented siRNA-layered inorganic hydroxide nano hybrid represented by the following formula (1):

[Formula 1]

[M (II) _{1- x} M (III) _x (OH) ₂ ] ^{X +} [S] [T]

Wherein M (II) represents a divalent metal cation and M (III) represents a trivalent metal cation, x is a number between 0.1 and less than 0.5, S is an siRNA, and [T] is a tumor targeting polyfunctional ligand. .

The present invention also provides a method comprising the steps of: (a) adding a base aqueous solution to an aqueous solution containing a divalent metal salt and a trivalent metal salt to prepare a precipitated layered inorganic hydroxide; (b) a siRNA-layered inorganic hydroxide nanohybrid by mixing and stirring the siRNA-containing solution with the solution containing the layered inorganic hydroxide prepared in step (a). Forming a sieve; And (c) binding a tumor marker specific multifunctional ligand to the hybrid to prepare a target-oriented siRNA-layered inorganic hydroxide nano hybrid. To provide.

The present invention also provides a pharmaceutical composition for treating tumors containing the nanohybrid and a method for producing the same.

According to the present invention, the target-oriented siRNA-layered inorganic hydroxide nanohybrid increases the in vivo stability of the siRNA, and the target-oriented multifunctional ligand which can specifically bind the tumor marker improves tumor specific delivery efficiency. It can be used as a composition to improve the efficiency and accuracy of the treatment of various diseases by showing the tumor therapeutic activity of siRNA at relatively low concentrations, as well as a new type of siRNA delivery system for basic research in biotechnology and the medical industry. It can be usefully used.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein and the experimental methods described below are well known and commonly used in the art.

The present invention relates to siRNA-layered inorganic hydroxide nanocomposites having target orientation.

As used herein, the term "nanohybrid" means that siRNA is bound to a layered inorganic hydroxide by intermolecular interaction. The kind of intermolecular attraction (e.g., electrostatic attraction, hydrophobic attraction, hydrogen bond, covalent bond (e.g., disulfide bond), van der Waals bond, ionic bond, etc.) may be variously selected because it is not particularly limited. In addition, the term "nano hybrid" should be understood to include a form in which a target-oriented polyfunctional ligand is bound by intermolecular attraction on an siRNA-layered inorganic hydroxide nanohybrid.

In one aspect, the present invention relates to a target-oriented siRNA-layered inorganic hydroxide nano hybrid represented by Formula 1 below:

[Formula 1]

[M (II) _{1- x} M (III) _x (OH) ₂ ] ^{X +} [S] [T]

Where M (II) represents a divalent metal cation, M (III) represents a trivalent metal cation, x is a number less than 0.1 to less than 0.5, S is an siRNA and T is a tumor targeting polyfunctional ligand.

In the present invention, RNA-induced in vivo enzyme complexes in which double-stranded siRNA oligomers are involved in their gene inhibition, even when combined with target-oriented layered metal hydroxides at long nucleotide lengths, such as molecular weight levels of 30,000, are used. Although it can reliably bind to sexual silencing complexes (RISCs), the normal 19 nucleotides of 10,000 siRNAs are structurally linked by target-directed multifunctional ligands when combined with intracellular enzyme complexes that aid in gene inhibition. It may be advantageous to introduce bonds that can degrade these conjugates in vivo or in the cell, since their stability may be compromised. Thus, the oligo strand of the siRNA is preferably selected between 10,000 and 30,000 molecular weight. If the siRNA oligo strands have a molecular weight in the above range, it will comprise 19 to 30, preferably 19 to 23 nucleotides. At this time, the siRNA is c-myc, c-myb, c-fos, c-jun, c-raf, c-src, bcl-2 or VEGF (vascular endothelial growth factor), VEGF-B, VEGF-C, It is preferred to use siRNAs derived from VEGF-D, PIGF, or survivin, but are not necessarily limited thereto.

In addition, when the siRNA is introduced, disulfide bonds that are degraded intracellularly by glutathione present in the cytoplasm, acid-degradable bonds that can be effectively degraded in an acidic environment after being introduced into the cell, introduced into the cell It may be desirable to introduce ester bonds, anhydride bonds, and enzyme-degradable bonds that can be degraded just before being introduced into the cells by enzymes present around the specific cell.

Cancer is generally associated with a slowing of apoptosis, active and spontaneous cell death, and a change in the cell cycle, so suppressing the cell cycle or restoring apoptosis is a new method for treating tumors. Is emerging. Tumors that inhibit apoptosis are known to express apoptosis inhibitor proteins (IAPs), and electrical IAPs directly inhibit the activity of apoptosis-causing enzymes or related transcription factors. It is known to exhibit its action in a manner that modulates the activity of phosphorus NF-kB. Recent studies have revealed that survivin, one of the IAPs, is associated with tumors. Survivin is a protein that is commonly expressed in most neoplastic tumors or transformed cell lines that have been tested to date. Survivin is known to be expressed at a constant level even in tumors with persistent mutations. (Ambrosini G, et al., Nat. Med., 3 (8): 917-921, 1997).

By binding to mRNA transcribed from a gene encoding survivin, siRNA capable of inactivating electric mRNA is introduced directly into tumor cells, thereby inhibiting the expression of survivin in tumor cells or By inhibiting activity, a method of effectively treating cancer cells has been attracting attention (Korea Patent Registration 10-0848665). Double-stranded siRNA of the present invention can bind to mRNA transcribed from a gene encoding survivin and inhibits survivin expression in cells.

Preferred embodiments of the siRNA of the present invention may be siRNA capable of complementary binding to the mRNA encoding survivin, which can suppress the expression of survivin, which is commonly expressed in almost all tumors.

Specifically, siRNA capable of complementary binding to the mRNA encoding the survivin may be a nucleotide sequence as shown in Table 1 below.

[Table 1]

Sequence SEQ ID NO: 5’-AAGGAGAUCAACAUUUUCA-3 ’ SEQ ID NO: 1 5’-UAGGAAAGGAGAUCAACAU-3 ’ SEQ ID NO: 2 5’-AGGAAAGGAGAUCAACAUU-3 ’ SEQ ID NO: 3 5’-AGGAAAGGAGAUCAACAUU-3 ’ SEQ ID NO: 4 5’-GGAAAGGAGAUCAACAUUU-3 ’ SEQ ID NO: 5 5’-GAAAGGAGAUCAACAUUUU-3 ’ SEQ ID NO: 6 5’-AAAGGAGAUCAACAUUUUC-3 ’ SEQ ID NO: 7 5’-AGGAGAUCAACAUUUUCAA-3 ’ SEQ ID NO: 8 5’-GGAGAUCAACAUUUUCAAA-3 ’ SEQ ID NO: 9

In addition, the end groups of the sense or antisense of the siRNA may be substituted with other functional groups. For example, as an example of the functional group, the hydroxyl group of 3 'may be substituted as an amine group, sulfhydryl group, phosphate group, or the like. SiRNA according to the present invention may be further provided with a tumor cell selective ligand at its terminal. Preferably, the ligand may be a cell specific antibody, a cell selective peptide, a cell growth factor, folic acid, galactose, mannose, alzidi, transferrin, or the like. These ligands can be introduced into the end groups of the siRNA through bonds such as disulfide bonds, amide bonds, ester bonds, and the like.

