KR20110114952A - Gene delivery system - Google Patents

Abstract

The present invention relates to a gene delivery system comprising: (a) a phospholipid liposome having a double membrane structure with a cationic peptide bound to its surface; And (b) a complex comprising (i) a nucleotide molecule as a cargo, and (ii) a cationic peptide as a cell penetration peptide, wherein the complex is encapsulated within the phospholipid liposome. encapsulation). The present invention utilizes two characteristics of the cationic peptide included in the gene carrier as a cell-penetrating peptide and a complex, and the cationic peptide used in the present invention forms a tight complex with a nucleotide molecule as a cargo to form liposomes. Nucleotide molecules can be encapsulated in a high loading efficiency, the cationic peptide bound to the liposome surface included in the present invention is excellent in cell-permeability and can deliver the carrier to the cytoplasm, and the intracellular absorption rate is excellent. The present invention can also be applied as an anticancer or anticancer agent sensitizing composition.

Description

Gene Delivery System

The present invention relates to a gene carrier, a method for producing the gene carrier, an anticancer composition or an anticancer sensitizing composition.

Gene therapy is a new approach to the treatment of disease and uses genetic material as a therapeutic agent. Applied in accordance with the practice, the therapy involves expression of exogenously introduced genes for functional replacement of missing or missing genes, and has been used to treat inherited and acquired diseases. Gene therapy can be extended to control endogenous gene expression using genetic materials such as antisense oligodeoxynucleotides (ODNs), small interfering RNAs (siRNAs), and ribozymes.

However, most gene therapy strategies have been found to be inadequate for systemic applications due to the very short half-life of the genetic material in the bloodstream and difficulty in crossing cell membranes. Thus, the success of gene therapy depends on the delivery of the nucleic acid therapeutic agent in the formulation of ODN or the entire gene (plasmid DNA) to the site of cellular action required. As a result, the development of an efficient non-invasive transport strategy is required.

The transcription factor decoy provides a potential approach to down-regulating transcription factors that are over-expressed during the onset of cancer, in in vitro and in It is known to be effective in inhibiting gene transcription in vivo . One such transcription factor decoy is an antisense ODN having a sequence corresponding to the consensus sequence of the NF-κB binding site. The ODN acts by blocking NF-κB binding to the promoter region of the target gene, thereby stopping the transcription of proliferation-related genes [1, 2]. ODN, which offers the benefit of high specificity and minimal toxicity, has been reported as a promising approach to optimizing chemotherapy for urinary carcinoma [3]. In order for ODN to down-regulate gene expression, ODN must first penetrate into target cells [4]. So, two major obstacle remaining following the use of ODN: incompetence and in the ODN negatively charged particles striking to pass through the cell membrane Instability of naked ODN in vivo .

Cell-penetrating peptides (CPPs) that can efficiently cross biological membranes and enter the cytoplasm [5,6] are in It is possible to deliver multiple cargoes [7] with high efficiency in vitro and in vivo . Importantly, CPPs generally exhibit low toxicity [8]. Since CPPs are not integrated into the genetic targets of the genetic material they deliver, CPPs have not been shown to cause oncogenic activation [9]. CPP has been extensively studied and has been successfully used for intracellular delivery of small drug molecules such as synthetic macromolecules [11], micelles, micelles, liposomes [2, 12, 13] and nanoparticles [14, 15]. Successful use of CPPs for intracellular delivery of numerous types of drug carriers will offer huge potential for novel therapeutics [10].

Because of the multiple positive charges of CPP, CPP can cause electrostatic interactions and has been used as polymerization / condensing agents. In addition, CPP may be covalently attached to the delivery vehicle to improve vector internalization as a whole [8]. One preferred method is to polymerize oligonucleotides with positively charged PEI, which resulted in significant toxicity of the product [16]. Nona-arginine peptides (9R), which do not exhibit cytotoxicity, are very suitable candidate peptides for use in polymerization. Drug encapsulation efficiency is an important aspect of drug delivery because encapsulation promotes efficient treatment by protecting cargo during delivery. This is especially true if the cargo is an ODN that is susceptible to degradation by nucleases [17]. Synthetic vectors are an attractive choice as inclusion vectors because they provide opportunities for improved safety, greater flexibility and ease of manufacture [18].

Throughout this specification many patent documents are referenced and their citations are indicated. The disclosures of the cited patent documents are incorporated by reference herein in their entirety, and the level of the technical field to which the present invention belongs and the contents of the present invention are more clearly described.

The inventors have sought to develop nucleotide carriers using cationic peptides that act as cell penetrating peptides (CPPs) and complex agents. As a result, when a phospholipid liposome having a double-membrane structure in which a cationic peptide and a nucleotide complex are encapsulated and a cationic peptide is bonded to a surface thereof is prepared, a hard complex is formed by a cationic peptide to form a nucleotide. The present invention was completed by confirming that the encapsulation efficiency was increased and the cell-permeability was improved by the cationic peptide bound to the surface of the liposome to increase the intracellular uptake rate.

Accordingly, it is an object of the present invention to provide a gene delivery system.

Another object of the present invention is to provide a method for producing a gene carrier.

Another object of the present invention to provide an anticancer composition.

Another object of the present invention to provide an anticancer sensitizing composition.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to one aspect of the present invention, the present invention provides a gene delivery system comprising: (a) a phospholipid liposome having a double membrane structure with a cationic peptide bound to its surface; And (b) a complex comprising (i) a nucleotide molecule as a cargo, and (ii) a cationic peptide as a cell penetration peptide, wherein the complex is encapsulated within the phospholipid liposome. encapsulation).

The inventors have sought to develop nucleotide carriers using cationic peptides that act as cell penetrating peptides (CPPs) and complex agents. As a result, when a phospholipid liposome having a double-membrane structure in which a cationic peptide and a nucleotide complex are encapsulated and a cationic peptide is bonded to a surface thereof is prepared, a hard complex is formed by a cationic peptide to form a nucleotide. Encapsulation efficiency was increased, the cell-permeability was improved by the cationic peptide bound to the surface of the liposomes was confirmed to increase the intracellular uptake rate.

According to a preferred embodiment of the present invention, the cationic peptide used in the gene carrier of the present invention is indirectly bound to the surface of the phospholipid liposome through a hydrophilic polymer bound to the surface of the phospholipid liposome.

As used herein, the term “liposome” refers to a lipid carrier made by forming a closed lipid bilayer. Liposomes are lipid bilayers that are biocompatible and biphilic, allowing them to pass through hydrophobic membranes with hydrophilic material inside.

As used herein, the term “phospholipid” means a phospholipid capable of forming liposomes, unless otherwise specified, and refers to lecithin (phosphatidyl choline), phosphatidyl serine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol and sphingomyelin Including but not limited to. Lecithin can be obtained from eggs, soybeans and other plants, most preferably soybean lecithin. Synthetic, semi-synthetic and natural lecithins that can be used in the present invention include soybean lecithin, distearoylphosphatidylcholine, hydrogenated soybean lecithin, egg lecithin, dioleoylphosphatidylcholine, hydrogenated egg lecithin, dielaidoylphosphatidylcholine, diuretic Palmitoylphosphatidylcholine and dimyristoylphosphatidylcholine.

As used herein, the term “encapsulation” refers to encapsulation to enclose a delivery material and to efficiently incorporate it in vivo.

Cationic peptides used in the gene carriers of the present invention are used in the present invention as cell penetrating peptides (CPPs) and to form complexes with cargoes. When cationic peptides act as cell-penetrating peptides, they bind to the surface of the phospholipid liposomes and are encapsulated within the phospholipid liposomes when they form a complex with a cargo.

As used herein, the term “cell penetrating peptide (CPP)” refers to a peptide having the ability to carry a cargo of a subject of delivery into a cell in vitro and / or in vivo, and the term “cell penetrating peptide (CPP)”. Cell membrane permeation domain ”.

Cationic peptides used as cell-penetrating peptides and complexes in the present invention are preferably composed of human immunodeficiency virus (HIV) -Tat, 6-40 amino acid residues, and at least three of the amino acid residues are lysine, arginine or lysine. An arginine peptide, KALA or protamine, more preferably consisting of 8-15 amino acid residues and at least three of said amino acid residues are lysine, arginine or a peptide that is lysine and arginine, even more preferably 8-10 amino acids At least six of said amino acid residues are peptides consisting of lysine, arginine or lysine and arginine, most preferably arginine peptides consisting of 9 arginine residues. The amino acid used may include not only L-form, but also amino acids of D-form.

