KR20140052550A - Complex for nucleic acid delivery - Google Patents

Abstract

The present invention relates to a nucleic acid delivery complex, and provides a nucleic acid delivery complex comprising a nucleic acid to be delivered, a nucleic acid delivery peptide comprising an HMGB1 A box peptide, and a peptide micelle encapsulating the HMGB1 A box peptide in the form of micelles. The nucleic acid delivery complex of the present invention can be used for the development of a gene carrier and a gene therapy agent for gene therapy because the nucleic acid delivery complex can introduce the nucleic acid to be delivered into the cell with high efficiency and is safe because of low cytotoxicity.

Description

Complex for nucleic acid delivery < RTI ID = 0.0 >

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a nucleic acid delivery complex, and more particularly, to a nucleic acid delivery complex using a nucleic acid delivery peptide and a peptide micelle together.

With the development of DNA recombination technology, techniques for expressing foreign genes in cells or in vitro have been commercialized. Using such techniques, observation of cell control, mass production of recombinant proteins, cloning of genes, Such as inhibition or activation of cell control or activation.

Among them, gene therapy, which is a treatment for treating a gene in a specific cell of a patient for the treatment of a disease caused by abnormalities of a specific gene of a patient, has been presented as a new treatment method for diseases. Research is being conducted.

A key element of gene therapy is the gene of interest and the gene delivery (or delivery material or system) that can deliver it safely and efficiently to the body. Among them, the selection of a gene delivery system relates to a problem of effectively delivering a target gene to a target organ, and a problem in which a desired gene is introduced into a suitable cell without rejection to induce expression, thereby obtaining a desired therapeutic effect. Indeed, the development of new delivery systems for the success of gene therapy is recognized as a major challenge in the field of gene therapy.

Conventionally known gene delivery systems include viral vectors prepared by modifying a virus having an infectious effect on a host to eliminate pathogenicity and introducing a transfer gene, and adenovirus, retrovirus, And adeno-associated virus (AAV) vectors. These viral vectors are highly efficient in transducing genes because of their infectious nature, but they are highly likely to mutate when inserted into the chromosome of the host, and therefore, risk factors such as the possibility of infectious virus generation and induction of inflammation in the host Its use is limited.

In order to overcome the problems of such viral vectors, a nonviral gene delivery system developed as an alternative method is a method of transferring a gene into a cell by a physicochemical transfer method, wherein a lipid and a cationic lipid carrier and a polymer carrier are representative Yes. These non-viral vectors are biodegradable, have high biostability, are non-immunogenic, and are easy to use since there is no restriction according to the size of the transgene.

However, the non-viral vector is degraded in the body after intracellular transduction, and the efficiency with which the gene is transferred to the cytoplasm is low, so that the rate of transferring the gene to the nucleus is low, so that the gene transfer efficiency is low as compared with the viral vector.

Specifically, the carrier comprising the liposome and the cationic lipid is relatively simple in operation, storage and quality control, has a good in vitro delivery efficiency, has a lower antigenicity and safety than the viral vector, The gene delivery efficiency is low and the expression period itself is short, which is ineffective, and clinical cases are not accumulated. The cationic lipid transporter is based on the principle that lipid-stable micelles are formed and cationic on the surface, so that gene therapy drugs, mainly negative-charged DNA or RNA, are easily incorporated and introduced into cells. On the other hand, a gene delivery method using a peptide having a specific sequence available for gene binding and delivery has been reported. Although this is advantageous over viral vectors, there is a disadvantage in that the intracellular structural stability to be provided for gene transfer is weak, resulting in a problem of inefficiency for gene transfer.

In other words, the viral vector has excellent gene transfer efficiency, but when inserted into the chromosome of the host, there is a high possibility of mutation, and there are many risk factors such as infectious virus generation and induction of inflammation in the host. The non-viral vector used to improve the above problems has a disadvantage that the gene transfer efficiency is low. To improve the disadvantage, the cationic polymer, cationic peptide or cationic liposome is different from the viral vector There is a problem of cytotoxicity on the side, and the transfer efficiency is still low to commercialize.

Therefore, there is an urgent need to develop a safe non-viral carrier which can deliver genes into cells with high efficiency to be commercialized, and which is free from toxicity in cells.

It is an object of the present invention to provide a safe nucleic acid delivery system which is capable of delivering a nucleic acid to be delivered within a cell with high efficiency, but without cytotoxicity.

In order to accomplish the above object, the present invention provides a nucleic acid delivery complex comprising a nucleic acid to be delivered, a nucleic acid delivery peptide comprising HMGB1 A box peptide, and a peptide micelle encapsulating the HMGB1 A box peptide in the form of micelles.

The nucleic acid delivery complex according to the present invention can be used for the development of a gene carrier and a gene therapy agent for gene therapy because it can introduce the nucleic acid to be delivered into cells with high efficiency while having low cytotoxicity and safety.

However, the effects of the present invention are not limited to the above-mentioned effects, and other effects not mentioned can be clearly understood by those skilled in the art from the following description.

