KR101495298B1 - AMPHIPHILIC BLOCK COPOLYMER CONTAINING siRNA AS A HYDROPHILIC BLOCK AND POLYION COMPLEX CONTAINING THE SAME - Google Patents

Abstract

The present invention relates to an amphiphilic block copolymer having a nucleic acid molecule as a hydrophilic block, a process for its preparation, a multi-ion complex comprising the amphiphilic block copolymer and a cationic carrier, a magnetic assembly comprising the multi- To a nucleic acid delivery composition. More specifically, the nucleic acid molecules are converted into hydrophilic blocks to increase the charge density and flexibility, thereby forming a multi-ion complex more effectively. By introducing a hydrophobic block, a cationic carrier and a denser and more stable multi- Thereby increasing the efficiency of delivery into cells.

Description

TECHNICAL FIELD The present invention relates to an amphiphilic block copolymer having a small interfering ribonucleic acid (siRNA) as a hydrophilic block and a multi-ion complex comprising the same. BACKGROUND ART [0002]

The present invention relates to an amphiphilic block copolymer having a nucleic acid molecule as a hydrophilic block and a multi-ion complex comprising the amphiphilic block copolymer.

Small interfering ribonucleic acid (siRNA) is a sense / antisense double-stranded ribonucleic acid consisting of 19 to 25 nucleotides. (Messenger RNA: mRNA) of a target gene having a nucleotide sequence complementary to the antisense strand, and functions to inhibit gene expression by degrading messenger ribonucleic acid.

Since the small interfering ribonucleic acid has a high specificity for selectively suppressing the expression of a specific gene and a high activity for effectively controlling the gene expression even in a small amount, a great deal of attention has been paid to a novel therapeutic agent capable of overcoming the limitations of conventional drugs It is getting. Knowing the nucleotide sequence of a specific gene, the small interfering ribonucleic acid can ultimately target all genes, so its potential is enormous. For this reason, many research institutes, pharmaceutical companies and biotechnology companies are investing heavily in the development of therapeutic agents using small interfering ribonucleic acid.

Despite these possibilities, however, there are still many problems to be solved until small interfering ribonucleic acid is applied to clinical practice. Since the small interfering ribonucleic acid is a macromolecule with a negative charge, it is not only very low in cell membrane permeability, but also the small interfering ribonucleic acid is easily degraded by the degrading enzyme in the blood. Due to these properties, delivery of small interfering ribonucleic acid to desired tissues or cells without side effects is the biggest obstacle for therapeutic agents.

In order to overcome this problem, anionic small interfering ribonucleic acid forms a cationic transporter and polyion complex to protect small interfering ribonucleic acid from degradation enzymes, Is increasingly used. The size of the multi-ion complex of recently reported small interfering ribonucleic acid is 400-500 nanometers (M. Breunig, C. Hozsa, U. Lungwitz, K. Watanabe, I. Umeda, H. Kato, A. Goepferich, Release, 130 (2008) 57-63), 350-450 nanometers (KA Howard, SR Paludan, MA Behlke, F. Besenbacher, B. Deleuran, J. Kjems, Mol Ther, 17 It is very difficult to find a structure having a stable structure with a diameter of 100 nm or less. This is because the low anion charge density and the long persistence length of the small interfering ribonucleic acid make it difficult to effectively form a multi-ion complex with the cationic carrier and thus the size of the complex is relatively large. In addition, there is a problem that the structure is very unstable in an in vivo condition in which a large number of ions exist depending on electrostatic attraction alone (P. Kebbekus, DE Draper, P. Hagerman, Biochemistry, 34 (1995) 4354-4357; H Mok, TG Park, Biopolymers, 89 (2008) 881-888).

To solve this problem, it is possible to form a more stable complex by increasing the molecular weight of the cationic carrier or increasing the charge density of the positive charge, but the toxicity of the cationic carrier is often a problem (HT Lv, SB Zhang, B. Wang, SH Cui, J. Yan, J Control Release, 114 (2006) 100-109). Therefore, there is a urgent need for a new method for increasing the stability of the nanostructure without causing toxicity, thereby enhancing the efficiency of ribonucleic acid delivery.

The size and stability of multi-ion complexes are very important parameters for in vivo delivery of small interfering ribonucleic acid. However, due to the inherent limitations of the small interfering ribonucleic acid itself described above, it is very difficult to find an improvement method.

Recently, in addition to studies for improving the structure of the cationic transporter, studies are underway to modify the structure of the small interfering ribonucleic acid itself to improve the delivery efficiency. Sticky small interfering ribonucleic acid (AL Bolcato-Bellemin, ME Bonnet, G. Creusatt, P. Erbacher, JP Behr, P Natl Acad) prepared by adding an extra complementary nucleic acid base to the end of small interfering ribonucleic acid Sci. USA, 104 (2007) 16050-16055), a small interfering ribonucleic acid multimeric siRNA in which a small interfering ribonucleic acid is introduced by introducing a disulfide bond, thereby increasing the molecular weight and flexibility of the small interfering ribonucleic acid, (Lee, JH Park, TG Park, Nat Mater, 9 (2010) 272-278; SH Lee, BH Chung, TG Park, YS Nam, H. Mok, Accounts Chem Res, 45 (2012) 1014-1025). However, the small interfering ribonucleic acid-based constructs form multi-ion complexes only by ionic interaction, which is problematic in forming dense and stable nanoparticles. In addition, since the resultant product is composed of several kinds of small interfering ribonucleic acid constructs, it is difficult to prepare a specimen having a constant physical property, and its structure is not easy to analyze, It is difficult to apply the method to the present invention.