In the present invention, the layered inorganic hydroxide has a layered crystal structure and has anion exchange capacity. This is because the hydroxide layer of the layered inorganic hydroxide has a positive charge and an anion is present between the layers to compensate for this, and the interlayer anion may be replaced with another anionic species. It may be characterized by represented by the formula (2):

[Formula 2]

_{[M (Ⅱ) 1- x M} (Ⅲ) x (OH) 2] X + [A n -] X / n? yH ₂ O

Where M (II) represents a divalent metal cation, M (III) represents a trivalent metal cation, A is an anionic species, n is the number of charges of the anion, x is a number less than 0.1 to 0.5 and y is 0 Is more than positive.

In the present invention, the divalent metal cation is selected from the group consisting of Mg ^{2 +} , Ca ^{2 +} , Co ^{2 +} , Cu ^{2 +} , Ni ^{2 +} and Zn ²⁺ , wherein the trivalent metal cation is Al ^{3 +} , Cr ^{3 +,} Fe ^{3 +,} Ga ^{3 +,} in ^{3 +,} V ^{3 +,} and Ti ^{3 +} is selected from the group, the anion consists of the _{^{CO 3 2 -, NO 3 -}} , Cl -, OH -, O ^{2 -} can be characterized as being selected from the group consisting of ^-, and SO ₄ ^2. The ratio of the divalent metal cation to the trivalent metal cation may be adjusted to 2: 1, 3: 1 and 4: 1 to form a layered inorganic hydroxide having a controlled layer charge. The divalent metal cation, the trivalent metal cation, and the anion are not limited to the above types, and may include all of those known as layered inorganic hydroxides in the art.

In the present invention, the "tumor target oriented polyfunctional ligand" means a tumor specific binding component that is additionally bound to the siRNA-layered inorganic hydroxide nanohybrid to confer target orientation. Examples of such tumor specific binding components include antigens, antibodies, RNA, DNA, hapten, avidin, streptavidin, neutravidin, protein A, protein G, lectin ), Selectin, radioisotope labeled biomaterials, biomaterials that can specifically bind to tumor markers, and the like.

Here, the target oriented multifunctional ligand means a material including (i) an attachment region, (ii) a cross-linking region, and (iii) an active ingredient region. Hereinafter, the multifunctional ligand will be described in more detail.

The "attachment region" means a portion, preferably a terminal, of a spacer or target-oriented polyfunctional ligand comprising a functional group capable of surface modification to attach to a layered inorganic hydroxide. Therefore, the attachment region preferably includes a functional group having high affinity with the surface of the layered inorganic hydroxide, and may be variously selected according to the material of the layered inorganic hydroxide. Adhesion regions include, for example, aminosilane, epoxysilane, vinylsilane, -COOH, -NH ₂ , -SH, -CONH ₂ , -PO ₃ H, -PO ₄ H, -SO ₃ H, -SO ₄ H or -OH can do.

The term "crosslinking region" refers to "end of the attachment region" and "end of the target-oriented polyfunctional ligand" that includes a functional group capable of crosslinking with a portion of the multifunctional ligand adjacent to the surface-modified layered inorganic hydroxide. By "crosslinking" is meant that the multifunctional ligand is bound by intermolecular interaction with the end of the attachment region of the surface-modified layered inorganic hydroxide located in close proximity. The types of intermolecular attraction (e.g., hydrophobic attraction, hydrogen bonds, covalent bonds (e.g. disulfide bonds), van der Waals bonds, ionic bonds, etc.) are not particularly limited. It can be variously selected according to. The cross-linking region, for example, _{-SH, -NH 2, -COOH, -OH} , -NR 4 + X -, - epoxy (epoxy), - ethylene (ethylene), - acetylene (acetylene) - sulfonate (sulfonate) , -Nitrate, or phosphonate may be included as a functional group. The functional group of the cross-linking region may vary depending on the type of the terminal of the attachment region and the terminal of the active ingredient and the formula thereof.

The intermolecular bonds may be either non-degradable bonds or degradable bonds. At this time, the non-degradable bond is an amide bond or phosphate bond, and the degradable bond is a disulfide bond, an acid decomposable bond, an ester bond, anhydride bond, a biodegradable bond, or Enzyme degradable bonds, but are not necessarily limited thereto.

The "active ingredient region" means a portion of a tumor specific binding component or target-oriented multifunctional ligand including a functional group capable of crosslinking with an attachment region, preferably a terminal located opposite the attachment region.

Here, the tumor specific binding component is antigen, antibody, RNA, DNA, hapten, avidin (avidin), streptavidin (streptavidin), neutravidin, protein A, protein G, lectin (lectin) , But not limited to, selectin, radioisotope labeled components, and substances capable of specifically binding tumor markers. As the tumor specific binding component ligand, preferably, cell specific antibodies, cell selective peptides, cell growth factors, folic acid, galactose, mannose, algidi, transferrin, and the like may be used. The active ingredient will be suitably ligand or antibody capable of binding to the tumor. Preferably, the ligand may be a cell specific antibody, a cell selective peptide, a cell growth factor, folic acid, galactose, mannose, alzidi, transferrin, or the like.

The present invention provides a nano hybrid in which the target-oriented siRNA-layered inorganic hydroxide nano hybrid is bound to a therapeutic agent by specifically binding to a tumor. That is, in the present invention, the "target oriented siRNA-layered inorganic hydroxide nano hybrid" is surrounded by a target-oriented polyfunctional ligand in which the siRNA-layered inorganic hydroxide nano hybrid is comprised of an attachment region, a cross-linking region, and an active ingredient region. In the active ingredient region, a nano hybrid is bound to a substance capable of specifically binding to a tumor marker.

In a preferred embodiment of the present invention, folic acid having a carboxyl terminus is selectively used as a target-oriented multifunctional ligand constituting the target-oriented nanohybrid, selectively responding to a folate receptor overexpressed in tumor cells. That is, the attachment region is the silane portion of the aminosilane, the cross-linking region is the peptide region where the amine end portion of the aminosilane and the carboxyl end of the folic acid are bound, and the active ingredient region is a region that is sensitive to the folate receptor.