FIG. 1 is an exemplary diagram illustrating a manner in which the nucleic acid transfer peptide and the peptide micelle are bound to a nucleic acid to be delivered.
FIG. 2 (A) shows the result of confirming whether the DNA forms a complex with TAT-HMGB1 Abox peptide and R3V6 peptide micelle. FIG. 2 (B) shows whether DNA forms a complex with HMGB1 Abox peptide and R3V6 peptide micelle (C) shows the result of confirming whether or not siRNA forms a complex with HMGB1 Abox peptide and R3V6 peptide micelle.
FIG. 3 is a graph showing the results of experiments in which the nucleic acid delivery efficiency of the nucleic acid delivery complex was determined while the ratio (A) of the HMGB1 Abox peptide to the ratio (B, C) of the R3V6 peptide micelle was different.
FIG. 4 is a graph showing the results of comparing DNA (A) and siRNA (B) delivery efficiencies of the nucleic acid delivery complex of the present invention with various conventional nucleic acid delivery materials.
FIG. 5 is a graph showing the results of experiments in which cytotoxicity of the nucleic acid delivery complex of the present invention when the DNA (A) or the siRNA (B) was delivered into cells.
FIG. 6 is a result of an experiment to confirm the specific nucleic acid transfer efficiency of lung cells of the nucleic acid delivery complex of the present invention.

First, terms used in the specification of the present invention will be described.

The term 'peptide' used in the present invention means a polymer in the form of a chain in which 4 to 1000 amino acid residues are bonded to each other by peptide bonding, and includes' polypeptide ',' oligopeptide 'and' It is possible to intermix with 'protein'.

The 'nucleic acid' referred to in the present invention means a polymer compound in which a nucleotide, which is a chemical monomer composed of three elements of base, sugar and phosphoric acid, is linked in a chain form through a plurality of phosphate ester bonds, Quot; means " DNA, '' RNA, '' oligonucleotide, '' polynucleotide, '' aptamer, '' plasmid, The nucleic acid includes a derivative in which an oxygen atom or the like contained in a phosphoric acid moiety or an ester moiety in the nucleic acid structure is substituted with another element such as a sulfur atom or a fluorine atom or an alkyl group such as a methyl group. In addition, the nucleic acid may be chemically or biochemically modified, or may comprise a derivative of a non-natural or nucleotide base, as is apparent to those skilled in the art, including both the synthesized form and the copolymer, sense strand and antisense strand .

In addition, 'siRNA' referred to in the present invention refers to a double-stranded RNA molecule that prevents translation of a target mRNA. The siRNA includes both dsRNA and shRNA. The 'dsRNA' refers to two RNA molecule constructs consisting of one strand and another strand having a sequence complementary thereto. The 'shRNA' refers to an siRNA having a stem-loop structure comprising first and second regions complementary to each other, sense and antisense strands.

In addition, 'cell penetrating peptide (CPP)' referred to in the present invention means a peptide capable of delivering cargo, which is a target to be delivered, in cells in vitro or in vivo, It is possible to intermix with the 'transmembrane domain'.

The term "contact" as used herein refers to that a nucleic acid delivery complex containing a target nucleic acid is in contact with a eukaryotic or prokaryotic cell. By this contact, the target nucleic acid in the nucleic acid delivery complex is transferred into the eukaryotic or prokaryotic cell .

In addition, in the present invention, the terms 'transport', 'penetration', 'transport', 'transmission', 'transmission', or 'pass' are mutually intermingled with respect to 'introducing' the nucleic acid to be delivered into a cell.

Hereinafter, the present invention will be described in detail.

The present invention relates to a nucleic acid to be delivered; Nucleic acid delivery peptides; And peptide < RTI ID = 0.0 > micelles. ≪ / RTI >

The nucleic acid delivery complex of the present invention comprises a nucleic acid to be delivered, a nucleic acid transfer peptide and a peptide micelle. The nucleic acid transport peptide is bound to the target nucleic acid through charge interaction, and the peptide micelle encapsulates the nucleic acid and the nucleic acid transport peptide in the form of micelles through charge interaction or hydrophobic interaction.

The nucleic acid to be delivered is a nucleic acid to be introduced into a cell, and refers to a polymer in which a plurality of nucleotides are linked to each other by a phosphodiester bond and the molecule has a negative charge as a whole. The nucleic acid has a negative charge as a whole due to the negative charge of the phosphate group participating in the phosphoric acid diester bond. Preferably, the nucleic acid to be delivered is DNA or RNA consisting of a single strand or double strand, and the target nucleic acid is more preferably a DNA consisting of a single strand or a double strand, and the target nucleic acid is a double stranded DNA Most preferably, but not exclusively.

The nucleic acid transfer peptide includes a HMGB1 A box peptide. The HMGB1 A box peptide comprises the amino acid sequence of SEQ ID NO: 1. The amino acid sequence of SEQ ID NO: 1 is not particularly limited, but is preferably encoded by the nucleotide sequence of SEQ ID NO: 2. The HMGB1 A box contains a large number of positively charged amino acids, so that the molecule is positively charged as a whole. Accordingly, the HMGB1 A box molecule can be bound to the target nucleic acid that is negatively charged through charge interaction.