Hydrophilic polymers used in the present invention include a variety of hydrophilic polymers known in the art, preferably polyethylene glycol, polyvinylpyrrolidone, polyoxazoline, polypropylene glycol, copolymers of polyethylene glycol and polypropylene glycol, Chitosan, dextran, cellulose, heparin, alginate or monoesterified derivatives thereof, more preferably polyethylene glycol, polyvinylpyrrolidone, chitosan, dextran, alginate, and most preferably polyethylene glycol.

As used herein, the term “antisense oligonucleotide” refers to DNA or RNA or derivatives thereof that contain a nucleotide sequence complementary to a sequence of a particular mRNA, which binds to a complementary sequence within the mRNA and translates the mRNA into a protein. There is a characteristic that inhibits. Antisense nucleotide sequence of the present invention means a DNA or RNA sequence that is complementary to the mRNA of the target gene and capable of binding to the mRNA of the target gene, and translates the mRNA of the target gene, translocation into the cytoplasm, maturation or May inhibit essential activity for all other global biological functions. The antisense oligonucleotide has a length of 6 to 100 bases, preferably 8 to 60 bases, and more preferably 10 to 40 bases.

The antisense oligonucleotides can be modified at one or more bases, sugars or backbone positions to enhance efficacy (De Mesmaeker et al., Curr Opin Struct Biol . , 5 (3): 343-55, 1995). Oligonucleotide backbones can be modified with phosphorothioates, phosphoroesters, methyl phosphonates, short chain alkyls, cycloalkyls, short chain heteroatomics, heterocyclic intersaccharide bonds, and the like. In addition, antisense oligonucleotides may include one or more substituted sugar moieties. Antisense oligonucleotides may comprise modified bases. Modified bases include hypoxanthine, 6-methyladenine, 5-me pyrimidine (particularly 5-methylcytosine), 5-hydroxymethylcytosine (HMC), glycosyl HMC, gentobiosil HMC, 2-aminoadenine, 2 Thiouracil, 2-thiothymine, 5-bromouracil, 5-hydroxymethyluracil, 8-azaguanine, 7-deazaguanine, N6 (6-aminohexyl) adenine, 2,6-diaminopurine, etc. There is this.

The term “aptamer” refers to an oligonucleotide (generally an RNA molecule) that binds to a specific target. Preferably, “aptamer” in the context of the present invention means oligonucleotide aptamer (eg, RNA aptamer). For general information about aptamers, see Bock LC et al., Nature 355 (6360): 5646 (1992); Hoppe-Seyler F, Butz K "Peptide aptamers: powerful new tools for molecular medicine". J Mol Med . 78 (8): 42630 (2000); Cohen BA, Colas P, Brent R. "An artificial cell-cycle inhibitor isolated from a combinatorial library". Proc Natl Acad Sci USA. 95 (24): 142727 (1998).

The term "siRNA" refers to a short double-chain RNA that can induce RNA interference (RNAi) through the cleavage of a particular mRNA. It consists of a sense RNA strand having a sequence homologous to the mRNA of the target gene and an antisense RNA strand having a sequence complementary thereto. siRNA is provided as an efficient gene knock-down method or gene therapy because it can inhibit the expression of the target gene.

siRNAs are not limited to the complete pairing of double-stranded RNA pairs paired with RNA, but paired by mismatches (the corresponding bases are not complementary), bulges (there are no bases corresponding to one chain), etc. May be included. The total length is 10 to 100 bases, preferably 15 to 80 bases, most preferably 20 to 70 bases. The siRNA terminal structure can be blunt or cohesive, as long as the expression of the target gene can be suppressed by the RNAi effect. The cohesive end structure is possible in both a three-terminal protruding structure and a five-terminal protruding structure. The number of protruding bases is not limited. For example, the number of bases may be 1 to 8 bases, preferably 2 to 6 bases. The full length of the siRNA herein is expressed as the sum of the length of the central double chain portion and the length constituting the single chain protrusion of both ends. In addition, siRNA is a low-molecular RNA (e.g., a natural RNA molecule such as tRNA, rRNA, viral RNA or artificial RNA) in the protruding portion of one end to the extent that can maintain the expression inhibitory effect of the target gene Molecules). The siRNA terminal structure does not need to have a cleavage structure at both sides, and may be a stem loop type structure in which one terminal portion of the double chain RNA is connected by a linker RNA. The length of the linker is not particularly limited as long as it does not interfere with pairing of stem portions. The siRNA of the present invention has an antisense RNA strand comprising a sequence complementary to the target gene or target nucleotide to act specifically on the target gene.

The term “shRNA” refers to 50-70 nucleotides in single strands, in Stem-loop structure in vivo . Long RNAs of 19-29 nucleotides complementary to both loop regions of 5-10 nucleotides form base pairs to form a double stranded stem.

The term “miRNA (microRNA)” regulates gene expression and refers to a single stranded RNA molecule consisting of 21-23 nucleotides in length. miRNAs are oligonucleotides that are not expressed intracellularly and have a short stem-loop structure. miRNAs are homologous, in whole or in part, with one or more mRNAs (messenger RNAs) and inhibit target gene expression through complementary binding to the mRNAs.

The term “ribozyme” is a type of RNA that has the same function as an enzyme that recognizes and cleaves itself on the base sequence of a particular RNA. Ribozyme is a complementary nucleotide sequence of a target messenger RNA strand consisting of a region that binds with specificity and a region that cleaves the target RNA.

The term “DNAzyme” is a single-stranded DNA molecule with enzymatic activity. DNAzyme (10-23 DNAzyme) consisting of 10-23 nucleotides cleaves RNA strands at specific positions under physiologically similar conditions. 10-23 DNAzyme cleaves between any exposed purine and pyrimidine without base pairing. 10-23 DNAzyme (DNAzyme) is an active site of the enzyme consisting of 15 conserved sequences (5'-GGCTAGCTACAACGA-3 ') and 7- to recognize RNA substrates to the left and right of the enzyme. It consists of an RNA substrate binding domain consisting of eight DNA sequences.

As used herein, the term “double-stranded ODN corresponding to a transcript binding sequence” refers to a double-stranded oligonucleotide that binds to a transcription factor in a sequence specific manner to inhibit transcriptional activity and “decoy oligonucleotides”. Used interchangeably with The length of the double-stranded oligonucleotides is preferably from 6 to 50 nucleotides in full length, more preferably from 10 to 40 nucleotides, most preferably from 15 to 30 nucleotides. The transcription factor is a transcription factor associated with cancer, preferably NF-κB, AP-1, STAT and steroid receptors, more preferably NF-κB.

According to another preferred embodiment of the invention, the nucleotide molecule of the carrier complexed with the cationic peptide in the present invention is an antisense oligonucleotide, aptamer, siRNA, shRNA, miRNA, ribozyme, DNAzyme or transcription factor Double-stranded ODN corresponding to the binding sequence, more preferably double-stranded ODN corresponding to the binding sequence of the NF-κB transcription factor, most preferably sequence listing It is a double-chain oligonucleotide consisting of the first sequence and the second sequence.

According to another preferred embodiment of the present invention, the complex of the present invention has a ratio of the charge (negative charge: positive charge) of the nucleotide molecule as the cargo and the cationic peptide as the cell penetration peptide is 1: 1. It is formed from -12, more preferably 1: 4-9, most preferably 1: 5-7. If the charge ratio is exceeded, there is a problem that the transfer efficiency of the gene is reduced.

According to another preferred embodiment of the present invention, the gene carrier of the present invention has an average size of 80-200 nm, more preferably 90-150 nm, most preferably 100-120 nm.

As used herein, the term “zeta potential” refers to a numerical value representing the overall tendency of the surface charge of liposomes dispersed in water, and that if the zeta potential is negative, it is highly likely that liposomes of negative charge are dispersed in water. If the zeta potential is zero, it means that the liposomes no longer have charges repulsing with each other, which increases the likelihood of aggregation and precipitation into larger particles.

According to a preferred embodiment of the present invention, since the zeta potential value of the gene carrier of the present invention is -30 mV to -10 mV, and has a negative value, the gene carriers of the present invention do not generate aggregation with each other. .

According to a preferred embodiment of the invention, the surface of the phospholipid liposome of the gene carrier of the invention further comprises a targeting ligand.

Targeting ligands suitable for the present invention may include any known in the art, for example antibodies, aptamers, polypeptides, peptides, ligands, lectins, sugars, lipids, glycolipids or nucleic acids may be used as the targeting ligand. have.