Folic acid (FA) is a nutrient that plays an important role in the folate cycle, a mechanism for producing genes in cells, and is known to play an important role in cell differentiation. In general, cancer cells require a large amount of folic acid (or folate) for rapid cell differentiation. To this end, cancer cells tend to overexpress folate receptors on cell membranes and consume large amounts of folic acid. In particular, in some breast cancer cells, such as KB cells, the folate receptor is overexpressed compared to normal cells, so folate may act as a ligand for recognizing these cancer cells. Ligands that recognize cancer cells may include antibodies, aptamers, etc. in addition to chemicals such as folate, but folate has the advantage of ligand in that it has no immune side effects and is accessible at a relatively low price. Big. Therefore, several recent studies have shown efforts to increase the affinity of the drug carrier for cancer cells by using folate as a ligand. In particular, studies on attaching ligands to the surface of drug carriers such as liposomes (Gabizon, A. et al., Adv. Drug Delivery Rev. (2004) 56: 1177-1192) and PEG-DSPE (polyethyelenglycol-disterarolyl phosphatidylethanolamine) A study to increase the infection efficiency of the loaded DNA by attaching a ligand to the terminal of the polymer drug carrier (Hattori, Y. et al., J. Contorlled Rel., (2004) 97: 173-183) or pNIPAM (poly ( (Nayak, S. et al., J. Am. Chem. Soc., (2004) 126: 10258-10259) to target cells by attaching folate to hydrogel-type drug carriers such as Nisopropylacrylamide). Representative example.

The nanohybrid of the present invention is used as a gene therapy for treating various diseases associated with tumors, such as gastric cancer, lung cancer, breast cancer, ovarian cancer, liver cancer, bronchial cancer, nasopharyngeal cancer, laryngeal cancer, pancreatic cancer, bladder cancer, colon cancer and cervical cancer. Can be.

In tumor disease cells such as the above, they express and / or secrete certain substances which are produced little or no in normal cells, which are generally termed "tumor markers". Nanocomposites made by binding a substance capable of specifically binding such tumor markers to siRNA-layered inorganic hydroxides can be usefully used for the treatment of tumors. Various tumor markers are known in the art, as well as substances that can specifically bind to them. Preferably, the ligand may be a cell specific antibody, a cell selective peptide, a cell growth factor, folic acid, galactose, mannose, alzidi, transferrin, or the like.

In the present invention, the tumor markers may be classified into ligands, antigens, receptors, and nucleic acids encoding them according to a mechanism of action.

When the tumor marker is "ligand", a substance capable of specifically binding to the ligand may be introduced as a target-oriented multifunctional ligand component of the nanohybrid according to the present invention, which may specifically bind to the ligand. Any receptor or antibody would be suitable. Examples of ligands and receptors that can specifically bind to the present invention include synaptotagmin C2 (synaptotagmin C2), phosphatidylserine, annexin V, phosphatidylserine, integrin and Its receptor, Vascular Endothelial Growth Factor (VEGF) and its receptor, angiopoietin and Tie2 receptor, somatostatin and its receptor, vasointestinal peptide and its receptor It doesn't happen.

When the tumor marker is an "antigen", a substance capable of specifically binding to the antigen can be introduced as a target-oriented active ingredient of the nanohybrid according to the present invention, and an antibody capable of specifically binding to the antigen is suitable. something to do. Examples of antigens and antibodies that specifically bind to the present invention include carcinoembryonic antigens (colon cancer marker antigens), Herceptin (Genentech, USA), and HER2 / neu antigens (HER2 / neu antigens-breast cancer markers). Antigen) and Herceptin, prostate-specific membrane antigen (prostate cancer marker antigen) and rituxan (IDCE / Genentech, USA).

A representative example where the tumor marker is a "receptor" is a folic acid receptor expressed in ovarian cancer cells. A substance capable of specifically binding to the receptor (folic acid in the case of a folic acid receptor) may be introduced as a target-oriented multifunctional ligand of the nanohybrid according to the present invention, or a ligand capable of specifically binding to the receptor or Antibodies, preferably antibodies, will be suitable.

Herein, the antibody has a property of selectively and stably binding only to a specific target, -NH _{2 of} lysine, -SH of cysteine, -COOH of aspartic acid and glutamic acid in the Fc region of the antibody are the target-directed activity of the nanocomposite. This is because it can be usefully used to bind component region functional groups. Such antibodies can be obtained commercially or prepared according to methods known in the art. In general, a mammal (eg, mouse, rat, goat, rabbit, horse or sheep) is immunized one or more times with an appropriate amount of antigen. After a period of time when the titer reaches an appropriate level, it is recovered from the serum of the mammal. The recovered antibody can be purified using known processes if desired and stored in a frozen buffered solution until use. Details of this method are well known in the art.

On the other hand, "nucleic acid" includes RNA and DNA encoding the aforementioned ligand, antigen, receptor or part thereof. Nucleic acid having a specific base sequence can be detected using a nucleic acid having a base sequence complementary to the base sequence because the nucleic acid has a feature that forms a base pair between complementary sequences as known in the art . Nucleic acid having a nucleotide sequence complementary to the nucleic acid encoding the enzyme, ligand, antigen, receptor can be used as a target-oriented active ingredient of the nano-hybrid according to the present invention.

In addition, the nucleic acid has a functional group such as -NH ₂ , -SH, -COOH at the 5'- and 3'- terminal can be usefully used to bind to the functional group of the active ingredient. Such nucleic acids can be synthesized by standard methods known in the art, for example using automated DNA synthesizers (such as those available from BioSearch, Applied Biosystems, etc.). As an example, phosphorothioate oligonucleotides can be synthesized by the methods described in Stein et al. Nucl. Acids Res. 1988, vol. 16, p. 3209. Methylphosphonate oligonucleotides can be prepared using controlled free polymeric supports (Sarin et al. Proc. Natl. Acad. Sci. USA 1988, vol. 85, p.7448).

In the present invention, the nanoparticles preferably have a particle size of 10 to 350 nm, more preferably 50 to 200 nm. This allows the target-oriented nanohybrid to selectively be sensitized to the receptor of the multifunctional ligand, i.e., the tumor marker, and then to be effectively nested in the cell, and to prevent capillary clogging and physical impact on the cell when administered in vivo in the future. It is for. If the target-oriented nanohybrid is too small to reach 50 nm, it may enter the cell in a large amount and give a physical shock.

In the present invention, the siRNA content in the target-oriented siRNA-layered inorganic hydroxide nanohybrid may be 1 to 50% by weight.

In another aspect, the present invention provides a method for preparing a precipitated layered inorganic hydroxide, the method comprising: (a) dropping a base aqueous solution into an aqueous solution containing a divalent metal salt and a trivalent metal salt to prepare a precipitated layered inorganic hydroxide; (b) mixing and stirring the siRNA-containing solution with the solution containing the layered inorganic hydroxide prepared in step (a) to form a siRNA-layered inorganic hydroxide nano hybrid; And (c) binding a tumor marker specific multifunctional ligand to the hybrid to prepare a target-oriented siRNA-layered inorganic hydroxide nanohybrid, the preparation of a target-oriented siRNA-layered inorganic hydroxide nanocomposite It is about a method.

 In addition, after the step (c), (d) preparing a pharmaceutical composition for treating the tumor containing the nano- hybrid comprising the step of formulating by adding one or more pharmaceutically acceptable carrier to the nano- hybrid It is about a method.