The nucleic acid transfer peptide may further include a transmembrane domain as a target-oriented peptide at the N-terminal or C-terminal of the HMGB1 A box peptide. In particular, the transmembrane domain is preferably included at the N-terminus of the HMGB1 A box peptide. The transmembrane domain may be any of those used in the art. For example, the transmembrane domain may be HIV-derived TAT. The transmembrane domain may be included in the nucleic acid delivery peptide together with the HMGB1 A box peptide to enhance the cell membrane permeability of the nucleic acid delivery complex.

The peptide micelle comprises a polypeptide having an amino acid sequence represented by the following formula (1).

[Chemical Formula 1]

R _n -X _m

In the above formula (1), R is arginine (Agr), X is a hydrophobic amino acid, n is an integer of 1 to 7, preferably 2 to 5, and m is an integer of 2 to 20, preferably 5 to 10 .

The peptide micelles are based on the cationic amino acid arginine (Arg). The arginine forms a charge interaction with the negatively charged nucleic acid, thereby binding the peptide micelle to the target nucleic acid. The number n of the arginine residues may be an integer of 1 to 7, preferably an integer of 2 to 5, but is not limited thereto. Even when only one arginine residue is included to constitute the peptide micelle, the peptide micelle can be bound to the target nucleic acid. However, when the number of arginine residues is more than 7, the total length of the unit peptide forming the peptide micelle becomes too long, and the intracellular toxicity is increased. That is, if the total length of the peptide micelle is too long, it may cause cytotoxicity due to the fact that the peptide does not decompose even after the delivery of the nucleic acid to be transferred and adheres to a membrane of some cell membranes or cell organelles, will be. Therefore, it is important that the peptide micelles are designed to minimize the number of arginines within a range that does not exhibit cytotoxicity.

The hydrophobic amino acids are such that the peptide micelles spontaneously aggregate in an aqueous solution to form the structure of the micelles. That is, the micelle structure of the peptide micelle is formed by aggregation of hydrophobic amino acid moieties forming a hydrophobic interaction with each other in an aqueous solution. This is a spontaneous reaction to lower the entropy of the peptide micellar molecules in aqueous solution. Therefore, the hydrophobic amino acid may be any one or more selected from the group consisting of isoleucine (Ile), Valine, Leu, Phe, Cys, More preferably, the hydrophobic amino acid is alanine (Ala) or valine (Val), but is not limited thereto. The number of residues m of the hydrophobic amino acid is preferably an integer of 2 to 20, more preferably the number of residues m of the hydrophobic amino acid is an integer of 5 to 10, but is not limited thereto. When the number of residues m of the hydrophobic amino acid is less than 2, there is a problem that the length of the hydrophobic portion is too short to allow the peptide micelle to form a micelle structure. When the number of residues m of the hydrophobic amino acid exceeds 20, There is a problem that the cytotoxicity is intensified.

FIG. 1 is an exemplary diagram illustrating a manner in which the nucleic acid transfer peptide and the peptide micelle are bound to a nucleic acid to be delivered.

Referring to FIG. 1, a nucleic acid transfer peptide containing an HMGB1 A box is first bound to a target nucleic acid through charge interaction (FIG. 1 (A)). Then, in the nucleic acid to be delivered, the arginine portion of the peptide micelle is coupled to the exposed portion of the peptide micelle through charge interactions, and the hydrophobic amino acid portion of the peptide micelle is exposed to the outside (FIG. 1 (B). Herein, when the peptide micelle molecule is further added, the micelle structure in which the arginine moiety is exposed to the outside through the hydrophobic interaction with the peptide micelle molecule to which the hydrophobic moiety of the externally exposed peptide micelle is additionally added is added (Fig. 1 (C)). The nucleic acid delivery complex formed as described above may introduce the nucleic acid to be transferred into the cell.

In order for the nucleic acid delivery complex to exhibit optimal nucleic acid delivery efficiency, the nucleic acid delivery peptide and the peptide micelle should be mixed with the target nucleic acid at a proper ratio. In the mixing ratio, the nucleic acid delivery peptide is preferably contained in an amount of 2 to 5 parts by weight based on 1 part by weight of the nucleic acid to be delivered. The peptide micelle is preferably added in an amount of 10 parts by weight By weight to 30 parts by weight.

In a specific embodiment of the present invention, a mixture of two siRNAs having the nucleotide sequence of pCMV-Luc plasmid or SEQ ID NO: 6 and SEQ ID NO: 7 is mixed with HMGB1-Abox peptide (nucleic acid transfer peptide) and R3V6 (peptide micelle) (See Table 2 and Fig. 2). ≪ tb > < TABLE > The thus prepared nucleic acid delivery complex was treated with Raw264.7 cells to confirm that the mixture of the pCMV-Luc plasmid as the delivery target nucleic acid or the two siRNAs having the nucleotide sequence of SEQ ID NO: 6 and SEQ ID NO: 7 was introduced at a high efficiency 3).

Further, in a specific embodiment of the present invention, a mixture of two siRNAs having the nucleotide sequence of pCMV-Luc plasmid or SEQ ID NO: 6 and SEQ ID NO: 7 is mixed with various ratios of HMGB1-Abox peptide (nucleic acid transfer peptide) and R3V6 To prepare a nucleic acid delivery complex, and treated with Raw264.7 cells to calculate an optimum ratio indicating optimal nucleic acid delivery efficiency (see FIG. 3). As a result, when the nucleic acid delivery peptide is contained in an amount of 2 to 5 parts by weight based on 1 part by weight of the nucleic acid to be delivered and the peptide micelle is contained in a proportion of 10 to 30 parts by weight, Respectively.