More preferably, the targeting ligand used in the present invention is a peptide having a Bipodal Peptide Binder (BPB) structure comprising: (i) a parallel formed interstrand non-covalent bond; A structure stabilizing region comprising antiparallel or parallel and antiparallel amino acid strands; And (ii) target binding region I and target binding region II, each of which is bound to both ends of the structure stabilization site and comprises randomly selected n and m amino acids, respectively.

In the BPB structure, the target binding site I and the target binding site II play a targeting role under binding to the target. Preferably, target binding site I and target binding site II bind to cell surface molecules. For example, BPB as a targeting ligand used in the present invention may be selected from CD cells of T cells (eg, CD1a, CD1b, CD1c, CD1d, CD1e, CD2-CD244 receptors), cancer cell specific surface proteins (eg, Her2 / neu receptors), Ion channels, receptors (e.g., VEGF receptors, G-protein coupled receptors, etc.), Fibronectin extra domain B (ED-B), vascular cell adhesion molecule-1 (VCAM1), nicotinic acetylcholine receptor (nAchR), epihelial cells (EpCAM) Adhesion Molecule), BTA (Bladder Tumor Antigens), estrogen receptor, progesterone receptor, PSA (Prostate-Specific Antigen) and the like.

The basic strategy of BPB is to connect a peptide that is bound to a target at both ends of a rigid peptide backbone. In this case, the rigid peptide backbone acts to stabilize the overall structure of the bipodal peptide provider and enhances the binding of target binding site I and target binding site II to the target molecule. Structural stabilization sites usable in the present invention include parallel, antiparallel or parallel and antiparallel amino acid strands, interstrand hydrogen bonds, electrostatic interactions, hydrophobic interactions, van der Waals interactions, pi-py Protein structure motifs in which non-covalent bonds are formed by interaction, cation-pi interaction, or a combination thereof. Non-covalent bonds formed by hydrogen bonds, electrostatic interactions, hydrophobic interactions, van der Waals interactions, pi-pi interactions, cation-pi interactions, or combinations thereof between the strands are the rigidity of the structure stabilization site. Contribute to.

According to a preferred embodiment of the present invention, interstrand non-covalent bonds at the structure stabilization site include hydrogen bonds, hydrophobic interactions, van der Waals interactions, pi-pi interactions or combinations thereof.

Optionally, there may be a covalent bond at the structured stabilization site. For example, disulfide bonds can be formed at the structured stabilization site to further increase the firmness of the structure stabilized site. The increase in firmness by such covalent bonds is given in consideration of the specificity and affinity of the bipodal peptide binder for the target.

According to a preferred embodiment of the invention, the amino acid strands of the structure stabilization site are linked by a linker. As used herein, the term "linker" as used to refer to the strands refers to the material that connects the strands. For example, when β-hairpin is used as a structure stabilization site, the turn sequence on the β-hairpin serves as a linker, and when leucine zipper is used, a substance connecting two C-terminus of leucine zipper (for example, , Peptide linkers) serve as linkers.

Linkers link parallel, antiparallel or parallel and antiparallel amino acid strands. For example, at least two strands (preferably two strands) aligned in parallel fashion, at least two strands (preferably two strands) aligned in antiparallel fashion, and at least three strands aligned in parallel and antiparallel fashion. The linker (preferably three strands) is connected by the linker.

According to a preferred embodiment of the invention, the linker is a turn sequence or peptide linker.

According to a preferred embodiment of the present invention, the turn sequence is β-turn, γ-turn, α-turn, π-turn or ω-loop (Venkatachalam CM (1968), Biopolymers , 6, 1425-1436; Nemethy G and Printz MP. (1972), Macromolecules , 5, 755-758; Lewis PN et al., (1973), Biochim . Biophys . Acta , 303, 211-229; Toniolo C. (1980) CRC Crit . Rev. Biochem . , 9, 1-44; Richardson JS. (1981), Adv . Protein Chem. , 34, 167-339; Rose GD et al., (1985), Adv . Protein Chem . , 37, 1-109; White EJ and Poet R.-Milner (1987), TIBS, 12, 189-192; Wilmot CM and Thornton JM. (1988), J. Mol . Biol . , 203, 221-232; Milner-White EJ. (1990), J. Mol . Biol . , 216, 385-397; Pavone V et al. (1996), Biopolymers , 38, 705-721; Rajashankar KR and Ramakumar S. (1996), Protein Sci . , 5, 932-946). Most preferably, the turn sequence used in the present invention is β-turn.

When β-turn is used as the turn sequence, it is preferably a type I, type I ', type II, type II', type III or type III 'turn sequence, more preferably type I, type I', type II, type II 'turn sequences, even more preferably type I' or type II 'turn sequences, and most preferably type I' turn sequences (BL Sibanda et al., J. Mol . Biol . , 1989) . , 206, 4, 759-777; BL Sibanda et al., Methods Enzymol. , 1991, 202, 59-82).

According to another preferred embodiment of the present invention, it can be used as a turn sequence in the present invention is described in H. Jane Dyson et al., Eur . J. Biochem . 255: 462-471 (1998), which is incorporated herein by reference. Available for the turn sequence include the following amino acid sequences: X-Pro-Gly-Glu-Val; Ala-X-Gly-Glu-Val (X is selected from 20 amino acids).

According to one embodiment of the invention, when a β-sheet or leucine zipper is used as the structural stabilization site, it is preferred that the peptide linker connects two strands arranged in parallel or two strands arranged in an antiparallel manner. desirable.

Peptide linkers can be used in any known in the art. The sequence of a suitable peptide linker can be selected in consideration of the following factors: (a) the ability to be applied to flexible extended conformation; (b) the ability not to create secondary structures that interact with biological target molecules; And (c) the absence of hydrophobic residues or residues with charges that interact with the biological target molecule. Preferred peptide linkers include Gly, Asn and Ser residues. Other neutral amino acids such as Thr and Ala can also be included in the linker sequence. Suitable amino acid sequences for linkers are described in Maratea et al., Gene 40: 39-46 (1985); Murphy et al., Proc . Natl . Acad Sci . USA 83: 8258-8562 (1986); US Pat. Nos. 4,935,233, 4,751,180 and 5,990,275. The peptide linker sequence may consist of 1-50 amino acid residues.

According to a preferred embodiment of the present invention, the structure stabilization site is a β-hairpin, a linker linked β-sheet or a linker leucine zipper, more preferably the structure stabilized site is a β-hairpin or linker linked β-sheet And most preferably β-hairpin.

As used herein, the term “β-hairpin” refers to the simplest protein motif comprising two β strands, the two β strands representing an antiparallel alignment with each other. In this β-hairpin the two β strands are generally linked by turn sequences.

Preferably, the turn sequence applied to the β-hairpin is a type I, type I ', type II, type II', type III or type III 'turn sequence, more preferably type I, type I', type II , A type II 'turn sequence, even more preferably a type I' or type II 'turn sequence, and most preferably a type I' turn sequence. In addition, X-Pro-Gly-Glu-Val; Alternatively, a turn sequence represented by Ala-X-Gly-Glu-Val (X is selected from 20 amino acids) can also be used for β-hairpins.

According to an exemplary embodiment of the invention, the type I turn sequence is Asp-Asp-Ala-Thr-Lys-Thr, the type I 'turn sequence is Glu-Asn-Gly-Lys, and the type II turn sequence is X- Pro-Gly-Glu-Val; Or Ala-X-Gly-Glu-Val (X is selected from 20 amino acids) and the type II 'turn sequence is Glu-Gly-Asn-Lys or Glu-D-Pro-Asn-Lys.

Peptides with β-hairpin formulations are well known in the art. Tryptophan zipper, as disclosed in, for example, US Pat. No. 6,914,123 and Andrea G. Cochran et al., PNAS, 98 (10): 5578-5583, template-fixed β- disclosed in WO 2005/047503. Β-hairpin variants disclosed in Hairpin Memetic, US Pat. No. 5,807,979 are well known. In addition, peptides with β-hairpin formulations are described in Smith & Regan (1995) Science 270: 980-982; Chou & Fassman (1978) Annu. Rev. Biochem. 47: 251-276; Kim & Berg (1993) Nature 362: 267-270; Minor & Kim (1994) Nature 367: 660-663; Minor & Kim (1993) Nature 371: 264-267; Smith et al. Biochemistry (1994) 33: 5510-5517; Searle et al. (1995) Nat. Struct. Biol. 2: 999-1006; Haque & Gellman (1997) J. Am. Chem. Soc. 119: 2303-2304; Blanco et al. (1993) J. Am. Chem. Soc. 115: 5887-5888; de Alba et al. (1996) Fold. Des. 1: 133-144; de Alba et al. (1997) Protein Sci. 6: 2548-2560; Ramirez-Alvarado et al. (1996) Nat. Struct. Biol. 3: 604-612; Stanger & Gellman (1998) J. Am. Chem. Soc. 120: 4236-4237; Maynard & Searle (1997) Chem. Commun. 1297-1298; Griffiths-Jones et al. (1998) Chem. Commun. 789-790; Maynard et al. (1998) J. Am. Chem. Soc. 120: 1996-2007; And Blanco et al. (1994) Nat. Struct. Biol. 1: 584-590, which is incorporated herein by reference.