One embodiment of the present invention provides an amphiphilic block copolymer comprising a hydrophilic block and a hydrophobic block, wherein the hydrophilic block comprises a nucleic acid molecule. The amphiphilic block copolymer can form a multi-ion complex more effectively by increasing the charge density and flexibility by including a nucleic acid molecule such as siRNA as a hydrophilic block in a hydrophilic block. By forming a hydrophobic block, The hydrophobic interaction between the amphiphilic block copolymer and the cationic carrier can form a more dense and stable multi-ion complex with the cationic carrier and increase the efficiency of delivery into the cell.

Another embodiment provides a method for preparing an amphiphilic block copolymer having a nucleic acid molecule as a hydrophilic block.

Another embodiment provides a multi-ion complex comprising the amphiphilic block copolymer and a cationic carrier.

Another embodiment provides a magnetic assembly comprising the multi-ion complex.

Another example provides a composition for nucleic acid delivery comprising at least one selected from the group consisting of the amphiphilic block copolymer, the multi-ion complex, and the self-assembly.

However, the technical problem to be solved by the present invention is not limited to the above-mentioned problems, and other matters not mentioned can be clearly understood by those skilled in the art from the following description.

An embodiment of the present invention provides an amphiphilic block copolymer having a structure represented by the following formula (1).

[Chemical Formula 1]

A1-B-A2

Wherein A1 and A2 are the same or different nucleic acid molecules each as a hydrophilic block,

B is a hydrophobic block.

The nucleic acid molecule may have a single stranded or double stranded structure. In the case of double stranded, the hydrophobic block may be connected to one of the double strands of the nucleic acid molecule.

Another embodiment provides a multi-ion complex comprising a cationic carrier conjugated to the amphiphilic block copolymer and the amphiphilic block copolymer by electrostatic attraction or hydrophobic interaction.

Another embodiment is a method for producing a nucleic acid molecule, comprising: introducing a hydrophobic unit compound having a functional group into one end of a nucleic acid molecule to prepare two nucleic acid molecules to which a hydrophobic unit compound having a functional group is connected; And forming a chemical bond in the functional period of each hydrophobic unit compound linked to the nucleic acid molecule to form a hydrophobic block connected through a functional period of the hydrophobic unit compound to prepare a polymer having a nucleic acid molecule-hydrophobic block-nucleic acid molecule Wherein the amphiphilic block copolymer has the structure of formula (1)

[Chemical Formula 1]

A1-B-A2

Wherein A1 and A2 are the same or different nucleic acid molecules each as a hydrophilic block,

B is a hydrophobic block.

The nucleic acid molecule may have a single stranded or double stranded structure. In the case of double stranded, the hydrophobic block may be connected to one of the double strands of the nucleic acid molecule.

Another embodiment provides a magnetic assembly comprising the multi-ion complex.

Yet another embodiment provides a nucleic acid delivery composition comprising at least one selected from the group consisting of the amphiphilic block copolymer, the multi-ion complex, and the self-assembly.

Hereinafter, the present invention will be described in detail.

As used herein, "amphipathic" refers to a property having a water-soluble region and a hydrophobic region simultaneously in one molecular structure.

In order to efficiently transfer a nucleic acid molecule, for example, siRNA into cells, stable nanoparticles of 100 nm or less must be formed, and the conventional techniques have a great difficulty in meeting these basic requirements. The present invention suggests a simple method to solve these problems simultaneously, and it is proposed to form a nanoconjugate for siRNA delivery based on a multi-ion complex through self-assembly (Fig. 1) It is to satisfy all the important factors simultaneously.

First, it has the effect of reducing the size of the multi-ion complex by increasing the charge density of the siRNA. A block copolymer having siRNA as a hydrophilic block has a charge density per molecule of 2 times that of a single siRNA. Therefore, as the charge density increases, the block copolymer having the siRNA as a hydrophilic block can form a more stable ion-intermolecular bond with the cationic carrier, thereby forming a small and stable multi-ion complex.

Second, it has the effect of reducing the size of the multi-ion complex by increasing the flexibility of the siRNA. The persistence length of the double-stranded RNA is 65 to 80 nm, and the length of the siRNA is 5 to 6 nm, which is very stiff and has a lack of flexibility. Unlike siRNA alone, where the lack of flexibility prevents effective polyion complex formation, the amphiphilic block copolymer into which the siRNA is introduced has the effect of increasing the flexibility by introducing a hydrophobic moiety between the siRNAs, Contributes to the formation of a small and stable multi-ion complex.