In the present invention, the layered inorganic hydroxide of step (a) is represented by the following formula (2), can be easily prepared by the coprecipitation method

[Formula 2]

_{[M (Ⅱ) 1- x M} (Ⅲ) x (OH) 2] X + [A n -] X / n? yH ₂ O

Where M (II) represents a divalent metal cation, M (III) represents a trivalent metal cation, A is an anionic species, n is the number of charges of the anion, x is a number less than 0.1 to 0.5 and y is 0 Is more than positive.

In Formula 2, M (III) may optionally have a trivalent cation corresponding thereto, and may not be present at all. When M (II) ions and M (III) ions coexist as shown in Formula 3, the excess M (III) ions may interfere with the formation of a layered structure, so that M (III) ions It is preferable to exist below 50 mol%.

The ratio of the divalent metal cation to the trivalent metal cation may be adjusted to 2: 1, 3: 1 and 4: 1 to form a layered inorganic hydroxide having a controlled layer charge.

The divalent metal salt is a ^{Mg 2 +, Ca 2 +,} Co 2 +, Cu 2 +, Ni 2 +, Zn ^{2 +,} or the like, and a ^{_{cation, NO 3 -, Cl -,}} OH -, O 2-, SO ₄ ^{2 -,} CO ₃ ^{2 -} or a salt compound having succinate as an anion may be used, but is not limited thereto. The trivalent metal salt is a ³⁺ Al, Cr ^{+ 3,} Fe ^{+ 3,} Ga ^{+ 3,} In ^{+ 3,} V ^{+ 3,} or Ti ^{+ 3} and so on to a cation, and ^{NO 3 -, Cl -, OH} -, O ^2-, SO ₄ ^{2 -,} CO ₃ ^{2 -} or a salt compound having succinate as an anion may be used, but is not limited thereto. In the case of the Mg metal salt, Mg (NO ₃ ) ₂ , MgCl ₂ , MgSO ₄ , or a hydrate thereof may be used, and in the case of Al metal salt, Al (OH) ₃ , Al (NO ₃ ) ₃ , Al ₂ (SO ₄ ) ₃ , or a hydrate thereof can be used. The divalent metal cation, the trivalent metal cation, and the anion are not limited to the above types, and may include all of those known as layered inorganic hydroxides in the art.

Coprecipitation can add a base to induce precipitation. Suitable bases can be used, for example, sodium hydroxide, potassium hydroxide, magnesium hydroxide, calcium hydroxide or ammonia. The pH of the reaction solution is 5 to 12, preferably 6 to 10, and the reaction temperature is 0 ° C to 100 ° C, preferably 15 ° C to 30 ° C. The reaction time is preferably 10 minutes or more. In addition, during the reaction, it is preferable to continuously add nitrogen or an inert gas to react.

In the manufacturing process, the layered inorganic hydroxide may be prepared from various synthetic factors such as i) the temperature of the reaction solution, ii) the concentration of the reaction solution, iii) the mixing ratio between the metal cations, iv) the temperature of the wash water, and v) the drying temperature. It can represent particle size and shape.

In the present invention, in the step (b), siRNA-layered inorganic hydroxide nano-hybrid manufacturing process, the co-precipitation method of forming a nano-hybrid by co-precipitating the siRNA-containing solution together with the preparation of the layered inorganic hydroxide ( co-precipitation method) The ion-exchange method was prepared in which a layered inorganic hydroxide was formed, followed by mixing and stirring the siRNA-containing solution and introducing the interlayer of the layered inorganic hydroxide by ion exchange to form nano hybrids. Calcination-reconstruction method of calcination of the layered inorganic hydroxide followed by reconstitution by addition of siRNA-containing solution to form a nano hybrid, or exfoliation of the prepared layered inorganic hydroxide in the form of a sheet. Exfoliation, followed by the addition of siRNA-containing solutions and recombination to form nanohybrids. reassembling method).

In the method of preparing a nano hybrid according to the present invention by the co-precipitation method, preparing a solution of siRNA and a divalent / 3 valent metal salt and adding a base to the solution to titrate the pH to 6 to 10 to obtain a precipitate. It includes.

In the method for preparing a nanocomposite according to the present invention by the ion exchange method, a step of preparing a solution of a divalent / 3 valent metal salt is added to a base of the solution of the divalent / 3 valent metal salt, and the pH is adjusted to 6 to 10 to obtain a layer. Forming an epitaxial inorganic hydroxide precipitate and introducing an siRNA-containing solution to the formed precipitate to introduce siRNA between layers of the precipitate by ion exchange with anions present between the layers of the precipitate.

In the method for preparing a nanocomposite according to the present invention by the calcination-reconstruction method, preparing a solution of a divalent / 3 valent metal salt by adding a base to the solution of the divalent / 3 valent metal salt, the pH is adjusted to 6 to 10. Forming a layered inorganic hydroxide precipitate by heat treatment at a temperature within 250 to 500 ℃ for at least 1 hour, preferably at 400 ℃ for 4 hours to remove the interlayer anion of the formed precipitate and siRNA Adding to the calcined precipitate to the containing solution and reconstituting by stirring.

In the method for preparing a nano hybrid according to the present invention by the exfoliation-recombination method, preparing a solution of a divalent / 3 valent metal salt by adding a base to the solution of the divalent / 3 valent metal salt, the pH is adjusted to 6 to 10. Titration to form a layered inorganic hydroxide precipitate by replacing the interlayer anions of the formed precipitate with anionic ions of a long carbon chain or exfoliating the layered inorganic hydroxide into a single sheet of sheet using a specific solvent, preferably Dispersing in a formamide solution at 0.05% by weight and stirring for 2 days to exfoliate, and adding siRNA-containing solution to the exfoliated colloidal solution and stirring to recombine.

In the above production method, the solvent for preparing the divalent / 3 valent metal salt solution, base solution or siRNA containing solution is not particularly limited as long as it is a solvent capable of dissolving both the siRNA and the metal salt without being involved in the reaction. For example, a mixed solvent of distilled water, ethanol, distilled water and ethanol may be used.

In the preparation method, the reaction between the layered inorganic hydroxide and siRNA is not particularly limited, and can be generally performed at room temperature, preferably at a siRNA denaturation temperature, and can be obtained by reacting for about 10 minutes to 7 days. have. The addition ratio of each of the reactants required for the reaction is not particularly limited, and may be added in a molar ratio of siRNA: layered inorganic hydroxide (%) from 10:90 to 90:10, and thus, the layered inorganic hydroxide of siRNA. The rate of introduction can be controlled.

In addition, after the siRNA is delivered into tumor cells, in the presence of a metal cation decomposed from the layered inorganic hydroxide, the interaction of the phosphate group and the metal cation of the double stranded siRNA may negatively affect the activity of the siRNA in insoluble form. (Duguid J et al. , Biophys . J. 65: 1916-1928, 1993). Ethylene diamine tetra acetic acid (EDTA), which strongly forms a chelate bond with a divalent / trivalent metal cation, was added together with siRNA in the preparation of the siRNA-layered inorganic hydroxide nano hybrid (b) to form a nano hybrid. When used in tumor therapy, it may play a role in increasing siRNA activity by reducing interactions with metal cations, which are degradation products of layered metal hydroxides, after being introduced into cells, in weakly acidic conditions.