When a peptide having β-hairpin conformation is used as the structure stabilization site, most preferably a tryptophan zipper is used.

According to a preferred embodiment of the present invention, the tryptophan zipper used in the present invention is represented by the following general formula (I):

Formula I

X ₁ -Trp (X ₂ ) X ₃ -X ₄ -X ₅ (X ' ₂ ) X ₆ -X ₇

X ₁ is Ser or Gly-Glu, X ₂ and X ' ₂ are independently of each other Thr, His, Val, Ile, Phe or Tyr, X ₃ is Trp or Tyr and X ₄ is Type I, Type I' , Type II, type II 'or type III or type III' turn sequences, X ₅ is Trp or Phe, X ₆ is Trp or Val, and X ₇ is Lys or Thr-Glu.

More preferably, in Formula I, X ₁ is Ser or Gly-Glu, X ₂ and X ' ₂ are independently of each other Thr, His or Val, X ₃ is Trp or Tyr, and X ₄ is Type I , Type I ', type II or type II' turn sequence, X ₅ is Trp or Phe, X ₆ is Trp or Val, and X ₇ is Lys or Thr-Glu.

Even more preferably, in Formula I, X ₁ is Ser or Gly-Glu, X ₂ and X ' ₂ are independently of each other Thr, His or Val, X ₃ is Trp, X ₄ is Type I, type I ', type II or type II' turn sequence, X ₅ is Trp, X ₆ is Trp and X ₇ is Lys or Thr-Glu.

Even more preferably, in Formula I, X ₁ is Ser, X ₂ and X ' ₂ are Thr, X ₃ is Trp, X ₄ is a type I' or type II 'turn sequence, and X ₅ is Trp, X ₆ is Trp and X ₇ is Lys.

Most preferably, in Formula I, X ₁ is Ser, X ₂ and X ' ₂ are Thr, X ₃ is Trp, and X ₄ is a type I' turn sequence (ENGK) or a type II 'turn sequence (EGNK) ), X ₅ is Trp, X ₆ is Trp and X ₇ is Lys.

The β-hairpin peptides usable as structural stabilization sites in the present invention are peptides derived from the B1 domaine of protein G, ie GB1 peptides.

When GB1 peptide is used in the present invention, the structural stabilization site is preferably represented by the following general formula II:

Formula II

X ₁ -Trp-X ₂ -Tyr-X ₃ -Phe-Thr-Val-X ₄

X ₁ is Arg, Gly-Glu or Lys-Lys, X ₂ is Gln or Thr, X ₃ is a Type I, Type I ', Type II, Type II' or Type III or Type III 'turn sequence, X ₄ is Gln, Thr-Glu or Gln-Glu.

More preferably, the structural stabilization site of Formula II is represented by the following Formula II ':

Formula II

X ₁ -Trp-Thr-Tyr-X ₂ -Phe-Thr-Val-X ₃

X ₁ is Gly-Glu or Lys-Lys, X ₂ is a type I, type I ', type II, type II' or type III or type III 'turn sequence, and X ₃ is Thr-Glu or Gln-Glu .

Β-hairpin peptides usable as structural stabilization sites in the present invention are HP peptides. When the HP peptide is used in the present invention, the structural stabilization site is preferably represented by the following general formula III:

General formula Ⅲ

X ₁ -X ₂ -X ₃ -Trp-X ₄ -X ₅ -Thr-X ₆ -X ₇

X ₁ is Lys or Lys-Lys, X ₂ is Trp or Tyr, X ₃ is Val or Thr, and X ₄ is Type I, Type I ', Type II, Type II' or Type III or Type III 'turn. Sequence, X ₅ is Trp or Ala, X ₆ is Trp or Val, and X ₇ is Glu or Gln-Glu.

Another β-hairpin peptide that can be used as a structural stabilization site in the present invention is represented by the following general formula (IV):

Formula IV

X ₁ -X ₂ -X ₃ -Trp-X ₄

X ₁ is Lys-Thr or Gly, X ₂ is Trp or Tyr, X ₃ is a Type I, Type I ', Type II, Type II' or Type III or Type III 'turn sequence, and X ₄ is Thr- Glu or Gly.

According to the present invention, a β-sheet connected by a linker may be used as the structural stabilization site. It is in an extended form of two amino acid strands, parallel or antiparallel, preferably antiparallel, in the β-sheet structure, and hydrogen bonds are formed between the amino acid strands.

In the β-sheet structure, two adjacent ends of two amino acid strands are connected by a linker. As the linker, various turn-sequences or peptide linkers described above may be used. If the turn-sequence is used as a linker, the β-turn sequence is most preferred.

According to another variant of the invention, leucine zippers or leucine zippers linked by linkers may be used as structural stabilization sites. Leucine zippers are conservative peptide domains that cause parallel dimerization of two α-chains and are generally dimerized domains found in proteins involved in gene expression (“Leucine scissors”. Glossary of Biochemistry and Molecular Biology ( (1997) .Ed. David M. Glick.London: Portland Press; Landschulz WH, et al. (1988) Science 240: 1759-1764). Leucine zippers generally comprise a heptad repeat sequence, with the leucine residues located at the fourth or fifth. For example, leucine zippers that may be used in the present invention comprise the amino acid sequence of LEALKEK, LKALEKE, LKKLVGE, LEDKVEE, LENEVAR or LLSKNYH. Specific examples of leucine zippers used in the present invention are described in SEQ ID NO: 39. Each half of the leucine zipper consists of short α-chains with direct leucine contact between the α-chains. The leucine zipper in the transcription factor generally consists of a hydrophobic leucine zipper site and a basic site (site that interacts with the main groove of the DNA molecule). When the leucine zipper is used in the present invention, the basic site is not necessarily required. In the leucine zipper structure, two adjacent ends of two amino acid strands (ie, two α-chains) may be linked by a linker. As the linker, various turn-sequences or peptide linkers described above may be used, and preferably, peptide linkers which do not affect the structure of the leucine zipper are used.

Random amino acid sequences are joined to both ends of the structure stabilization site described above. The random amino acid sequence forms target binding site I and target binding site II. One of the greatest features of the present invention is to prepare a peptide binder in a bipodal manner by connecting target binding site I and target binding site II to both ends of the structure stabilization site. Target binding site I and target binding site II cooperatively bind to the target, thereby greatly increasing the affinity for the target.

The amino acid number n of the target binding site I is not particularly limited, preferably an integer of 2-100, more preferably an integer of 2-50, even more preferably an integer of 2-20, most preferably It is an integer of 3-10.

The amino acid number m of the target binding site II is not particularly limited, preferably an integer of 2-100, more preferably an integer of 2-50, even more preferably an integer of 2-20, most preferably It is an integer of 3-10.

Target binding site I and target binding site II may comprise different or identical number of amino acid residues, respectively. The target binding site I and the target binding site II may include different or identical amino acid sequences, and preferably include different amino acid sequences.

According to another aspect of the present invention, the present invention provides a method of preparing a gene delivery system, comprising the steps of: (a) (i) nucleotide molecules as cargo and (ii) cells Forming a complex comprising a cationic peptide as a cell penetration peptide; (b) mixing the phospholipid and the phospholipid having a cationic peptide bound thereto to form a phospholipid film; And (c) mixing and extruding the complex of step (a) and the phospholipid film of step (b) to obtain a liposome with the complex encapsulated.

Since the method of the present invention is a process for producing the gene carrier of the present invention described above, the common content between the two is omitted in order to avoid excessive complexity of the specification according to the repetitive description.

Detailed description of the method of the present invention for producing a gene carrier is as follows:

(a) carrying the target (cargo) and cell permeable peptides (cell penetration complex formation of peptide )

First, the present invention prepares a complex comprising (i) a nucleotide molecule as a cargo and (ii) a cationic peptide as a cell penetration peptide.

In the method of the present invention, a complex of a cargo and a cell penetration peptide is a nucleotide molecule as a cargo and a cell permeable peptide to form a complex by electrical interaction. As penetration peptides, cationic peptides are used to form highly stable complexes by electrical attraction by negative charges charged on nucleotide molecules and positive charges on cationic peptides.