Third, hydrophobic interactions between amphiphilic block copolymers and amphiphilic block copolymers and amphiphilic block copolymers other than electrostatic attraction are introduced to improve the structural stability of the multi-ion complex.

Fourth, siRNA constructs with one type of identical structure can be fabricated by simple methods to facilitate clinical applications. Conventional methods mainly produce siRNA constructs by using chemical crosslinkers in various steps. However, siRNA constructs can be prepared by simple method by using no chemical crosslinker as a block copolymer having siRNA as a hydrophilic block.

In order to overcome the inherent limitations of nucleic acid molecule delivery technology such as conventional siRNA, for example, difficulty in permeation of cell membranes and degradation by a degradation enzyme, a novel type 3 of nucleic acid molecule such as siRNA combined with a hydrophobic block ≪ / RTI > It is expected that the nucleic acid molecule such as siRNA and the cationic carrier will form a dense and stable multi-ion complex to solve the problems related to in vivo delivery.

More specifically, linking a pair of siRNAs and a hydrophobic block at an appropriate ratio not only leads to the formation of efficient electrostatic attraction due to increased charge density and flexibility, but also due to the hydrophobic interactions between the hydrophobic blocks, Stable and dense multi-ion complexes, and provides a very simple way to dramatically increase the intracellular delivery efficiency of siRNA. Since such siRNA block copolymers have one kind of the same structure, it is easy to analyze the physical properties and is easy to apply to clinical applications. In addition, it can be decomposed into siRNA having biologically active activity in a reducing environment inside the cell, and thus, it can effectively inhibit gene.

Therefore, it is possible to form a multi-ion complex more efficiently due to increase of charge density and flexibility compared with a nucleic acid molecule carrier such as siRNA in the past, and to form a stable multi-ion complex, An amphiphilic block copolymer having as a block is provided. More specifically, one embodiment provides an amphiphilic block copolymer having the structure of Formula 1:

[Chemical Formula 1]

A1-B-A2

Wherein A1 and A2 are the same or different nucleic acid molecules each as a hydrophilic block,

B is a hydrophobic block.

The nucleic acid molecule may have a single stranded or double stranded structure. In the case of double stranded, the hydrophobic block may be connected to one of the double strands of the nucleic acid molecule.

The nucleic acid molecule may be a deoxyribonucleic acid (DNA) or a ribonucleic acid (RNA). Specifically, it may be a nucleic acid molecule such as a siNA, siRNA, dsRNA, miRNA, or shRNA capable of mediating RNA interference (RNAi) for gene expression, and may be a siRNA.

For example, the nucleic acid molecule may be a nucleic acid molecule comprising 10 to 50 bases, such as a double-stranded siRNA. The nucleic acid molecule may be a nucleic acid molecule consisting of 15 to 30 bases, for example, a double-stranded siRNA, so as to be more suitable for exerting activity in a cell.

The hydrophobic block may be formed of a bond of a hydrophobic unit compound having a functional group. The hydrophobic unit compound means a unit constituting a hydrophobic block, specifically, two hydrophobic unit compounds may be combined to form a hydrophobic block. For example, the hydrophobic block may have a structure of the formula -b1-X-b2-, and b1 and b2 may be the same or different from each other. Examples of each of b1 and b2 are the same as 1) to 4).

1) C1 to C30 linear or branched alkyl, preferably C1 to C15 linear or branched alkyl, and specific examples thereof include methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, Decyl, undecyl, dodecyl, tridecyl, tetradecylpentadecyl.

2) C2 to C30 linear or branched hydrocarbon compounds containing one or more unsaturated bonds, preferably C2 to C15 linear or branched alkyl compounds containing an unsaturated bond, and specific examples include ethene, acetylene , Propene, propyne, butene, butene, butadiene, pentene, pentene, pentadiene, hexene.

3) a C 7 to C 30 hydrocarbon compound containing an aromatic ring, and specific examples thereof include benzene, toluene, quinine, styrene, ethylbenzene, and propylbenzene.

4) a C4 to C30 hydrocarbon compound containing a cyclic compound, preferably a C4 to C15 hydrocarbon compound containing a cyclic compound, and specific examples thereof include cyclopropane, cyclobutane, cyclopentane, cyclohexane , Cycloheptane, cyclooctane, methylcyclopentane, dimethylcyclopentane, methylethylcyclopentane, cyclopentene, and cyclohexadiene.

B1 and b2 may be the same or different from each other and may be the same or different from each other and may be the same or different from each other and may be selected from the group consisting of poly (lactide), PLA, poly (glycolic acid), PGA, poly (lactic-glycolic acid) (PLA), polycaprolactone (PCL), polymethyl methacrylate (PMMA), polyurethane (PU), and polystyrene (PS) May be at least one kind of polymer selected from the group consisting of At this time, the polymer may have a molecular weight of 500 to 500,000, preferably 1000 to 10,000.