In another aspect, the present invention relates to a pharmaceutical composition for tumor treatment containing the target-oriented siRNA-layered inorganic hydroxide nano hybrid as an active ingredient.

In the present invention, the pharmaceutical composition may include a pharmaceutically acceptable carrier or vehicle which is commonly used.

In the present invention, the tumor is not limited thereto, but may be breast cancer, lung cancer, oral cancer, pancreatic cancer, colon cancer, prostate cancer, ovarian cancer, or the like, and for example, oral cancer or lung cancer, as shown in the Examples.

Pharmaceutically acceptable carriers that can be used in the pharmaceutical composition for treating tumors of the present invention include, but are not limited to, ion exchange resins, alumina, aluminum stearate, lecithin, serum proteins (eg, human serum albumin), buffer substances (E.g. various phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids), water, salts or electrolytes (e.g. protamine sulfate, disodium hydrogen phosphate, carbohydrogen phosphate, sodium chloride and zinc salts) , Colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose based substrates, polyethylene glycol, sodium carboxymethylcellulose, polyarylates, waxes, polyethylene glycols and wool, and the like. The pharmaceutical composition for treating tumors of the present invention may further include a lubricant, a humectant, an emulsifier, a suspending agent, a preservative, and the like, in addition to the above components.

The nanohybrid-containing pharmaceutical compositions of the present invention can be formulated into suitable formulations using known techniques for clinical administration. For example, during oral administration, it may be mixed with an inert diluent or an edible carrier, sealed in hard or soft gelatin capsules, or pressed into tablets. For oral administration, the active compounds can be mixed with excipients and used in the form of intake tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers and the like. In addition, various formulations, such as for injection and parenteral administration, can be prepared according to techniques known in the art or commonly used techniques.

The nanocomposite-containing tumor therapeutic pharmaceutical composition of the present invention may be prepared by additionally containing one or more pharmaceutically acceptable carriers in addition to the above-described active ingredients for administration. Pharmaceutically acceptable carriers should be compatible with the active ingredients of the present invention and may be used in combination with saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol and one or more of these components. Other conventional additives such as antioxidants, buffers, bacteriostatics, etc. may be added as necessary. In addition, diluents, dispersants, surfactants, binders and lubricants may be additionally added to formulate injectable formulations such as aqueous solutions, suspensions, emulsions and the like. Furthermore, it may be preferably formulated according to each disease or component by a suitable method in the art or using a method disclosed in Remington's Pharmaceutical Science (Recent Edition), Mack Publishing Company, Easton PA.

The pharmaceutical composition for the treatment of a nano hybrid containing tumor according to the present invention may be administered through a route commonly used in the pharmaceutical field, and parenteral administration is preferred, for example, oral, intravenous, intramuscular, intraarterial, Administration may be by intramedullary, intradural, intracardiac, transdermal, subcutaneous, intraperitoneal, intestinal, sublingual or topical routes of administration.

In one embodiment, the nanocomposite-containing tumor therapy pharmaceutical composition according to the present invention can be prepared in an aqueous solution for parenteral administration. Preferably, buffer solutions such as Hanks' solution, Ringer's solution, or physically buffered saline may be used. Aqueous injection suspensions can be added with a substrate that can increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol or dextran.

In the present invention, the formulation may be selected from the group consisting of tablets, capsules, solutions, injections, ointments and syrups, in the case of formulations of the injections, it may be in the form of solutions, suspensions or emulsions Can be.

Preferred nano hybrid-containing tumor therapeutic pharmaceutical compositions of the present invention may be in the form of sterile injectable preparations as sterile injectable aqueous or oily suspensions. Such suspensions can be formulated according to techniques known in the art using suitable dispersing or wetting agents (eg Tween 80) and suspending agents. Sterile injectable preparations may also be sterile injectable solutions or suspensions (eg solutions in 1,3-butanediol) in nontoxic parenterally acceptable diluents or solvents. Vehicles and solvents that may be used include mannitol, water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile, nonvolatile oils are conventionally employed as a solvent or suspending medium. For this purpose any non-irritating non-volatile oil can be used including synthetic mono or diglycerides.

Alternatively, in addition to the final preparation for injection or infusion, it may be in the form of a lyophilized or sterile powder, which may be mixed with a solvent, for example water, immediately prior to administration to produce the final preparation for injection or infusion.

The nanocomposite-containing oncology pharmaceutical compositions of the present invention can be determined by one of ordinary skill in the art based on common patient symptoms and disease severity. It may also be formulated in various forms, such as powders, tablets, capsules, solutions, injections, ointments, syrups, and the like, and may also be provided in unit-dose or multi-dose containers, such as sealed ampoules and bottles. .

The dosage of the nano hybrid-containing pharmaceutical composition of the present invention varies depending on the weight, age, sex, health condition, diet, time of administration, administration method, excretion rate and severity of the disease, etc. of the patient, A person of ordinary skill can easily decide.

In the present invention, the dosage of siRNA-layered inorganic hydroxide nanocomposites capable of complementary binding to a gene encoding survivin is weight kg, depending on the age, sex, symptoms, administration method or prophylaxis of the patient. Parenteral administration may be from 0.05 to 0.1 μg on the basis of sugar siRNA. Dosage levels for patients with specific symptoms may vary by those skilled in the art depending on the patient's weight, age, sex, health condition, diet, time of administration, method of administration, and the like.

Hereinafter, the present invention will be described in more detail by way of examples. These examples are intended to illustrate the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited to these examples.

Example 1: Preparation of Targeted siRNA-Layered Inorganic Hydroxide Nanocomposites

1-1. NO ₃ -Layered inorganic hydroxide manufacture

_{?? Mg (NO 3) 2} H 2 O (0.2 M) and _{Al (NO 3) 3 H 2} O (0.1 M) a carbonate ion (CO ₃ ^{2 -)} has been removed was dissolved in distilled water, NaOH solution (1 The mixture was titrated at a pH of 9 to 10 to obtain a layered inorganic hydroxide crystal formed by precipitation. The layered inorganic hydroxide crystals were stirred at 100 ° C. for 16 hours, washed to remove unreacted salt, and then lyophilized to obtain a NO ₃ -layered inorganic hydroxide.

1-2. Preparation of siRNA-Layered Inorganic Hydroxide Nanocomposites

The layered inorganic hydroxide obtained in 1-1 was redispersed in distilled water, and siRNA (Bionia, KR, SEQ ID NO: 1) capable of complementarily binding to a gene encoding survivin in this dispersion solution was dissolved in distilled water. The solution was added (siRNA: layered inorganic hydroxide = 3: 1 weight ratio), stirred at 37 ° C for 2 days, washed to remove unreacted siRNA, and then the nanohybrid was inserted between layers of layered inorganic hydroxide. A sieve was obtained. The nano hybrid manufacturing process was performed under a nitrogen atmosphere in order to prevent the production of carbonate ions (CO ₃ ^2- ) by carbon dioxide in the air.