In forming the complex in the method of the present invention, a binder is required to provide a surrounding environment necessary to promote the binding between the nucleotide molecule of the carrier and the cationic peptide, which binder has a pH value of 6.5 Preferred is a buffer solution of -8.5. On the other hand, according to known techniques, for the proper binding between the nucleotide molecule of the carrier and the cationic peptide, a special material is required or a complicated process is required. However, in the present invention, the complex formation step is achieved by a simple method of incubating the nucleotide molecules and the cationic peptides at a suitable temperature for a period of time in a buffer solution having a suitable pH value.

According to a preferred embodiment of the present invention, the binder is a phosphate buffer, HEPES buffer, MOPS buffer, various cell cultures without the addition of serum (e.g. DMEM (Dulbecco's modified minimum essential medium), Ham's F12, CMRL 1066, RPMI 1640, IMDM (Iscove's modified Dulbecco's medium, etc.), more preferably HEPES buffer and most preferably HEPES buffer-glucose solution.

The ratio of the proper charge (negative charge: positive charge) of the nucleotide molecule and the cationic peptide in the complex is 1: 1-12, more preferably 1: 4-9, most preferably 1: 5-7.

(b) formation of phospholipid film

Then, the phospholipid and the phospholipid in which the cationic peptide is bound to the surface are mixed to form a phospholipid film.

In the process of the invention a phospholipid film is prepared to enclose the pre-formed composite. Phospholipids used to prepare the phospholipid film include various phospholipids known in the art as described above, preferably 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POP), cholesterol and POPG (1-palmitoyl-2-oleoyl-sn-glycero-3-phospho- (1'-rac-glycerol)), more preferably a molar ratio of 4: 3: 3 POPC, cholesterol and POPG, most preferably the phospholipid has a charge ratio (positive charge: negative charge: negative charge) of 6: 1: 6.

The gene carrier of the present invention is characterized by a formulation in which a cell permeation peptide is bound to a liposome surface in order to increase cell permeability, and mixes the phospholipid with the cationic peptide. The phospholipid to which the cationic peptide is bound in the method of the present invention is preferably cationic peptide-DSPE (1,2-distearoyl-sn-glycero-3-phosphoethanolamine).

According to a preferred embodiment of the present invention, the phospholipid in which the cationic peptide is bound to the surface used in the method of the present invention is indirectly connected to the surface of the phospholipid through the hydrophilic polymer bound to the surface of the phospholipid. And cationic peptide-hydrophilic polymer-DSPE (1,2-distearoyl-sn-glycero-3-phosphoethanolamine) may be further mixed to form the phospholipid film.

The phospholipid mixture is then dried under vacuum and freeze-dried to produce a dried phospholipid film.

(c) mixing and extruding the composite of (a) and the phospholipid film of (b) to encapsulate the composite; To give a liposome

According to the method of the present invention, self-liposomes are formed by rehydrating and mixing the complex and the phospholipid film to obtain liposomes. In order to form liposomes, a high pressure homogenizer, a sonicator, a microfluidizer, an extrusion apparatus, or a french press is preferably used. Preferably, a sonicator and an extrusion apparatus are used.

Preferably, the mixture is sonicated and reacted at 3-10 ° C. for 2-8 hours, followed by extrusion through a membrane to produce liposomes of uniform size (average size: 80-130 nm). Is carried out.

Various extruders known in the art may be used to carry out the extrusion process, preferably using hand-held extruders. Since the extrusion process is performed to obtain a uniformly dispersed liposome, the pore size of the membrane used in the extrusion process may vary depending on the size of the liposome to be obtained, and preferably a membrane having a pore size of 100 nm ( membrane).

Gene carriers produced by the methods of the invention have an average size of 80-130 nm, more preferably 90-120 nm, most preferably 100-110 nm, and the loading efficiency of the complex encapsulated in liposomes is about 100%, more preferably 80-98% and most preferably 90-98%. This high loading efficiency is shown because the nucleotide molecule and the cationic peptide have previously formed a tight complex.

According to another aspect of the present invention, the present invention provides an anticancer composition comprising: (a) a phospholipid liposome having a double membrane structure with a cationic peptide bound to its surface; And (b) (i) double-stranded ODN corresponding to the binding sequence of the NF-κB transcription factor as a cargo, and (ii) cationic as a cell penetration peptide. Complex comprising a peptide (complex), the complex is encapsulated (encapsulation) inside the phospholipid liposomes.

According to another aspect of the present invention, the present invention provides an anticancer sensitizing composition comprising: (a) a phospholipid liposome having a double membrane structure with a cationic peptide bound to its surface; And (b) (i) double-stranded ODN corresponding to the binding sequence of the NF-κB transcription factor as a cargo, and (ii) cationic as a cell penetration peptide. Complex comprising a peptide (complex), the complex is encapsulated (encapsulation) inside the phospholipid liposomes.

The anticancer or anticancer sensitizing composition is characterized by using a double-stranded ODN corresponding to the binding sequence of the NF-κB transcription factor as a cargo to achieve an anticancer effect. Since the gene carrier of the present invention is used, the contents common between the two are omitted in order to avoid excessive complexity of the specification according to the repetitive description.

The composition of the present invention itself comprises a double-stranded ODN (decoy oligonucleotide) corresponding to the binding sequence of the NF-κB transcription factor and thus binds to the NF-κB transcription factor in a sequence specific manner. It can be used for gene therapy that can treat NF-κB transcription factor-related cancer disease by inhibiting transcriptional activity, and can be used as drug and gene delivery system by additionally enclosing anticancer agent inside liposomes. After administering the anticancer agent, the composition of the present invention can be used to sensitize cancer cells.

The disease or condition that can be treated by the composition of the present invention is cancer, preferably gastric cancer, lung cancer, breast cancer, ovarian cancer, liver cancer, bronchial cancer, nasopharyngeal cancer, laryngeal cancer, pancreatic cancer, bladder cancer, colon cancer, colon cancer, cervical cancer , Brain cancer, prostate cancer, bone cancer, skin cancer, thyroid cancer, parathyroid cancer or ureter cancer.

Anticancer agents encapsulated in addition to the liposomes of the present invention or used for sensitizing cancer cells are cisplatin, carboplatin, procarbazine, mechlorethamine, cyclophosphamide, Ifosfamide, melphalan, chlorambucil, bisulfan, nitrosourea, diactinomycin, daunorubicin, doxorubicin ), Bleomycin, plicomycin, mitomycin, etoposide, tamoxifen, paclitaxel, transplatinum, 5-fluorine Preference is given to 5-fluorouracil, adriamycin, vincristin, vinblastin or methotrexate.

Pharmaceutically acceptable carriers included in the pharmaceutical compositions of the present invention are those commonly used in the preparation, such as lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, Calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, and the like It doesn't happen. In addition to the above components, the pharmaceutical composition of the present invention may further include a lubricant, a humectant, a sweetener, a flavoring agent, an emulsifier, a suspending agent, a preservative, and the like. Suitable pharmaceutically acceptable carriers and agents are Remington's Pharmaceutical Sciences (19th ed., 1995).

The pharmaceutical composition of the present invention may be administered orally or parenterally, preferably parenterally, and in the case of parenteral administration, it may be administered by intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, transdermal administration, or the like. have.

Suitable dosages of the pharmaceutical compositions of the present invention may vary depending on factors such as the formulation method, mode of administration, age, weight, sex, morbidity, condition of food, time of administration, route of administration, rate of excretion and response to response of the patient. Can be. On the other hand, the oral dosage amount of the pharmaceutical composition of the present invention is preferably 0.001-100 mg / kg (body weight) per day.

The pharmaceutical composition of the present invention may be formulated into a unit dose form by formulating it using a pharmaceutically acceptable carrier and / or excipient according to a method which can be easily carried out by a person having ordinary skill in the art to which the present invention belongs. Or by intrusion into a multi-dose container. In this case, the formulation may be in the form of a solution, suspension or emulsion in an oil or an aqueous medium, or may be in the form of extracts, powders, granules, tablets or capsules, and may further include a dispersant or stabilizer.

The features and advantages of the present invention are summarized as follows:

(a) The present invention is the first technology using two characteristics of a cationic peptide included in a gene carrier as a cell-penetrating peptide and a combination agent.

(b) The cationic peptide used in the present invention can form a tight complex with a nucleotide molecule as a cargo to encapsulate the nucleotide molecule in a liposome with high loading efficiency.