X may be an enzyme-decomposable bond, an acid-decomposable bond or a reducing agent-decomposable bond, for example, a disulfide bond (-SS-), an ester bond (-CO-O-), an anhydride bond (-CO- ), A carbamate bond (-O-CO-N-), a carbonate bond (-ORO-CO-), an amide bond (-CO-NH-), a urethane bond (-NH-COO-), a phosphodiester bond -O-OP-O-) and a hydrazone bond (-CNNR), but the type of the bond is not limited. The amphiphilic block copolymer having the structure of formula (1) can be formed by the above-mentioned bonding.

With respect to the A1-B-A2 linkage, B can be linked to one strand of each nucleic acid molecule of A1, A2, and if the nucleic acid molecule is a double strand, it can be linked via a sense strand or an antisense strand end. For example, when the nucleic acid molecule is an siRNA, B may be connected to the sense strand of the siRNA or to the end of the antisense strand, and preferably to the 3 'end of the sense strand.

With respect to the above-mentioned b1-X-b2 linkage (X), the functional groups of b1 and b2 are bonded to each other and the bond (X) is as described above. Examples of the functional group forming the bond (X) include a sulfhydryl group (-SH), an alcohol (-OH), a carboxylic acid (-COOH) amine group (-NH ₂ ), a chloride (-Cl), a bromide Br), iodine (-I), aldehyde (-COH), azide (-N ₃ ), acrylate, vinyl group or acetylene group. For example, when the functional group at the terminal of b1 and b2 is a sulfhydryl group, they may be connected through a disulfide bond (i.e., X = disulfide bond), and in another example, functional groups at the terminals of b1 and b2 may be connected to an alcohol When it is a carboxylic acid (-COOH), it can be linked via an ester linkage (i.e., X = ester linkage).

Further, in order to increase the efficiency of transfer into target cells and stability in serum A cell-specific substance or polymer may be further bound to the end of the nucleic acid molecule. For example, a cell-specific ligand or a hydrophilic polymer may be bonded. The further joined sites are nucleic acid molecule ends that are not linked to the hydrophobic block. For example, the siRNAs of A1 and A2 in the structure of A1-B-A2 may further comprise a cell-specific material at the opposite end connected to B.

Specific examples of the cell-specific substance or polymer to be added include peptides, antibodies, cell growth factors, sugars, transferins, and polyethylene glycol (PEG).

(SEQ ID NO: 1, TACHQHVRMVRP), a breast cancer-specific peptide (SEQ ID NO: 2, VPWMEPAYQRFL), and a peptide specific for a breast cancer. The peptide of the present invention binds specifically to a cell surface molecule, Or colon cancer specific peptide (SEQ ID NO: 3, VHLGYAT) Lt; / RTI >

The antibody may be an anti-HER2 antibody, an anti-insulin receptor antibody, or an anti-CD22 antibody, and the cell growth factor may be folic acid, epithelial growth factor (EGF), or fibroblast growth factor (FGF) May be galactose or mannose.

The PEG can be used to improve stability in serum. Although not particularly limited thereto, the molecular weight of the PEG may be in the range of 200 to 10,000.

Unlike conventional siRNA multijunctions, which are mixtures of components having various molecular weights and thus have poor reproducibility of studies, the amphiphilic block copolymers according to the present invention are much simpler to manufacture and have a better chemical structure , But also has the advantage of achieving high charge density and flexibility of the multi-junction body. Further, the hydrophobic block contained in the amphiphilic block copolymer is based on a new mechanism in which the property of being dissolved in an aqueous solution is reduced, and the hydrophobic interaction between the hydrophobic blocks imparts a property of aggregating or self-assembling to each other.

The amphiphilic block copolymer is characterized by being capable of forming a stable and high density polyion complex having a diameter of 100 nm or less by spontaneous self-assembly when mixed with a cationic carrier.

Accordingly, another embodiment of the present invention provides a multi-ion complex comprising a cationic carrier conjugated to the amphiphilic block copolymer and the amphiphilic block copolymer by electrostatic attraction or hydrophobic interaction.

The ratio of the cation of the cationic carrier and the charge of the anion of the amphipathic block copolymer (anion of the cationic carrier / cationic / amphipathic block copolymer) may be from 0.5 to 1000. Accordingly, the content of the cationic carrier and the amphiphilic block copolymer in the multi-ion complex can be adjusted within the range of the charge amount.

In order to solve problems of intracellular delivery efficiency and structural instability in vivo, the multi-ion complex preferably has a size of 50 to 500 nanometers, and is used for maximizing intracellular delivery efficiency and in vivo structural stability And most preferably 50 to 200 nanometers.

In addition, the hydrophobic blocks of the amphiphilic block copolymer can cause hydrophobic interactions between the hydrophobic blocks, resulting in the formation of high density multi-ion complexes.

For example, the cationic carrier that is a constituent of the multi-ion complex may be at least one member selected from the group consisting of a cationic peptide, a cationic lipid, and a cationic polymer.