In addition, in a specific embodiment of the present invention, when the nucleic acid delivery complex of the present invention is used alone or in combination with a nucleic acid delivery peptide and a peptide micelle, or a poly-L-lysine PLL), PEI25K (polyethylenimide 25K), lipofectamine, and the like. As a result, it was confirmed that the nucleic acid delivery complex of the present invention had remarkably superior DNA delivery efficiency as compared with the case where each of the nucleic acid delivery peptide and the peptide micelle was used alone (see FIG. 4), and the conventional nucleic acid delivery material poly- (PLL), PEI25K (polyethylenimide 25K), and lipofectamine (see FIG. 4). In addition, the nucleic acid delivery complex of the present invention may be prepared by using nucleic acid transfer peptide and peptide micelle alone or in combination with conventional nucleic acid delivery materials such as poly-L-lysine (PLL), PEI25K (polyethylenimide 25K) (See FIG. 5), it was confirmed that the nucleic acid delivery complex of the present invention is a safe substance which is not toxic to cells. In addition, in vitro experiments showed that the nucleic acid delivery complex of the present invention has a significantly higher gene transfer efficiency into lung cells than lipofectamine, nucleic acid transfer peptide or peptide micelles (see FIG. 6). From the above results, it can be seen that the nucleic acid delivery complex of the present invention can efficiently transfer nucleic acid molecules such as DNA and siRNA into cells without cytotoxicity by using nucleic acid transfer peptide and peptide micelle together. Therefore, the nucleic acid delivery complex of the present invention can be usefully used as a gene delivery system capable of delivering a target nucleic acid into a cell with high efficiency and safety.

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

It is to be understood, however, that the following examples are intended to assist the understanding of the present invention and are not intended to limit the scope of the present invention.

Expression and purification of nucleic acid delivery peptides

<1-1> Expression and purification of HMGB1-Abox nucleic acid delivery peptide

A pET21a vector (Novagen, USA) in which the polynucleotide of SEQ ID NO: 2 encoding the HMGB1-Abox peptide of SEQ ID NO: 1 was inserted was transformed into E. coli BL21. Single clones of the transformed strains were taken and cultured in 10 ml of LB (10 g / L tryptone, 5 g / L yeast extract, and 10 g / L NaCl) containing 50 mg / ml of ampicillin (Fluka Chemical, Switzerland) And cultured at 37 DEG C for 18 hours with stirring at 220 rpm. 2 ml of the culture solution was inoculated into 2 L of LB culture medium containing 50 mg / ml of ampicillin and cultured at 37 ° C. until an OD600 of about 0.8 was reached. Then, IPTG (Isopropyl-β -D-Thiogalactopyranoside (Sigma Chemical, USA) was added and the mixture was stirred for 6 hours to induce the expression of HMGB1-Abox protein. The cultures thus cultured were centrifuged at 4 ° C at 5000 rpm for 10 minutes to recover the cells. The recovered cells were suspended in 100 ml of cell lysis buffer (50 mM Na ₂ H ₂ PO ₄ , 300 mM NaCl, 10 mM imidazole) (pH 8.0), and incubated with Lysozyme (Sigma Chemical, USA) Was added and the mixture was gently stirred on ice for 30 minutes. The agitated mixture was disrupted with a sonicator (Cell Disruptor 200, Branson Ultrasonics Corp., USA), centrifuged at 10,000 g for 30 minutes at 4 캜, and the supernatant was recovered. The supernatant recovered as described above was loaded onto a column (Glass Econo Column, 1.5 x 30 cm) (Bio-Rad, USA) filled with nikel resin (Invitrogen, USA) at a rate of 0.5 ml / min Expressed HMGB1-Abox protein was extracted. The HMGB1-Abox protein-bound column was washed with 100 ml of a buffer solution (50 mM Na ₂ H ₂ PO ₄ , 300 mM NaCl, 20 mM imidazole) (pH 8.0) to obtain a nonspecifically bound protein Respectively. Thereafter, washings were repeated with increasing concentrations of imidazole to separate and purify the HMGB1-Abox protein.

<1-2> Expression and purification of TAT-HMGB1-Abox nucleic acid delivery peptide

(TAT-HMGB1-Abox) was obtained in the same manner as in Example <1-1>, except that pET21a vector (Novagen, USA) into which the polynucleotide of SEQ ID NO: 4 encoding the TAT- Proteins were separated and purified.

Preparation of nucleic acid delivery complexes

<2-1> Preparation of Luc-DNA: HMGB1-Abox: R3V6 complex

0.5 μg of the pCMV-Luc plasmid containing the luciferase gene in PBS and 1.5 μg of the HMGB1-Abox peptide isolated and purified in Example <1-1> were added and reacted at room temperature for 30 minutes to obtain Luc-DNA : HMGB1-Abox complex was first prepared.

2.5 μg and 5.0 μg of R3V6 peptide micelle (Peptron, Korea) having the amino acid sequence of SEQ ID NO: 5 was added to the above Luc-DNA: HMGB1-Abox complex at a ratio of 15 Min to prepare various nucleic acid delivery complex Luc-DNA: HMGB1-Abox: R3V6 complexes having different ratios of R3V6, respectively.