(c) The cationic peptide bound to the surface of liposomes included in the present invention has excellent cell-permeability and can deliver a delivery target to the cytoplasm, thereby providing excellent intracellular uptake.

(d) The present invention can be applied to gene delivery, anticancer or anticancer sensitizing compositions.

1A is a schematic diagram showing a process of manufacturing 9R-LS (9R / ODN). 9R-LS means a liposome conjugated to the surface 9R peptide, 9R / ODN means 9R peptide and ODN (oligonucleotide) complex and 9R-LS (9R / ODN) is the complex is encapsulated inside the liposome By liposomes conjugated to the surface of the 9R peptide.
1) form a 9R / ODN complex in aqueous phase buffer, 2) dry under vacuum and freeze-dried overnight to obtain a phospholipid film, to prepare liposome phospholipid films in separate vials, 3) The 9R / ODN complex phospholipid film was rehydrated in aqueous solution buffer, 4) PEG-stable liposomes encapsulated with 9R / ODN complexes, and 5) membrane extrusion was used to produce uniform liposomes.
FIG. 1B shows the structure of a liposome in which a 9R / ODN complex prepared in the above process is encapsulated and 9R is conjugated to a liposome surface.
2 shows the results of preparing 9R-LS (9R / ODN). Figure 2a is a gel photograph confirmed using agarose gel electrophoresis that the (+ /-) charge ratio of the 9R peptide and ODN complex is 5 or more. 2B is size exclusion chromatography (SEC) showing that the ODN loading efficiency is about 100%. If the 9R peptide is not previously complexed, the loading of ODN is negligible. FIG. 2C is a photograph showing the results of transmission electron microscopy (TEM) analysis showing membrane extrusion converting into a stable, nano-sized particle of spherical structure in an unstable dispersion of irregular structure.
3 shows the results of in vitro intracellular uptake of 9R-LS (9R / ODN) in U87MG human glioblastoma cells. After 1 hour of reaction with 9R-LS (9R / ODN) fluorescently labeled with rhodamine-phosphatidylethanolamine (rh-PE), the cells were fixed with paraformaldehyde and visualized by laser-scanning confocal microscopy. Intracellular uptake of 9R-LS particles (FIG. 3A) and intact (yellow) particles and dissociated particles by dual labeling experiments using Alexa 488-labeling 9R (blue) and Alexa 546-labeling ODN (red). Release and intracellular location (Figure 3b) was confirmed.
4 shows in vitro activity of 9R-LS (9R / ODN) against U87MG human glioblastoma cells. React cells for 4 hours with 9R-LS (9R / ODN) or without 9R-LS (9R / ODN) or with 9R-liposomes without control complex (control), followed by 50 nM PTX ( paclitaxel) or without PTX. Cell viability was assessed using the MTT assay as described in the Examples. Relative cell viability was calculated as a percentage of viability of cells treated with medium only. At relatively low NF-κB decoy ODN concentrations (50 nM), cell viability was reduced by treatment with 9R-LS (9R / ODN) (p <0.01) (FIG. 4A), and more co-treated with 50 nM PTX (P <0.02) (FIG. 4B), which shows a therapeutic benefit by combining with anticancer agents that inhibit the NF-κB pathway. Figure 4c is a photograph confirming the signal of cell death using a TUNEL assay. Apoptosis was evident in cells treated with 9R-LS (9R / ODN) (green spot signal) and significantly increased in the presence of 50 nM PTX.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, and the scope of the present invention is not limited by these examples in accordance with the gist of the present invention to those skilled in the art. Will be self explanatory.

Example

Materials and Methods

Experimental material

A single stranded 20-mer oligodeoxynucleotide (ODN) containing an NF-κB cis element was purchased from Bioneer Corporation (Korea). Equimolar amount of single strand sense (5'CCTTGAAGGGATTTCCCTCC-3 ') (SEQ ID NO: 1) and antisense (3'GGAACTTCCCTAAAGGGAGG-5') (SEQ ID NO: 1) at a final ODN concentration of 1.22 μg / μl (100 μM) Listing 2) Double stranded ODN was prepared by annealing the ODN. Thiolated 9R peptides (CGG-9R) with D-type 9R and 1-cysteine / 2-glycine spacers were purchased from Anigen (Korea). POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine), POPG (1-palmitoyl-2-oleoyl-sn-glycero-3-phospho- (1'-rac-glycerol)), DSPE (1 , 2-distearoyl-sn-glycero-3-phosphoethanolamine), PEG2000-DSPE (polyethylene glycol (2000) -DSPE) (ammonium salt), Mal-PEG2000-DSPE (N-maleimide-PEG2000-DSPE) (ammonium salt) and Vegetable cholesterol (Chol) was purchased from Avanti Polar Lipids (Alabaster, AL, USA). OliGreen Assay Kit was obtained from Invitrogen Corporation (Carlsbad, CA, USA). All other chemicals were reagent grade and used as purchased.

Complex Formation of ODN and 9R Peptides

An amount of ODN (50 μg) and various amounts of 9R peptide were each diluted in HEPES-buffered 5% glucose solution (pH 7.4) to a final volume of 250 μl. After 10 minutes of reaction at room temperature, the solution was rapidly mixed and vortexed simultaneously to obtain 500 μl of 9R / ODN complex having a positive charge / negative charge ratio of 1-12. The amount of 9R peptide was calculated by deriving the desired charge ratio, assuming that each 9R peptide had nine positive charges from the arginine guanidium group and each ODN molecule contained 40 negative charges from the phosphate group. Each mixture was analyzed using 2% agarose gel electrophoresis (100 V, 30 minutes) to determine the optimal charge ratio for complete complex formation.

Preparation of Anionic Liposomes

The 9R peptide and the NF-κB ODN were each diluted in HEPES-buffered 5% glucose solution (pH 7.4) to a final volume of 250 μl and allowed to react for 10 minutes at room temperature. The solution was mixed and vortexed to yield 500 μl of 9R / ODN complex with a positive to negative charge ratio of 6: 1. The 9R / ODN complex was added to an anionic phospholipid film of POPC / Chol / POPG / (molar ratio: 4: 3: 3) with a + /-/-charge ratio of 6: 1: 6. The mixture was briefly sonicated and reacted at 4 ° C. for 4 hours, then through a stack of two polycarbonate membranes (pore size: 100 nm) using a hand-held extruder (Avanti Polar Lipid, AL, USA). Extruded 11 times. Extrusion is important for obtaining uniformly dispersed liposomes. Liposomes with 9R-DSPE were added to the original liposome composition at a concentration of 2 wt%. Liposomes without 9R-DSPE but loaded with the NF-κB 9R / ODN complex served as controls. Attempts to prepare similar formulations loaded only with ODN have failed, meaning that 9R / ODN complex formation is absolutely required to increase loading efficiency. First, 9R / ODN-loading liposomes were purified using size-exclusion chromatography (CL-4B column). The column was prewashed three times with HBS buffer. To prevent non-specific binding of the formulation to the column, the column was first washed with 1 ml of 5% dextran. A total of 15 fractions in 1 ml were collected as samples (control liposomes and 9R / ODN-conjugated liposomes). The characteristics of the complex formulation, size distribution, zeta potential and loading efficiency of these formulation results were characterized.

Size distribution and zeta-potential

To obtain optimal scattering intensity, 9R-LS (9R peptide-conjugated liposomes) loaded with 9R / ODN was diluted with HBS. Hydrodynamic diameter and zeta potential were measured by electrophoretic light scattering using an ELS 8000 device (Otsuka Electronics Korea, Seoul, Korea).

Encapsulation Efficiency

9R-LS loaded with 9R / ODN was prepared as described above and the ODN content was analyzed using OliGreen (Invitrogen) for the fraction of Sepharose CL4B column eluent [17]. In summary, 100 μl of each fraction was treated with Triton-X 100 (1.0 wt%) and heparin (20 U). The emission of ODN was measured by the fluorescence intensity emitted by OliGreen after ODN binding, using UV excitation and emission wavelengths of 485 and 560 nm, respectively. The entrapment efficiency of ODN was calculated as the relative value of the area under the curve of the first peak to the total area under the curve. A formulation without 9R peptide was used as a control to confirm the effect on the encapsulation efficiency of the ODN complex by 9R peptide.

Transmission electron microscopy (TEM)

Aliquots of 9R-LS (9R / ODN) particles were applied to carbon-only mesh copper grids. After 5 minutes at room temperature, the filter paper was blotted and washed five times with water to remove excess sample. Samples were negatively stained by applying 10 μl of 1% uranyl acetate to water. After 2 minutes at room temperature, the samples were washed three times with water and air-dried at room temperature for 30 minutes. Samples were visualized by transmission electron microscopy (TEM) using a TECNAI F20 electron microscope (Philips Electronic Instruments Corp., Mahwah, NJ) performed at 200 kV.