The cationic peptide contributes to the formation of a multi-ion complex by binding to a nucleic acid molecule having a negative charge. It is a peptide as a cell-specific substance that specifically binds to a cell surface molecule and imparts cell specificity to the multi- Respectively. The cationic peptide may be a cationic peptide comprising amino acids with cationic nature, such as arginine, histidine, lysine. For example, the cationic peptide may be at least one selected from the group consisting of KALA peptide, polylysine, polyglutamic acid, and protamine.

Wherein the cationic lipid is selected from the group consisting of dioleoyl phosphatidylethanolamine, cholesterol dioleoyl phosphatidyl choline, and dioleoyl trimethylammoniumpropan. It can be more than a species.

The cationic polymer may be at least one selected from the group consisting of polyethylenimine, polyamine, polyvinylamine, chitosan, and polyamidoamine. The molecular weight of the cationic polymer is not particularly limited, but may be in the range of 1 kDa to 1000 kDa.

Since the multi-ion complex has a stabilized structure, a high density, and a small size, it is characterized by high intracellular delivery efficiency of siRNA.

In another embodiment of the present invention, there is also provided a method for producing a nucleic acid molecule, comprising: preparing a nucleic acid molecule having a functional group by introducing a hydrophobic unit compound having a functional group into one end of the nucleic acid molecule; And

Forming a chemical bond in the functional period of each hydrophobic unit compound linked to the nucleic acid molecule to form a hydrophobic block connected through a functional period of the hydrophobic unit compound to form a polymer having a nucleic acid molecule-hydrophobic block-nucleic acid molecule Wherein the amphiphilic block copolymer has a structure represented by the following formula (1): < EMI ID =

[Chemical Formula 1]

A1-B-A2

Wherein A1 and A2 are each the same or different nucleic acid molecule,

B is a hydrophobic block.

Wherein B is a hydrophobic block having a structure represented by -b1-X-b2-,

b1 and b2 are the same or different from each other, and each independently,

Linear or branched C1 to C30 alkyl,

A C2 to C30 linear or branched hydrocarbon compound containing at least one unsaturated bond,

C8 to C30 hydrocarbon compounds containing aromatic rings,

A C4 to C30 hydrocarbon compound containing a cyclic compound, and

Poly (lactide), PLA), poly (glycolic acid), PGA, poly (lactic-glycolic acid), PLGA), polycaprolactone , PCL), polymethyl methacrylate (PMMA), polyurethane (PU), and polystyrene (PS).

, ≪ / RTI >

X is a bond selected from the group consisting of a disulfide bond, an ester bond, an anhydride bond, a carbamate bond, a carbonate bond, an amide bond, a urethane bond, a phosphodiester bond and a hydrazone bond.

Descriptions of specific nucleic acid molecules and hydrophobic blocks are as described above.

The nucleic acid molecule may have a single stranded or double stranded structure. In the case of double stranded, the hydrophobic block may be connected to one of the double strands of the nucleic acid molecule.

The amphiphilic block copolymer of the A1-B-A2 type according to the present invention can form a multi-ion complex more effectively by increasing the charge density and flexibility by including the hydrophilic block of the siRNA, and by introducing the hydrophobic block, Hydrophobic interactions between the blocks can increase the efficiency of delivery into the cell by forming a more dense and stable multi-ion complex with the cationic transporter. In addition, while conventional siRNA-based constructs are simple mixtures of several types of constructs, the present invention is advantageous in that it is not only a single compound but also has a very simple and highly efficient production process and is easily applicable to clinical applications.

Thus, the amphiphilic block copolymer, which is an embodiment of the present invention, not only has a high gene transfer efficiency but also has a possibility as a therapeutic agent having a single structure. Further, the amphiphilic block copolymer enables application to various fields related thereto and ultimately, It is expected to contribute to life welfare.

Brief Description of the Drawings Figure 1 shows a self-assembling view of an amphipathic block copolymer having siRNA as a hydrophilic block and a multi-ion complex formed of a cationic carrier.
Fig. 2 is a schematic diagram for preparing an amphiphilic block copolymer having siRNA as a hydrophilic block according to an example of the present invention.
Fig. 3 shows the result of confirming the amphiphilic block copolymer and siRNA by acrylamide gel electrophoresis according to Example 1. Fig.
4 is a polyion complex ((a formed in accordance with Example 2) siRNA, (b) is C ₆ -siRNA block copolymer, (c) is C ₁₂ -siRNA block copolymer, (d) is a C ₂₄ - siRNA block copolymer) was observed with AFM.
FIG. 5 (a) is a graph showing the relative amount of siRNA introduced according to Example 2 by real-time polymerase chain reaction (PCR), (b) MDA- The results are shown in Fig.
6 (a) is a graph showing the expression amount of GFP by measuring the inhibition of the expression of GFP (target gene) according to Example 3 with a fluorescence analyzer, and FIG. 6 (b) 435 cells were observed with a confocal microscope.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, these are only for illustrating the present invention, and the scope of the present invention is not limited by these examples.