<2-2> Preparation of Luc-DNA: TAT-HMGB1-Abox: R3V6 complex

0.5 μg of the pCMV-Luc plasmid containing the luciferase gene in PBS and 2.0 μg of the HMGB1-Abox peptide isolated and purified in Example <1-2> were added and reacted at room temperature for 30 minutes to obtain Luc-DNA : TAT-HMGB1-Abox complex was first prepared.

2.5, and 5.0 μg of R3V6 peptide micelle (Peptron, Korea) having the amino acid sequence of SEQ ID NO: 5 was added to the Luc-DNA: TAT-HMGB1-Abox complex, For 15 minutes to prepare Luc-DNA: TAT-HMGB1-Abox: R3V6 complex, which is a nucleic acid delivery complex having various ratios of R3V6, respectively.

<2-3> Preparation of Luc-siRNA: HMGB1-Abox: R3V6 complex

The expression of the HMGB1-chimeric protein isolated and purified in Example <1-1> in 0.5 [mu] g of a mixture of two siRNAs having the nucleotide sequence of SEQ ID NO: 6 and SEQ ID NO: 7 knock- 1.5 μg of Abox peptide was added and reacted at room temperature for 30 minutes to prepare Luc-siRNA: HMGB1-Abox complex first.

The Luc-siRNA: HMGB1-Abox complex was treated at increasing ratios such as 0.5 μg, 2.5 μg, and 5.0 μg of R3V6 peptide micelle (Peptron, Korea) having the amino acid sequence of SEQ ID NO: Min to prepare various nucleic acid delivery complex Luc-siRNA: HMGB1-Abox: R3V6 complexes having different ratios of R3V6, respectively.

Confirmation of formation of nucleic acid delivery complex

<3-1> Gel-Retardation Assay

Each of the Luc-DNA: HMGB1-Abox: R3V6 complex, Luc-DNA: TAT-HMGB1-Abox: R3V6 complex and Luc-siRNA: HMGB1-Abox: R3V6 complex prepared in Example 2 was dissolved in 1% The gel was electrophoresed at 100V for 30 minutes, and the gel was irradiated with UV light to confirm formation of a complex. The higher the degree of binding of HMGB1-Abox (or TAT-HMGB1-Abox) peptide and R3V6 peptide micelle to the Luc-DNA or Luc-siRNA, the lower the mobility of the complex on the gel, And the degree of detection by the UV on the gel is lowered.

The mobility of Luc-DNA: HMGB1-Abox complex or Luc-DNA: HMGB1-Abox: R3V6 complex was significantly higher than that of Luc-DNA (pCMV-Luc plasmid) , And complexes were not well detected on the gel (when the amount of R3V6 was more than 5 times that of DNA) when the amount of R3V6 exceeded 2.5 占 퐂 (FIG. 2 (A)). From the above results, it can be seen that the Luc-DNA: HMGB1-Abox: R3V6 complex is properly formed in the embodiment <2-1>.

In the case of Luc-DNA: TAT-HMGB1-Abox: R3V6 complex, Luc-DNA: TAT-HMGB1-Abox complex or Luc-DNA: TAT-HMGB1-Abox: Luc-DNA (pCMV-Luc plasmid) The mobility of the R3V6 complex was markedly decreased and the complex was not well detected on the gel (when the amount of R3V6 was 10 times or more as high as that of DNA) as the amount of R3V6 exceeded 5.0 占 퐂 (FIG. 2B) . From the above results, it can be seen that the Luc-DNA: TAT-HMGB1-Abox: R3V6 complex is properly formed in the embodiment <2-2>.

Finally, in the case of the Luc-siRNA: HMGB1-Abox: R3V6 complex, the mobility of the siRNA (polynucleotide of SEQ ID NO: 6 and polynucleotide of SEQ ID NO: 7) was significantly reduced by HMGB1-Abox peptide added in different amounts , And no complex was detected on the gel while R3V6 was added (Fig. 2 (C)). From the above results, it can be seen that Luc-siRNA: HMGB1-Abox: R3V6 complex was properly formed in the above Example <2-3>.

<3-2> Zeta-size measurement

In order to confirm the size when the target nucleic acid was complexed with the HMGB1-Abox peptide and the R3V6 peptide micelle, 0.5 μg of the pCMV-Luc plasmid, 1.5 μg of the HMGB1-Abox peptide and 3 μg of the R3V6 peptide micelle The size of the complex prepared by adding 10 [mu] g was measured with a Zetasizer Nano ZS system (Malvern Instruments, UK).

As a result, it was confirmed that the Luc-DNA: HMGB1-Abox: R3V6 complex had a size of 228 ± 81 ㎚ in diameter and could be sufficiently delivered into cells (Table 1).