1-3. Preparation of Targeted SiRNA-Layered Inorganic Hydroxide Nanocomposites

In order to prepare a target-oriented siRNA-layered inorganic hydroxide nano hybrid in which a folic acid polyfunctional ligand is bound as an active ingredient, the siRNA-layered inorganic hydroxide prepared in the above 1-2 is first used as an aminosilane-attached siRNA-layered type. Inorganic hydroxide nano hybrids were synthesized. The siRNA-layered inorganic hydroxide was redispersed in ethanol and dried to evaporate the surface water, and then added to the toluene solution in which the amino propyl silane was dissolved, stirred at 60 ° C. for 6 hours, and washed, whereby the aminosilane was bound to the attachment region. siRNA-layered inorganic hydroxide nano hybrids were obtained. Aqueous redispersed solution of siRNA-layered inorganic hydroxide nanocomposite to which the aminosilane is bound, 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide (EDC), N-hydroxysuccinate, which is a reaction catalyst An aqueous solution in which imide (NHS) and triethylamine (ET ₃ N) were dissolved, and a solution in which folic acid (folate) was dissolved in dimethyl sulfoxide (DMSO) were prepared. To the siRNA-layered inorganic hydroxide nano hybrid dispersion, 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide aqueous solution and N-hydroxysuccinimide aqueous solution were added, followed by addition of folic acid solution, and then The pH is titrated to 9 using aqueous ethylamine solution. The reaction was stirred at 38 ° C. for 5 hours, washed with dimethylsulfoxide, distilled water and lyophilized to obtain a target-oriented siRNA-layered inorganic hydroxide nanohybrid with a folic acid polyfunctional ligand.

The surface reaction of the preparation of the target-oriented siRNA-layered inorganic hydroxide nanocomposite is primarily a surface-modified M (metal) -OH bond of the layered inorganic hydroxide, which is an adhesion region, and is secondary In the cross-linking region, the terminal amine reacts with the carboxyl group of folic acid to form MO-Si-peptide-folic acid, and the active component region is the terminal region sensitive to the folate receptor.

X-ray diffraction (Rigaku, D / Max 2200) analysis was performed to confirm the crystal structure of the nanocomposite prepared in Example 1. As a result, as shown in FIG. 2, the interlayer spacing of the layered inorganic hydroxide was about 7.9 Å and the typical lamellae structure into which nitrate anions were inserted. The interlayer spacing of the siRNA-layered inorganic hydroxide was about 25 Å. The anion was ion exchanged with siRNA and the siRNA was intercalated into the layer in a structure parallel to the hydroxide layer and expanded about 20 mm, resulting in a siRNA-layered inorganic hydroxide nanocomposite having a two-dimensional structure. The target-oriented siRNA-layered inorganic hydroxide also maintains the interlayer spacing even after the multi-functional ligand binding reaction, it can be seen that the target-oriented siRNA-layered inorganic hydroxide nanocomposite of the form inserted between the siRNA layers.

Transmission electron microscopy (JEOL JEM-2100F) analysis was performed to confirm the shape and particle size of the target-oriented siRNA-layered inorganic hydroxide nano hybrid prepared in Example 1. As a result, as shown in Figure 3, it can be seen that the target-oriented siRNA-layered inorganic hydroxide nano hybrids were made of hexagonal nanoparticles having an average size of 100 ± 20 nm.

Experimental Example 1 Analysis of Stability in Serum of Target-oriented siRNA-Layered Inorganic Hydroxide Nanocomposites

Serum stability of the target-oriented siRNA-layered inorganic hydroxide nano hybrids prepared in Example 1 was investigated.

A concentration of 10 µg was added to 90 µl of the stability reaction solution (including 10% of rat serum, Invitrogen) based on siRNA, and left at 37 ° C. After a predetermined time (0, 0.5, 1, 2, 4, 6, 8, 10, 12 and 24 hours), dispense 12 μl each, immediately freeze at -70 ° C, and store for each hour 2.5 μl of the sample was subjected to gel electrophoresis (1% agarose gel) in Tris-Acetate (TAE) buffer to confirm whether siRNA was maintained in serum. Pure siRNA was used as a comparative group.

As a result, as shown in Fig. 4, pure siRNA was almost completely degraded after 8 hours of incubation in the reaction solution containing serum (Fig. 4 (a)). In contrast, no target-oriented siRNA-layered inorganic hydroxide nanohybrids were detected at all after 24 hours of nuclease-mediated siRNA degradation (Fig. 4 (b)). Sustained stabilization of the siRNA in the serum is characterized by the intercalation of the siRNA intercalated into the layered inorganic hydroxide with the cationic layer charge of the layered inorganic hydroxide and the anionic phosphate group of the siRNA with strong electrostatic attraction. This is believed to be achieved because it structurally masks access to the inner core of the sieve.

Example 2 In-Vitro Efficacy Test of Targeted siRNA-Layered Inorganic Hydroxide Nanocomposites

2-1. Tumor cell line culture and suppression of survivin expression in cell line

Human oral cancer cell line (KB, Korea Cell Line Bank), a tumor cell line, was used after incubation at 37 ° C. and CO ₂ conditions for at least 2 weeks in a folate-free medium to induce its maximum expression as cells overexpressing folate receptors. .

The KB cells RPMI 1640 medium (Welgene, KR) for each 1 x 10 ⁵ / seeded into 2ml 37 ℃, a back, Example 1 siRNA- layered inorganic hydroxide nano hybrid material, the target prepared from the culture in a CO ₂ incubator The directional siRNA-layered inorganic hydroxide nanohybrids were treated with cells at a concentration of 100 nM based on siRNA and incubated in a 37 ° C., CO ₂ incubator. After 6 hours, washed twice with RPMI 1640 medium, and then replaced with fresh RPMI 1640 medium and further incubated for 24 hours to suppress the expression of survivin in 37 ℃, CO ₂ incubator.

The cells treated with no treatment in the comparison group, the NO ₃ -layered inorganic hydroxide as the control group, and the cells treated with the siRNA-layered inorganic hydroxide nano hybrids were used as the experimental group. In addition, in order to confirm tumor specific cellular internalization by folic acid, which is a target-oriented ligand, the cells treated with the target-oriented siRNA-layered inorganic hydroxide nanohybrid after incubating the culture solution containing folic acid (1 mg / ml) for 24 hours. Was used.

Target oriented siRNA-layered inorganic hydroxide nanohybrids according to the present invention are permeated into cells through receptor-mediated tumor cells in which tumor markers, which are overexpressed as tumor markers (receptor-mediated endocytosis), do not express tumor markers. In tumor cells, both siRNA-layered inorganic hydroxide nanohybrids or targeted oriented siRNA-layered inorganic hydroxide nanohybrids are internalized by a mechanism called clathrin-mediated endocytosis to show tumor therapeutic effects. Judging. In addition, it can be seen that a large amount of nanohybrids are selectively introduced into cells in the case of using a nanocomposite to which a target-directed multifunctional ligand is attached. These results suggest that targeted oriented inorganic hydroxides can serve as tumor cell specific siRNA delivery mediators.