[Example]

Example  One. Amphipathic  Preparation of block copolymers

A sense-strand-hydrophobic block component-sense strand polymer was prepared using a sense strand ribonucleic acid substituted with a polymer having the same terminal, and then the antisense strand was bound to the strand by hydrogen bonding to form a siRNA as a hydrophilic block. Amphiphilic block copolymer was prepared.

The siRNA was selected as a siRNA targeting green fluorescent protein (GFP), the sense strand 5'-GCAAGCUGACCCUGAAGUUdTdT-3 '(SEQ ID NO: 4) and the antisense strand 5'-AACUUCAGGGUGAGCUUGCdTdT-3' (SEQ ID NO: 5).

Specifically, a carbon body (alkyl chain) having 3, 6, or 12 carbon atoms at the 3'- 100 nmol of DEPC (diethylprocarbonate) -treated water was added to 225 uL of DIPC (diethylprocarbonate) treated water, and 100 nmol of a sense strand ribonucleotide whose carbon end was substituted with a sulfhydryl group , And then 250 μL of 2 M DTT (dithiothreitol) and 25 μL of 0.1 M phosphate buffered saline (PBS) were added thereto, followed by reaction for 16 hours. After completion of the reaction, 50 μL of 3 M sodium acetate and 1500 μL of ethanol were added and left at -75 ° C. for 30 minutes. Then, the siRNA was precipitated at 13000 rpm for 20 minutes at 4 ° C by centrifugation. The supernatant was discarded and the precipitated ribonucleic acid was dissolved in water and used in the next reaction. Purified sense strand ribonucleic acid was oxidized in 25% DMSO (v / v) to produce sense strand ribonucleic acid duplexes. The reaction product was lyophilized, and then dissolved in water. The same amount of antisense strand ribonucleic acid was added to obtain double-stranded siRNA (C ₆ -, C ₁₂ -, C ₂₄ -dimer) -B-A2 type amphiphilic block copolymer).

The prepared amphiphilic block copolymer, siRNA (the sense strand is SEQ ID NO: 4 and the antisense strand is SEQ ID NO: 5, unless otherwise specified) was identified by acrylamide gel electrophoresis, The results are shown in Fig.

The left panel of FIG. 3 shows the result of synthesis of the amphiphilic block copolymer, wherein lane 1 is DNA size marker, lane 2 is siRNA, lanes 3,4,5 are siRNA block copolymer (C ₆ , C ₁₂ , C ₂₄ -dimer). The right panel shows that when treated with DDT, the disulfide bond is cleaved and the respective siRNA block copolymer is decomposed into siRNA monomers (siRNA monomers are introduced into one end of the siRNA when no specific mention is made) Respectively.

Example  2. Amphipathic  Block copolymers and Cationic  The multi-ion complex of the carrier

2-1: Preparation of multi-ion complex

The amphiphilic block copolymer (2 ug) and linear polyethyleneimine (2.66 ug) prepared in Example 1 were each prepared in 50 μl of phosphate buffer solution, and each solution was mixed to form a multi-ion complex at room temperature for 15 minutes.

2-2: AFM Observation of morphology using

The amphiphilic block copolymer of the A1-B-A2 type having the hydrophilic block as the siRNA prepared in Example 1 was mixed with the linear polyethyleneimine as a cationic transfer to form a small stable complex, The siRNA was also identified as a comparative group, and the results are shown in FIG. 4 and Table 1.

(B) is a C ₆ -siRNA block copolymer, (c) is a C ₁₂ -siRNA block copolymer, and (d) is a C ₂₄ -siRNA block copolymer. Ion complexes.

Multi ion complex
(b) Multi ion complex
(c) Multi ion complex
(d) size 106 ± 19 nm 89 ± 23 nm 87 ± 13 nm

As shown in FIG. 4 and Table 1, it was found that amphiphilic block copolymer of the A1-B-A2 type having siRNA as a hydrophilic block forms a smaller and more stable multi-ion complex than siRNA.

2-3: Block copolymers and Cationic  Of the multi-ion complex of the carrier Degree of inflow  Confirmation test

In order to compare with the siRNA whether the amphiphilic block copolymer of the form of A1-B-A2 having the hydrophilic block as the hydrophilic block was effectively introduced into the cell, the cationic transfer Chain polyethylenimine and an amphiphilic block copolymer, and then treated with MDA-MB-435 cells (Samyang Corp.), which is a stably expressing GFP gene, and cultured in a cell culture medium free of serum (fetal bovine serum) In DMEM (Dulbecco's Modified Eagle Medium) (Gibco) for 5 hours at 37 ° C under 5% carbon dioxide. As a comparative group, siRNAs were treated under the same conditions and cultured. Then, the siRNA introduced into the cell is synthesized by using a specially designed primer (ordered by Applied Biosystems), real-time polymerase chain reaction (RT-PCR) , And the results are shown in FIG. 5A.

The conditions of the RT-PCR were performed in accordance with the manual provided by Applied Biosystems. More specifically, 1) 95 ° C for 10 minutes, 2) 95 ° C for 15 seconds, 3) 60 ° C for 60 seconds (annealing and extension) 2) and 3) were performed in 40 cycles.