Avg. Size ± SE (nm) Avg. PDI Avg. Zeta
 potential (mV) PLL 173 ± 108 0.299 0.0572 ± 0.032 HMGB1-Abox 123 ± 9 0.0316 0.0269 0.019 R3V6 1241 ± 26 0.93 0.0687 0.046 Luc-DNA: HMGB1-Abox: R3V6 complex 228 ± 81 0.462 0.0327 + 0.016

Determination of optimal ratio of nucleic acid delivery complexes showing optimal delivery efficiency

<4-1> The optimal ratio of HMGB1-Abox peptide

In order to confirm the optimal ratio of the HMGB1-Abox peptide added when forming the nucleic acid delivery complex exhibiting the optimal delivery efficiency, 0.5 μg of the pCMV-Luc plasmid and 0.5 μg of the R3V6 peptide micelles were added to prepare Luc-DNA: HMGB1-Abox: R3V6 complex, and the addition amount of HMGB1-Abox peptide was 0.15 μg, 0.25 μg. 0.35,, 0.5,, 1.0,, 1.5,, 2.0 및 and 2.5 ㎍.

The Luc-DNA: HMGB1-Abox: R3V6 complex prepared as described above was treated with Raw264.7 cells (ATCC) and the amount of expressed luciferase was detected to confirm nucleic acid delivery efficiency. More specifically, Raw 264.7 cell 1 cultured in DMEM medium (Dulbecco's modified Eagle's medium) supplemented with penicillin, streptomycin 1%, and 10% serum for about 24 hours before treating Luc-DNA: HMGB1-Abox: R3V6 complex × 10 ⁵ cells were dispensed into each well of a 6-well plate and cultured at 37 ° C and 5% CO ₂ for about 18 hours until the cells grew to about 70% of the plate. After the culture, the culture medium was removed from each well, and 1 ml of the medium without serum was washed and dispensed. Two ml of the same medium was dispensed into each well, and the Luc-DNA: HMGB1-Abox: R3V6 complex was added And cultured for 4 hours at 37 ° C and 5% CO ₂ . After 4 hours, 2 ml of DMEM medium containing 1% penicillin, streptomycin and 10% serum was injected into each well, cultured for 24 hours at 37 ° C and 5% CO ₂ , and the medium was removed from each well And the cells were washed with 1 ml of PBS (pH 7.4). The washed cells were lysed with 200 [mu] l of lysis buffer. Then, each sample was well mixed and centrifuged at 13000 rpm for 3 minutes at 4 ° C to take the supernatant. 50 μl of the supernatant was added with 50 μl of luciferase substrate solution (Promega, USA), and the luciferase activity was measured with a luminometer (Detection System GmbH, Pforzheim, Germany).

As a result, when 2.0 의 of HMGB1-Abox peptide was added, it showed the highest luciferase activity and showed the optimal nucleic acid delivery efficiency (Fig. 3 (A)).

From the above results, it can be seen that when the HMGB1-Abox peptide is contained in an amount of 2 parts by weight to 5 parts by weight based on 1 part by weight of the nucleic acid, the optimal nucleic acid delivery efficiency is shown.

<4-2> Optimal ratio of R3V6 peptide micelles

<4-2-1> Optimal ratio of R3V6 peptide micelles for DNA delivery

In order to confirm the optimal ratio of the R3V6 peptide micelle added when forming the nucleic acid delivery complex exhibiting the optimal delivery efficiency, 0.5 μg of the pCMV-Luc plasmid and 1.5 μg of HMGB1 -Abox peptide was added to prepare Luc-DNA: HMGB1-Abox: R3V6 complex, and the amount of R3V6 peptide micelle added was varied to 0.5, 2.5, 5.0, 7.5, 10, 12.5 and 15 μg. The Luc-DNA: HMGB1-Abox: R3V6 complex prepared as described above was treated with Raw264.7 cells (ATCC) and the amount of the expressed luciferase was detected in the same manner as in Example <4-1> And the transfer efficiency was confirmed.

As a result, when 10 μg of R3V6 peptide micelle was added, it showed the highest luciferase activity and showed an optimal nucleic acid delivery efficiency (FIG. 3 (B)).

From the above results, it can be seen that the optimal nucleic acid delivery efficiency is shown when R3V6 peptide micelle is contained in an amount of 10 parts by weight to 30 parts by weight based on 1 part by weight of the nucleic acid.

<4-2-2> Optimal ratio of R3V6 peptide micelles for RNA delivery

The Luc-siRNA: HMGB1-Abox: R3V6 complex was prepared by adding 0.5 μg of Luc-siRNA (SEQ ID NO: 6 and SEQ ID NO: 7) and 1.5 μg of HMGB1-Abox peptide in the same manner as in Example <2-3> The addition amount of R3V6 peptide micelles was varied to 0.5, 2.5, 5.0, 7.5, 10 and 15 μg. The thus-prepared Luc-siRNA: HMGB1-Abox: R3V6 complex was treated with Raw264.7 cells (ATCC) into which luciferase was introduced by introducing a pCMV-Luc plasmid into the cell suspension to inhibit the expression of luciferase. Was detected by the same method as in Example < 4-1 >, and the nucleic acid delivery efficiency was confirmed.

As a result, when 10 占 퐂 of R3V6 peptide micelle was added, it showed the lowest luciferase activity and showed the optimal nucleic acid delivery efficiency (Fig. 3 (C)).

From the above results, it can be seen that the optimal nucleic acid delivery efficiency is shown when R3V6 peptide micelle is contained in an amount of 10 parts by weight to 30 parts by weight based on 1 part by weight of the nucleic acid.