2-2. RT - PCR On by Survival mRNA Quantitative Analysis of

To determine whether tumor cell suppression actually results from a decrease in the amount of intracellular survivin mRNA, the RNA extraction kit (RNeasy mini kit, Qiagen, Germany) was used to monitor the concentration of survivin mRNA in total RNA. It was quantified by the following method by -PCR (Real-time PCR) analysis.

That is, 1 μg of total RNA of each sample was mixed with 1 μl of oligo-dT18 (500 ng / μl) and 2 μl of dNTP (2.5 mM each), reacted at 70 ° C. for 10 minutes, and then cooled on ice for 5 minutes, 0.5 μl reverse superscriptase (200 U / μl), Invitrogen), 2 μl 10 × reaction buffer, 0.5 μl RNase inhibitor and an appropriate amount of sterile water were mixed to adjust the total volume to 20 μl, followed by 42 ° C. Reaction was carried out for 15 minutes at, and 5 minutes at 95 ℃, 5 minutes at 4 ℃ to give each cDNA. Then, 1 μl of each cDNA, 10 μl of 2X SYBR Green Master Mix from RT-PCR system (Applied Biosystems Prism 7900 Sequence Detection System, Applied Biosystems, USA), each of the forward primers specific for survivin and GAPDH (10 pM). ) 0.4 μl and reverse primer (10 pM) 0.4 μl were mixed, followed by RT-PCR (2 minutes at 50 ° C., 10 minutes at 95 ° C., 30 seconds at 95 ° C., 30 seconds at 60 ° C., 72 ° C.). 40 repetitions of 30 seconds). In this case, the sequence of the forwarding and reverse primers used is as follows:

Survivin specific forward primer: 5'-CCTTCACATCTGTCACGTTCTCC-3 '(SEQ ID NO: 10)

Survivin specific reverse primer: 5'-ATCATCTTACGCCAGACTTCAGC-3 '(SEQ ID NO: 11)

GAPDH specific forward primer: 5'-GGTGAAGGTCGGAGTCAACG-3 '(SEQ ID NO: 12)

GAPDH specific reverse primer: 5'-ACCATGTAGTTGAGGTCAATGAAGG-3 '(SEQ ID NO: 13)

After the completion of PCR, using the cDNA standard curve, the amount of each obtained survivin PCR product and the amount of GAPDH PCR product were measured, and the measured value of survivin was divided by the measured value of GAPDH and survived. The relative empty expression level was calculated, and the expression reduction rate of survivin mRNA was compared.

The results are shown in FIG. 5, in the case of treatment of siRNA-layered inorganic hydroxide nanohybrids in KB cells, 40% survivin expression reduction, and treatment of target-oriented siRNA-layered inorganic hydroxide nanohybrids 60% reduced survivin expression.

Through this, the siRNA-layered inorganic hydroxide nano hybrid and the target-oriented siRNA-layered inorganic hydroxide nano hybrid of the present invention not only reduce the mRNA level of survivin, but also decrease the expression of survivin tumor cells. It could be confirmed that it can directly induce the growth inhibition of.

Targeted siRNA-layered inorganic hydroxide nano hybrids according to the present invention increased the in vivo stability of siRNA, the target-directed multi-functional ligand that can specifically bind tumor markers improve tumor specific delivery efficiency It has been shown that it can be used as a composition to improve the efficiency and accuracy of the treatment of various diseases by showing the tumor therapeutic activity of siRNA at relatively low concentrations. The new type of siRNA delivery system is a basic research and medical industry for biotechnology. It has been found that the invention is a useful invention that can be used very usefully.

As described above in detail the present invention, it will be apparent to those skilled in the art that such specific descriptions are merely preferred embodiments, and thus the scope of the present invention is not limited thereto. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

1 is a schematic diagram of a reaction for preparing a target-oriented siRNA-layered inorganic hydroxide nanohybrid.

2 is an X-ray diffraction diagram of a target-oriented siRNA-layered inorganic hydroxide nanohybrid. ((a): NO ₃ -layered inorganic hydroxide, (b): siRNA-layered inorganic hydroxide, (c): target oriented siRNA-layered inorganic hydroxide)

3 is a transmission electron microscope image of a target-oriented siRNA-layered inorganic hydroxide nanohybrid.

Figure 4 is an electrophoretic image showing the degree of degradation of siRNA over time in the presence of serum proteins for the evaluation of blood stability of siRNA and target-oriented siRNA-layered inorganic hydroxide nanohybrids. ((a): pure siRNA, (b): target oriented siRNA-layered inorganic hydroxide)

FIG. 5 is a graph showing mRNA levels of target-directed siRNA-layered inorganic hydroxide nanohybrids inhibiting the expression of survivin in tumor cells. (a): comparative group, (b): NO ₃ -layered inorganic hydroxide, (c) siRNA-layered inorganic hydroxide, (d): target oriented siRNA-layered inorganic hydroxide, folic acid containing medium, (e) : Target-oriented siRNA-layered inorganic hydroxide, medium without folic acid)

<110> Nanohybrid <120> Active siRNA-Inorganic Layered Double Hydroxide Nanohybrid, a          Process for Preparing the Same and a Pharmaceutical Composition          Comprising the Same for Treating Targeted Tumor <130> P09-B100 <160> 13 <170> KopatentIn 1.71 <210> 1 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> siRNA derived from survivin <400> 1 aaggagauca acauuuuca 19 <210> 2 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> siRNA derived from survivin <400> 2 uaggaaagga gaucaacau 19 <210> 3 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> siRNA derived from survivin <400> 3 aggaaaggag aucaacauu 19 <210> 4 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> siRNA derived from survivin <400> 4 aggaaaggag aucaacauu 19 <210> 5 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> siRNA derived from survivin <400> 5 ggaaaggaga ucaacauuu 19 <210> 6 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> siRNA derived from survivin <400> 6 gaaaggagau caacauuuu 19 <210> 7 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> siRNA derived from survivin <400> 7 aaaggagauc aacauuuuc 19 <210> 8 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> siRNA derived from survivin <400> 8 aggagaucaa cauuuucaa 19 <210> 9 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> siRNA derived from survivin <400> 9 ggagaucaac auuuucaaa 19 <210> 10 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> survivin specific forwarding primer <400> 10 ccttcacatc tgtcacgttc tcc 23 <210> 11 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> survivin specific reverse primer <400> 11 atcatcttac gccagacttc agc 23 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GAPDH specific forwarding primer <400> 12 ggtgaaggtc ggagtcaacg 20 <210> 13 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> GAPDH specific reverse primer <400> 13 accatgtagt tgaggtcaat gaagg 25  

Claims (21)