As shown in FIG. 5A, it was confirmed that the amphipathic block copolymer of the form of A1-B-A2 having siRNA as a hydrophilic block was more effectively introduced into cells than siRNA. In particular, it was found that an amphiphilic block copolymer having a carbon number of 12 (C ₁₂ -dimer) as a hydrophobic block was more efficiently introduced into cells than an amphipathic block copolymer having another hydrophobic block. This shows the importance of introducing appropriate hydrophobic blocks.

2-4: Confocal  Inflow experiment using a microscope

Fluorescently labeled linear polyethyleneimine, the multi-ion complex (C ₂₄ -dimer) and siRNA prepared in Example 2.1 were treated with MDA-MB-435 cells for 5 hours (same as in Example 2-3) The cells were fixed and observed with a confocal microscope. A control without any treatment was also observed with a confocal microscope, and the results are shown in FIG. 5B.

As shown in FIG. 5B, it was found that amphiphilic block copolymer of the form of A1-B-A2 having siRNA as a hydrophilic block was more infiltrated into cells than siRNA.

Example  3. siRNA On by GFP  Expression inhibition measurement

MDA-MB-435 cells were treated for 5 hours with the multi-ion complexes prepared in Example 2-1 and the multi-ion complex prepared by binding siRNA and linear polyethyleneimine to each other (the same conditions as in Example 2-3) ) And fresh DMEM medium supplemented with 10% serum. After 48 hours, the expression pattern of GFP gene was quantified using a fluorescence analyzer. A control without any treatment was also confirmed with a fluorescence analyzer, and the results are shown in FIG.

FIG. 6 (a) is a graph showing the expression of GFP by measuring the inhibition of GFP expression with a fluorescence analyzer, and FIG. 6 (b) is a graph showing MDA-MB-435 cells observed with a confocal microscope.

As shown in FIG. 6, the multi-ion complexes prepared in Example 2-1 inhibited the target gene much more effectively than the multi-ion complexes prepared by combining siRNA with linear polyethyleneimine. Furthermore, it has been found that a multi-ion complex with an amphipathic amphiphilic block copolymer containing ₁₂ carbon-containing hydrophobic blocks (C ₁₂ -dimer) is much more complex than a multi-ion complex with an amphipathic block copolymer containing different carbon strands Effectively inhibiting the gene. This indicates that the introduction of appropriate hydrophobic blocks is important.

It will be understood by those skilled in the art that the foregoing description of the present invention is for illustrative purposes only and that those of ordinary skill in the art can readily understand that various changes and modifications may be made without departing from the spirit or essential characteristics of the present invention. will be. It is therefore to be understood that the above-described embodiments are illustrative in all aspects and not restrictive.

<110> KOREA ADVANCED INSTITUTE OF SCIENCE AND TECHNOLOGY <120> AMPHIPHILIC BLOCK COPOLYMER CONTAINING siRNA ASA HYDROPHILIC          BLOCK AND POLYION COMPLEX CONTAINING THE SAME <130> DPP20126703KR <160> 5 <170> Kopatentin 2.0 <210> 1 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> liver cancer-specific peptide <400> 1 Thr Ala Cys His Gln His Val Arg Met Val Val Pro   1 5 10 <210> 2 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> breast cancer-specific peptide <400> 2 Val Pro Trp Met Glu Pro Ala Tyr Gln Arg Phe Leu   1 5 10 <210> 3 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> colorectal cancer-specific peptide <400> 3 Val His Leu Gly Tyr Ala Thr   1 5 <210> 4 <211> 23 <212> RNA <213> Artificial Sequence <220> <223> sense strand of siRNA <400> 4 gcaagcugac ccugaaguud tdt 23 <210> 5 <211> 23 <212> RNA <213> Artificial Sequence <220> <223> Antisense strand of siRNA <400> 5 aacuucaggg ugagcuugcd tdt 23

Claims (20)