Identification of transfection efficiency of nucleic acid delivery complex

<5-1> Check DNA transfection efficiency

 The nucleic acid delivery complex was prepared in the same manner as in Example <2-1> except that 0.5 μg of pCMV-Luc plasmid, 1.5 μg of HMGB1-Abox peptide and 10 μg of R3V6 peptide micelle were used to prepare Luc-DNA: HMGB1- Abox: R3V6 complex was prepared, and the efficiency of nucleic acid delivery was confirmed by detecting the activity of luciferase in the same manner as in Example <4-1>. Luc-DNA: HMGB1-Abox complex, Luc-DNA-R3V6 complex, Luc-DNA: Poly-L-Lysine and Luc- DNA: HMGB1-Abox: DNA: Lipofectamine.

As a result, the Luc-DNA: HMGB1-Abox: R3V6 complex exhibited significantly higher DNA transfer efficiency than the Luc-DNA: HMGB1-Abox complex and Luc-DNA-R3V6 complex and exhibited a higher transfer efficiency than the conventionally known PLL (Poly_L_Lysine) But the delivery efficiency thereof was found to be somewhat lower than that of lipofectamine, which has been widely used (Fig. 4 (A)).

<5-2> Identification of transfection efficiency of RNA

 (SEQ ID NO: 6 and SEQ ID NO: 7), 1.5 μg of HMGB1-Abox peptide, and 10 μg of R3V6 peptide micelles were added to each well in the same manner as in Example <2-3> , The Luc-siRNA: HMGB1-Abox: R3V6 complex was prepared and luciferase activity was detected in the same manner as in Example <4-2-2> to confirm the nucleic acid delivery efficiency. Luc-siRNA, HMGB1-Abox complex, Luc-siRNA-R3V6 complex, Luc-siRNA: Polyethylenimide 25K and Luc-siRNA: Lipofectamine were used for the nucleic acid delivery efficiency of the Luc-siRNA: HMGB1-Abox: R3V6 complex. .

As a result, the Luc-siRNA: HMGB1-Abox: R3V6 complex showed higher RNA delivery efficiency than the Luc-siRNA: HMGB1-Abox complex and the Luc-siRNA-R3V6 complex and exhibited higher delivery efficiency than the conventionally known PEI25K (Polyethylenimide 25K) But the delivery efficiency thereof was found to be somewhat lower than that of lipofectamine, which has been widely used (Fig. 4 (B)).

Cytotoxicity of nucleic acid delivery complexes

A nucleic acid delivery complex was prepared in the same manner as in Example <2-1> or Example <2-3> except that 0.5 μg of pCMV-Luc plasmid or 0.5 μg of Luc-siRNA (SEQ ID NO: 6 and SEQ ID NO: 7) , HMGB1-Abox: R3V6 complex and Luc-siRNA: HMGB1-Abox: R3V6 complex were prepared using 1.5 μg of HMGB1-Abox peptide and 10 μg of R3V6 peptide micelle, (ATCC), and MTT assay (MTT assay) was performed to analyze cytotoxicity. The cytotoxicity of Luc-DNA: HMGB1-Abox: R3V6 complex was compared with that of PLL (poly-L-lysine), PEI25K (Polyethylenimide 25K) and lipofectamine.

As a result, the Luc-DNA: HMGB1-Abox: R3V6 complex and the Luc-siRNA: HMGB1-Abox: R3V6 complex showed higher cell survival rate than PLL (Poly_L_Lysine), PEI25K (Polyethylenimide 25K) and lipofectamine, It was confirmed that the cytotoxicity was less than that of the known materials (Fig. 5).

Identification of target cell specificity of nucleic acid delivery complex

(SEQ ID NO: 6 and SEQ ID NO: 7) conjugated with 0.5 F of FITC, 1.5 의 of HMGB1-Abox peptide and 10 ㎍ of FITC-conjugated FITC conjugate were prepared in the same manner as in Example < Luc-siRNA: HMGB1-Abox: R3V6 complex was prepared using R3V6 peptide micelles and the complex was treated with mouse lung epithelial cell LA-4 (ATCC) and then analyzed by flow cytometry to determine whether FITC- The number of introduced cells was measured. Luc-siRNA, HMGB1-Abox complex, Luc-siRNA-R3V6 complex, Luc-siRNA: Polyethylenimide 25K and Luc-siRNA: Lipofectamine were used for the nucleic acid delivery efficiency of the Luc-siRNA: HMGB1-Abox: R3V6 complex. .

As a result, it was confirmed that the Luc-siRNA: HMGB1-Abox: R3V6 complex exhibiting a low cell introduction efficiency as compared with lipofectamine showed a particularly excellent cell introduction efficiency in lung cells (FIG. 6).

From the above results, it can be seen that the complex of the present invention exhibits particularly high nucleic acid delivery specificity to lung cells.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the scope of the invention is not limited to the disclosed exemplary embodiments. It will be possible to change it appropriately.