In the absorption of liposomes in vitro efficacy

Fluorescently labeled 9R-LS (9R / ODN) particles with rhodamine-phosphatidylethanolamine (rh-PE) were diluted at serum-free RPMI 1640 to a concentration of 0.5 μM ODN. Human U87MG glioblastoma cells at 10% RPMI 1640 supplemented with fetal bovine serum (FBS) to be approximately 80% confluence on cover slips (12x12 mm, Fisher Scientific, TX, USA). The medium was replaced with serum-free medium containing fluorescently labeled 9R-LS (9R / ODN). After incubation at 37 ° C. for 1 hour, cells were washed three times with PBS and fixed with 4% (w / v) paraformaldehyde. Nuclei were counter-stained with DRAQ5 (Biostatus, UK). Coverslips with fixed cells were mounted on a slide glaze with glycerol mounting medium (Dako) and examined using a Leica TCS SP2 AOBS confocal microscope (Mannheim, Germany) equipped with 25X and 60X oil-immersion lenses. It was. 488 and 543 nm laser lines for the transition of Cy2 and Cy3, respectively, were provided using an Ar laser and a HeNe laser.

Paclitaxel via NF-κB inhibition in in vitro sensitivity

9R-LS (9R / ODN) in In vitro efficacy was confirmed by assessing the NF-κB decoy ODN-mediated sensitivity of U87MG glioblastoma to paclitaxel chemotherapy. 9R-LS (9R / ODN) was diluted in serum-free RPMI 1640 to a 0.5 μM concentration of ODN. U87MG cells were cultured to about 50% confluence on 96-well plates of RPMI 1640 supplemented with 10% FBS. Treatment was started by replacing the complete medium with a serum-free medium containing the lipid formulation. The formulations were reacted for 4 hours at 37 ° C., then the cells were washed and reacted for an additional 24 hours at complete RPMI 1640 with paclitaxel at a concentration of 50 nM. After 24 hours of reaction with paclitaxel, cell viability was measured by MTT (3-4,5-dimethylthiazol-2-yl-2,5-diphenyl tetrazolium bromide) assay. In summary, 40 μl of MTT solution (2.5 mg / ml) was added to each well and the plate was allowed to react for 4 hours. The medium was washed, 200 μl of DMSO was added to each well and pipetted several times to ensure good mixing. Optical density (OD) was measured at 570 nm wavelength using a plate reader. Each condition was analyzed three times and the relative cell viability was calculated as the ratio of cell viability treated with medium only.

In vitro TUNEL Assay

24 hours prior to seeding the cells, the coverslips were “aged” by placing them in wells of 6-well plates and incubating in complete medium to enhance cell adhesion without chemical intervention. U87MG cells were seeded onto coverslips at about 50% confluency. Cells were treated with control liposomes or 9R / ODN-loaded liposome formulations. After treatment for 4 hours, the medium was washed and replaced with complete medium, and the cells were further incubated for 24 hours. For paclitaxel treatment, 50 nM PTX (paclitaxel) was included in the added complete medium. A terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay was performed using a TdT-FragEL DNA fragment detection kit (Promega, WI), and the sections were subjected to fluorescence microscopy (TCS SL, Leica, Heidelberg, Germany). ) Was investigated.

Experiment result

9R-modified liposomes loaded with 9R / ODN complexes

Modifications were made to the previously described processes [16, 18] to which the interaction of charges was applied and used to encapsulate 9R / ODN complexes preformed with liposomes. As can be seen in FIG. 1, the formulation was constructed by pre-condensation of a cation 9R peptide with a 20-mer double-stranded anion ODN. Complex formation between 9R peptide and ODN was confirmed using electrophoresis on 2% (w / v) agarose gel (FIG. 2A). When complexed with the 9R peptide, ODN cannot migrate through the gel due to size limitations. All ODNs were present in the wells with no free ODN shift at +/- charge ratio 5, meaning that all ODNs were completely complexed. Through these experiments, a +/- charge ratio of 6 was used to yield a 9R / ODN complex with a totally positive charge. By reacting with an anionic lipid membrane in an aqueous solution, the preformed and totally positively charged complex was enclosed in 9R peptide-modified liposomes (9R-LS) and the suspension was extruded through the membrane. The ratio of (+) in the peptide, (-) in ODN and (-) charge in POPG was 6: 1: 6.

Rehydration of the phospholipid film in an aqueous solution buffer containing the 9R / ODN complex produced a turbid suspension with a broad size distribution (478 ± 2) and a tendency to rapid aggregation and phase separation. However, membrane extrusion produced a transparent, stable suspension with a narrow, monomodal size distribution (106 ± 3, Table 1).

Size Distribution and Zeta Potential - Size distribution (nm) Zeta Potential (mV) LS- (9R / ODN) 125.9 ± 2.82 -23.41 ± 2.82 9R-LS (9R / ODN) 106.2 ± 3.44 -14.55 ± 1.65

Data represent mean ± sem (n ≧ 3).

These observations were consistent with the morphological analysis by transmission electron microscopy (TEM). TEM visualization after control staining showed follicular structure with a darkly stained core surrounded by a lightly stained coat, with the phospholipid membrane and the core showing the 9R / ODN complex, respectively (FIG. 2C). Size exclusion chromatography showed that almost all ODN was encapsulated in liposomes (because the ODN eluted in the pore volume), but only if the ODN first complexed with the 9R peptide (FIG. 2B). Only ODN could not be embedded in liposomes.

Thus, precomplexation of 9R and ODN is absolutely required for the preparation of 9R / ODN-coloaded anionic liposomes. Sequential charge interactions ensured efficient loading of the 9R / ODN complex into the anionic liposomes and resulted in about 100% loading efficiency of 2.5 wt% content. In addition, the use of anionic liposomes has shown that liposomes themselves will exhibit greatly reduced non-specific binding to the cell membrane due to the repulsive forces acting on the same charge.

In in vitro cell uptake

Fluorescently labeled 9R-LS (9R / ODN) liposomes were associated with and included cells during the 1 hour reaction time. Fluorescent signals were quickly detected as discrete particles in the cell membrane and in the cytoplasm. No accumulation in the nucleus was observed. On the other hand, cell affinity and intracellular uptake were the smallest in cells treated with the control formulation LS (9R / ODN), in which no 9R peptide was bound to the liposome surface. Means to allow uptake into cells. Co-localization of TMR and A488 showed that the particles exist in intact form and maintain their intensity (FIG. 2C).

Taken together, these results showed that 9R / ODN-loading liposomes were prepared using 9R peptide and 1: 6 total charge ratio (N / P) as CPP, and the inclusion of PEG2000-DSPE into anionic liposomes resulted in efficient intercellular delivery. It means that the system is provided.

In In vitro cytotoxicity assay

The cytotoxicity of the 9R-LS (9R / ODN) formulations was adjusted to the extent detectable, which was caused by the 9R / ODN core and not by the 9R-LS envelope. Treatment of U87MG cells with 9R-LS (9R / ODN) at 0.5 μM ODN concentration reduced cell viability by about 20% compared to untreated cells, but the same concentration of 9R-LS formulation without 9R / ODN resulted in cell survival. There was no effect on (FIG. 4B).

The NF-κB pathway has been involved in the development and progression of various cancers, and activation of this pathway has been known to induce resistance to anticancer treatments [19,20]. Thus, inhibition of the NF-κB pathway has been shown to be a promising strategy for sensitizing cancer cells to chemotherapeutic agents such as paclitaxel [21], which has been shown to be more efficient when NF-κB is inhibited with various blocking agents. In order to determine whether 9R-LS (9R / ODN) liposomes efficiently carry a cargo of decoy ODN containing an NF-κB cis-element [18], we have treated paclitaxel only. We tested the ability of phospholipid formulations to sensitize U87MG glioblastoma cells at concentrations of paclitaxel that were only slightly cytotoxic. Treatment of U87MG cells with 50 nM paclitaxel or 9R-LS (9R / ODN) reduced cell viability by about 20% compared to untreated cells. However, treatment with the same concentration of paclitaxel reduced the viability of cells pretreated with 9R-LS (9R / ODN) by about 50%. These results indicate that decoy ODN successfully inhibits NF-κB activation and that inhibition of NF-κB by decoy ODN in combination with paclitaxel is a promising approach for treating cancer.