An amphiphilic block copolymer having a structure represented by the following formula (1): &lt; EMI ID =
[Chemical Formula 1]
A1-B-A2
Wherein A1 and A2 are the same or different double-stranded nucleic acid molecule as the hydrophilic block,
B is a hydrophobic block having a structure of the formula -b1-X-b2-, connected to one strand of the double-stranded nucleic acid molecule,
B1 and b2 of the hydrophobic block are the same as or different from each other,
Each independently,
Linear or branched C1 to C30 alkyl,
A C2 to C30 linear or branched hydrocarbon compound containing at least one unsaturated bond,
C8 to C30 hydrocarbon compounds containing aromatic rings,
A C4 to C30 hydrocarbon compound containing a cyclic compound, and
Poly (lactide), PLA), poly (glycolic acid), PGA, poly (lactic-glycolic acid), PLGA), polycaprolactone , PCL), polymethyl methacrylate (PMMA), polyurethane (PU), and polystyrene (PS).
, &Lt; / RTI &gt;
X is a bond selected from the group consisting of a disulfide bond, an ester bond, an anhydride bond, a carbamate bond, a carbonate bond, an amide bond, a urethane bond, a phosphodiester bond and a hydrazone bond. The method according to claim 1,
Wherein the nucleic acid molecule is a ribonucleic acid (RNA). 3. The method of claim 2,
Wherein the ribonucleic acid is selected from the group consisting of siNA, siRNA, dsRNA, miRNA and shRNA. delete The method according to claim 1,
Wherein the nucleic acid molecule is comprised of 10 to 50 nucleic acids. delete The method according to claim 1,
The straight or branched alkyl of C1 to C30 is preferably methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, or tetradecylpentadecyl,
The C2 to C30 linear or branched hydrocarbon compound containing at least one unsaturated bond is selected from the group consisting of ethene, acetylene, propene, propyne, butene, butyne, butadiene, pentene, pentyne, pentadiene,
The C7 to C30 hydrocarbon compound containing the aromatic ring is benzene, toluene, quinine, styrene, ethylbenzene, or propylbenzene,
The C4 to C30 hydrocarbon compound containing the cyclic compound may be at least one selected from the group consisting of cyclopropane, cyclobutane, cyclopentane, cyclohexane, cycloheptane, cyclooctane, methylcyclopentane, dimethylcyclopentane, methylethylcyclopentane, cyclopentene, Hexadiene amphiphilic block copolymer. The method according to claim 1,
Wherein the polymer has a molecular weight of 500 to 500,000. The method according to claim 1,
1) a peptide selected from the group consisting of a liver cancer-specific peptide (SEQ ID NO: 1), a breast cancer-specific peptide (SEQ ID NO: 2) and a colon cancer-specific peptide (SEQ ID NO: 3);
2) an antibody selected from the group consisting of an anti-HER2 antibody, an anti-insulin receptor antibody, and an anti-CD22 antibody;
3) a cell growth factor selected from the group consisting of folic acid, epithelial cell growth factor and fibroblast growth factor;
4) a sugar selected from the group consisting of galactose and mannose;
5) transferrin; And
6) PEG (polyethylene glycol)
&Lt; / RTI &gt; wherein at least a portion of the nucleic acid molecule is attached to the nucleic acid molecule end. 10. An amphiphilic block copolymer according to any one of claims 1 to 3, 5, and 7 to 9; And a cationic carrier conjugated to the amphiphilic block copolymer by electrostatic attraction and hydrophobic interaction. 11. The method of claim 10,
Wherein the multi-ion complex is between 50 and 500 nanometers in size. 11. The method of claim 10,
Wherein the cationic carrier is at least one selected from the group consisting of a cationic peptide, a cationic lipid, and a cationic polymer. 13. The method of claim 12,
Wherein the cationic peptide is at least one selected from the group consisting of KALA peptide, polylysine, polyglutamic acid, and portamine. 13. The method of claim 12,
Wherein the cationic lipid is selected from the group consisting of dioleoyl phosphatidylethanolamine, cholesterol dioleoyl phosphatidyl choline, and dioleoyl trimethylammoniumpropan. Multiple ion complexes. 13. The method of claim 12,
Wherein the cationic polymer is at least one selected from the group consisting of polyethylenimine, polyamine, polyvinylamine, chitosan, and polyamidoamine. Introducing a hydrophobic unit compound having a functional group into one end of a double-stranded nucleic acid molecule to prepare a double-stranded nucleic acid molecule having a functional group-containing hydrophobic unit compound linked thereto; And
Forming a chemical bond in the functional period of each hydrophobic unit compound linked to the nucleic acid molecule to form a hydrophobic block connected through a functional period of the hydrophobic unit compound to form a polymer having a nucleic acid molecule-hydrophobic block-nucleic acid molecule Wherein the amphiphilic block copolymer has a structure of formula
[Chemical Formula 1]
A1-B-A2
Wherein A1 and A2 are double-stranded nucleic acid molecules which are the same or different from each other as a hydrophilic block,
B is a hydrophobic block having a structure of the formula -b1-X-b2-, connected to one strand of the double-stranded nucleic acid molecule,
b1 and b2 are the same or different from each other, and each independently,
Linear or branched C1 to C30 alkyl,
A C2 to C30 linear or branched hydrocarbon compound containing at least one unsaturated bond,
C8 to C30 hydrocarbon compounds containing aromatic rings,
A C4 to C30 hydrocarbon compound containing a cyclic compound, and
Poly (lactide), PLA), poly (glycolic acid), PGA, poly (lactic-glycolic acid), PLGA), polycaprolactone , PCL), polymethyl methacrylate (PMMA), polyurethane (PU), and polystyrene (PS).
, &Lt; / RTI &gt;
X is a bond selected from the group consisting of a disulfide bond, an ester bond, an anhydride bond, a carbamate bond, a carbonate bond, an amide bond, a urethane bond, a phosphodiester bond and a hydrazone bond. 17. The method of claim 16,
Wherein the nucleic acid molecule is a ribonucleic acid. delete 12. A magnetic assembly comprising the multi-ion complex of claim 10. A composition for nucleic acid delivery comprising the amphiphilic block copolymer of claim 1.

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