<110> Industry-University Cooperation Foundation Hanyang University <120> Complex for nucleic acid delivery <130> HY120053N <160> 7 <170> Kopatentin 1.71 <210> 1 <211> 67 <212> PRT <213> Homo sapiens <400> 1 Met Ser Ser Tyr Ala Phe Phe Val Gln Thr Cys Arg Glu Glu His Lys   1 5 10 15 Lys Lys His Pro Asp Ala Ser Val Asn Phe Ser Glu Phe Ser Lys Lys              20 25 30 Cys Ser Glu Arg Trp Lys Thr Met Ser Ala Lys Glu Lys Gly Lys Phe          35 40 45 Glu Asp Met Ala Lys Ala Asp Lys Ala Arg Tyr Glu Arg Glu Met Lys      50 55 60 Thr Tyr Ile  65 <210> 2 <211> 201 <212> DNA <213> Homo sapiens <400> 2 atgccatcat atgcattttt tgtgcaaact tgtcgggagg agcataagaa gaagcaccca 60 gatgcttcag tcaacttctc agagttttct aagaagtgct cagagaggtg gaagaccatg 120 tctgctaaag agaaaggaaa atttgaagat atggcaaaag cggacaaggc ccgttatgaa 180 agagaaatga aaacctatat c 201 <210> 3 <211> 92 <212> PRT <213> Artificial Sequence <220> <223> Recombinant polypeptide comprising both TAT and HMGB1-Abox <400> 3 Lys Leu Arg Lys Lys Arg Arg Gln Arg Arg Arg Glu Phe Met Gly Lys   1 5 10 15 Gly Asp Pro Lys Lys Pro Arg Gly Lys Met Ser Ser Tyr Ala Phe Phe              20 25 30 Val Gln Thr Cys Arg Glu Glu His Lys Lys Lys His Pro Asp Ala Ser          35 40 45 Val Asn Phe Ser Glu Phe Ser Lys Lys Cys Ser Glu Arg Trp Lys Thr      50 55 60 Met Ser Ala Lys Glu Lys Gly Lys Phe Glu Asp Met Ala Lys Ala Asp  65 70 75 80 Lys Ala Arg Tyr Glu Arg Glu Met Lys Thr Tyr Ile                  85 90 <210> 4 <211> 276 <212> DNA <213> Artificial Sequence <220> <223> Polynucleotide sequence coding for recombinant polypeptide          comprising both TAT and HMGB1-Abox <400> 4 aagttcgca aaaagcggag acagagacgc agggaattca tgggcaaagg agatcctaag 60 aagccgagag gcaaaatgtc atcatatgca ttttttgtgc aaacttgtcg ggaggagcat 120 aagaagaagc acccagatgc ttcagtcaac ttctcagagt tttctaagaa gtgctcagag 180 aggtggaaga ccatgtctgc taaagagaaa ggaaaatttg aagatatggc aaaagcggac 240 aaggcccgtt atgaaagaga aatgaaaacc tatatc 276 <210> 5 <211> 9 <212> PRT <213> Artificial Sequence <220> Polypeptide sequence of peptide micelle R3V6 <400> 5 Arg Arg Val Val Val Val Val Val   1 5 <210> 6 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> Polynucleotide sequence of 1st siRNA for the knock-down of the          luciferase expression <400> 6 ggccuuucac uacuccuacu u 21 <210> 7 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> Polynucleotide sequence of 2nd siRNA for the knock-down of the          luciferase expression <400> 7 guaggaguag ugaaaggccu u 21

Claims (11)

The target nucleic acid;
A nucleic acid delivery peptide comprising an HMGB1 A box peptide and coupled to said nucleic acid through charge interaction; And
And a peptide micelle comprising a polypeptide having an amino acid sequence of the following formula 1 and surrounding the nucleic acid and the nucleic acid transfer peptide in the form of micelle through charge interaction or hydrophobic interaction,
[Chemical Formula 1]
R _n -X _m
In Formula 1,
R is arginine (Agr)
X is a hydrophobic amino acid,
n is an integer of 1 to 7,
and m is an integer from 2 to 20. 2. The nucleic acid delivery complex of claim 1, wherein the HMGB1 A box peptide comprises the amino acid sequence of SEQ ID NO: 1. The nucleic acid delivery complex according to claim 1, wherein the nucleic acid transfer peptide further comprises a transmembrane domain at the N-terminus of the HMGB1 A box peptide. The nucleic acid delivery complex according to claim 3, wherein the transmembrane domain is TAT. The method of claim 1, wherein the hydrophobic amino acid is selected from the group consisting of isoleucine (Ile), Valine, Leu, Phe, Cys, Methionine and Alanine Nucleic acid delivery complex. The nucleic acid delivery complex according to claim 5, wherein the hydrophobic amino acid is alanine (Ala) or valine (Val). The nucleic acid delivery complex of claim 1, wherein n is an integer of 2 to 5. 2. The nucleic acid delivery complex according to claim 1, wherein m is an integer of 5 to 10. The method according to claim 1,
The nucleic acid transport peptide is contained in a ratio of 2 to 5 parts by weight based on 1 part by weight of the nucleic acid to be delivered;
Wherein the peptide micelle is contained in an amount of 10 to 30 parts by weight per 1 part by weight of the nucleic acid to be delivered. The nucleic acid delivery complex according to claim 1, wherein the target nucleic acid is DNA. The nucleic acid delivery complex according to claim 1, wherein the nucleic acid to be delivered is transferred to lung cells.

Images