Target-oriented siRNA-layered inorganic hydroxide nano hybrid represented by the following formula (1): [Formula 1] [M (II) _1-x M (III) _x (OH) ₂ ] ^{X +} [S] [T] In Formula 1, M (II) represents a divalent metal cation, M (III) represents a trivalent metal cation, x is a number less than 0.1 to 0.5, S is siRNA, T is aminosilane, epoxysilane , vinylsilane, -COOH, -NH ₂ , -SH, -CONH ₂ , -PO ₃ H, -PO ₄ H, -SO ₃ H, -SO ₄ H and -OH An attachment region attached by surface modification of the hydroxide; And at least one selected from the group consisting of phosphatidylserine, integrin receptor, Tie2 receptor, somatostatin receptor, vasointestinal peptide receptor, Herceptin, rituxan and folic acid so as to cross-link with a portion of the adhesion region and the other side to be combined with a tumor marker. Tumor-targeted multifunctional ligand comprising an active ingredient region containing. The target oriented siRNA-layered inorganic hydroxide nanohybrid according to claim 1, wherein the siRNA is derived from a survivin gene. The target-oriented siRNA-layered inorganic hydroxide nanohybrid according to claim 1, wherein the siRNA is represented by any one nucleotide sequence selected from the group consisting of SEQ ID NOs: 1 to 9. The method of claim 1, wherein the divalent metal cation is selected from the group consisting of Mg ^{2 +} , Ca ^{2 +} , Co ^{2 +} , Cu ^{2 +} , Ni ^{2 +} and Zn ^{2 +} , wherein the trivalent metal cation is Al ^{3 +,} Cr ^{+ 3,} Fe ^{+ 3,} Ga ^{+ 3,} in ^{+ 3,} V ^3+, and targeted siRNA- layered inorganic hydroxide nano hybrid material, characterized in that is selected from the group consisting of Ti ^{3 +.} The method of claim 1, wherein the tumor-targeting multifunctional ligand is an antigen, an antibody, RNA, DNA, hapten, avidin, streptavidin, neutravidin, protein A, protein G , Target-directed siRNA-layered type, characterized in that it can specifically bind with one selected from the group consisting of lectins, selectins, radioisotopes labeled biomaterials, and tumor receptors Inorganic hydroxide nano hybrids. The target-oriented siRNA-layered inorganic hydroxide nanohybrid according to claim 5, wherein the tumor receptor is selected from the group consisting of ligands, antigens, receptors, and nucleic acids encoding them. The method of claim 6, wherein the tumor receptor is C2, annexin V, integrin, VEGF, angiopoietin 1, angiopoietin 2, somatostatin, vasointestinal peptide, cancerous fetal antigen, Targeted siRNA-layered inorganic hydroxide nanohybrid, characterized in that it is selected from the group consisting of HER2 / neu antigen, prostate specific antigen and folic acid receptor. delete A pharmaceutical composition for the treatment of tumors comprising the siRNA-layered inorganic hydroxide nano hybrid of any one of claims 1 to 7 and a pharmaceutically acceptable carrier. 10. The carrier of claim 9 wherein the carrier is ion exchange resin, alumina, aluminum stearate, lecithin, serum protein, buffer material, water, salt, electrolyte, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose based substrates. , Polyethylene glycol, sodium carboxymethyl cellulose, polyarylate, wax, polyethylene glycol and wool, the pharmaceutical composition for tumor treatment, characterized in that at least one selected from the group consisting of. The method of claim 9 further comprising an additive selected from the group consisting of excipients, disintegrants, binders, lubricant suspending agents, surfactants, sweeteners, preservatives, lubricants, flavoring agents, thickeners, pH adjusting agents, wetting agents and mixtures thereof. Pharmaceutical composition for the treatment of tumors, characterized in that it comprises a. 10. The pharmaceutical composition of claim 9, wherein the formulation is selected from the group consisting of tablets, capsules, solutions, injections, ointments and syrups. 10. The pharmaceutical composition for treating tumors according to claim 9, wherein the formulation is an injection in the form of a liquid, suspension or emulsion. 10. The tumor treatment of claim 9, formulated for intravenous, intraperitoneal, intramuscular, intraarterial, oral, intracardiac, intramedullary, intradural, transdermal, intestinal, subcutaneous, sublingual or topical administration. Pharmaceutical composition for. The pharmaceutical composition of claim 9, formulated in unit-dose or multi-dose unit preparations. 10. The pharmaceutical composition for treating tumors according to claim 9, which contains 0.05 to 0.1 µg siRNA per kg of body weight to be treated. The pharmaceutical composition for treating tumors according to claim 9, wherein the tumor is oral cancer or lung cancer. A process for preparing the target-oriented siRNA-layered inorganic hydroxide nanocomposite of claim 1, comprising the following steps: (a) adding a base aqueous solution to an aqueous solution containing a divalent metal salt and a trivalent metal salt to prepare a precipitated layered inorganic hydroxide; (b) mixing and stirring the siRNA-containing solution with the solution containing the layered inorganic hydroxide prepared in step (a) to form a siRNA-layered inorganic hydroxide nano hybrid; (c) modifying the hybrid to form a region of attachment to the hybrid; And (d) target-directed attachment of a tumor-targeting multifunctional ligand to the hybrid by reacting the modified hybrid with a tumor specific binding component to form an active ingredient region cross-linked with the attachment region of the modified hybrid. Preparation of siRNA-Layered Inorganic Hydroxide Nanocomposites. The method of claim 18, wherein the layered inorganic hydroxide of step (a) is represented by the following formula (2): [Formula 2] _{[M (Ⅱ) 1- x M} (Ⅲ) x (OH) 2] X + [A n -]? X / n yH 2 O Where M (II) represents a divalent metal cation M (III) represents a trivalent metal cation A is an anionic species, n is the number of charges of the anion x is a number from 0.1 to less than 0.5 and y is a positive number greater than zero. The method of claim 19, wherein the divalent metal cation is selected from the group consisting of Mg ^{2 +} , Ca ^{2 +} , Co ^{2 +} , Cu ^{2 +} , Ni ^{2 +} and Zn ^{2 +} , wherein the trivalent metal cation is Al ^{3. +} , Cr ^{3 +} , Fe ^{3 +} , Ga ^{3 +} , In ³⁺ , V ^{3 +} , and Ti ^{3 +} , the anion is selected from CO ₃ ^{2 −} , NO ^{3 −} , Cl ⁻ , OH ⁻ , O ^2-, and SO ₄ ^{2 -} and wherein is selected from the group consisting of. A method for preparing a pharmaceutical composition for treating tumors, comprising the siRNA-layered inorganic hydroxide nanohybrid of claim 9, comprising the following steps: (a) adding a base aqueous solution to an aqueous solution containing a divalent metal salt and a trivalent metal salt to prepare a precipitated layered inorganic hydroxide; (b) mixing and stirring the siRNA-containing solution with the solution containing the layered inorganic hydroxide prepared in step (a) to form a siRNA-layered inorganic hydroxide nano hybrid; (c) binding a tumor marker specific multifunctional ligand to the hybrid to prepare a target-oriented siRNA-layered inorganic hydroxide nanohybrid; And (d) formulating the nano hybrid with a pharmaceutically acceptable carrier.

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