In vitro TUNEL Assay

As evidenced by the presence of green spot fluorescence in the nucleus of apoptotic cells, the TUNEL assay showed DNA fragmentation characteristics of cell death. Fluorescence microscopy images showed that green signals were present in cells treated with 9R-LS (9R / ODN) or 9R-LS (9R / ODN) and 50 nM PTX, but the fluorescence intensity was much higher in the latter group. On the other hand, cells treated with 50 nM PTX alone did not show cell death. These results were consistent with the results of the MTT assay, which showed that 50 nM PTX was not toxic to U87MG cells and substantially reduced survival after treatment with 9R-LS (9R / ODN) (FIG. 4C). .

Discussion

The results of this experiment are compatible with the following conclusions: First, cations 9R peptides can encapsulate olinucleotides in liposome carriers through tight complex formation. Second, the 9R peptide present on the surface of the liposome carrier can deliver the carrier and its cargo to the cytoplasm. Third, intercellular transport of NF-κB decoy ODN improved the anti-cancer efficacy of paclitaxel against U87MG glioblastoma cells.

An important characteristic of an efficient drug delivery system is the high drug loading efficiency, which is required to reduce the dosing cycle. In addition, the encapsulation can protect the transport object from disassembly before reaching the target. Conventional and inclusion of free DNA molecules into pegylated liposomes has been studied in several ways; However, in most cases only a low efficiency of about 50% was shown.

The 9R peptide has the advantage of not inducing cytotoxicity. In addition, complex formation with 9R peptides provides an easy and efficient way to increase ODN encapsulation efficiency. By using the 9R peptide as the complex and condensing agent, the encapsulation efficiency of ODN was significantly increased to about 95% compared to ODN that did not form a complex (5% or less). This high loading rate can be achieved by manipulating the N / P charge ratio of the 9R peptide and ODN; At a charge ratio (N / P) of 1: 6, the complex may have a somewhat positive charge. The net surface charge of the carrier plays an important role in determining gene transfer efficiency. Ideally, the positive zeta potential of the carrier can improve DNA condensation [23]. This helps the complex to be encapsulated with anionic liposomes.

Consistent with the properties of 9R as CPP, 9R peptide-conjugate liposomes showed improved cell permeability. Because of the total negative charge (on POPG) of the liposomes, the control liposomes did not show non-specific binding to U87MG cells because the repulsive forces act by negatively charged cell membranes. Therefore, we conclude that the cell-adhesion and permeation properties of liposomes are solely due to the effect of the 9R peptide.

Using ODN fluorescently labeled with Alexa 546, we were able to investigate the trafficking of ODN into cells. We found that ODN was present in U87MG cells even 24 hours after the reaction (data not shown). This does not mean that ODN was secreted rapidly or degraded by endosomes. Because ODN has been in the cytoplasm for a longer time, ODN could be effectively used in knockdown studies, requiring stable and persistent retention of the carrier in the cytoplasm. Using the Alexa 488-labeling 9R peptide in the double labeling study, we found that only the 9R peptide could penetrate the nucleus.

From these observations, we conclude that 9R-LS can efficiently deliver 9R / ODN complexes to cells. In addition, the inventors found that after the complexes were released into the cytoplasm, 9R / ODN complexes not maintained by strong covalent bonds dissociated in the cytoplasm. The 9R peptide then moved to the nucleus, but the ODN remained in the cytoplasm. If this interpretation is valid, nuclear targeting is likely to increase by covalently conjugating the carrier to the 9R peptide.

The NF-κB pathway is involved in the development and progression of various cancers, and activation of this pathway has been shown to induce resistance to anticancer treatments [19,20]. However, simply inhibiting NF-κB will not result in a firm cell death response. Rather, the inhibition tends to lower the threshold of cell death and will likely be used as an adjunct to other cancer therapies [21]. Thus, inhibition of the NF-κB pathway has been proposed as a promising strategy for sensitizing cancer cells to chemotherapeutic agents and for improving the efficacy of chemotherapeutic methods [22].

The possibility of 9R-LS (9R / ODN) was clearly demonstrated by inhibition of the NF-κB pathway of paclitaxel and improved cytotoxic effects in U87MG cancer cells. In addition, the improved anticancer efficacy of the combination treatment of 9R-LS (9R / ODN) and paclitaxel suggested a very interesting possibility that further improvements could be obtained by encapsulating paclitaxel inside the lipid membrane of 9R. Such formulations will also be effective in the treatment of various inflammatory diseases associated with the NF-κB pathway and may provide a promising approach for intracellular delivery of various therapeutic genetic material.

conclusion

We report a novel ODN delivery system in which 9R peptides act as cell-permeation and combination agents. ODN encapsulation efficiency has been greatly improved using 9R peptides, whereby ODN can be provided as an efficient delivery system for tumor treatment. The cell-permeability of 9R peptides can be complemented by tumor-specific targeting ligands to further improve the specificity and efficiency of the formulation.

Having described the specific part of the present invention in detail, it is apparent to those skilled in the art that the specific technology is merely a preferred embodiment, and 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 equivalents thereof.

References

[1] JS Gill, X. Zhu, MJ Moore, L. Lu, MJ Yaszwmski, AJ Windebank, Biomaterials . 2002 , 23 , 2773.

[2] S. Futaki, W. Ohashi, T. Suzuki, M. Niwa, S. Tanaka, K. Ueda, H. Harashima, Y. Sugiura, Bioconjug Chem 2001 , 12 , 1005.

[3] C. Bolenz, C. Weiss, M. Wenzel, U. Gabriel, A. Steidler, A. Becker, E. Herrmann, L. Trojan, MS Michel, J. Cancer Res . Clin . Oncol . 2009 , 135 , 679

[4] N. Dias, CA Stein, Mol . Cancer Ther . 2002 , 1 , 347

[5] C. Plank, W. Zauner, E. Wagner, Adv Drug Deliv Rev 1998 , 34 , 21.

[6] A. Prochiantz, Curr Opin Cell Biol 2000 , 12 , 400.

[7] KM Stewart, KL Horton, SO Kelley, Org Biomol Chem 2008 , 6 , 2242.

[8] Y. Wang, Y. Hou, H. Lee, J. Biochem . Bioph . Meth . 2007 . 70 , 579.

[9] V. Sebbage, Bioscience Horizons . 2009 , 2 , 64.

[10] J. Guo, UN Verma, RB Gaynor, EP Frenkel, CR Becerra, Clin . Cancer Res . 2004 , 10 , 3333.

[11] S. Deshayes, M. Morris, F. Heitz, G. Divita, Adv Drug Deliv Rev 2008 , 60 , 537.

[12] PA Wender, DJ Mitchell, K. Pattabiraman, ET Pelkey, L. Steinman, JB Rothbard, Proc Natl Acad Sci USA 2000 , 97 , 13003.

[13] C. Zhang, N. Tang, X. Liu, W. Liang, W. Xu, VP Torchilin, J. Control Release . 2006 , 112 , 229.

[14] WJ Kim, LV Christensen, S. Jo, JW Yockman, JH Jeong, YH Kim, SW Kim, Mol Ther 2006 , 14 , 343.

[15] P. Kumar, H. Wu, JL McBride, KE Jung, MH Kim, BL Davidson, SK Lee, P. Shankar, N. Manjunath, Nature 2007 , 448 , 39.

[16] YT Ko, R. Bhattacharya, U. Bickel, J Control Release 2009 , 133 , 230.

[17] MN Uddin, DP Do, SB Pai, S. Gayakwad, CW Oettinger, MJ D'Souza, Analyst . 2009 , 134 , 1483.

[18] DW Pack, AS Hoffman, S. Pun, PS Stayton, Nat . Dru Del . Rev. 2005, 4 , 581.

[19] M. Karin, Nature 2006 , 441 , 431.

[20] WE Naugler, M. Karin, Curr Opin Genet Dev 2008 , 18 , 19.

[21] M. Karin, Mol Carcinog 2006 , 45 , 355.

[22] OP Veiby, MA Read, Clin Cancer Res 2004 , 10 , 3262.

[23] XD Guo, F. Tandiono, N. Wiradharma, DY Khor, CG Tan, M. Khan, Y. Qian, YY Yang, Biomaterials . 2008 , 29 , 4838-4846

<110> Gwangju Institute of Science and Technology <120> Gene Delivery System <160> 2 <170> KopatentIn 1.71 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Oligodeoxynucleotide Sense Strand <400> 1 ccttgaaggg atttccctcc 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Oligodeoxynucleotide Antisense Strand <400> 2 ggagggaaat cccttcaagg 20

Claims (17)

Gene delivery systems, including:
(a) a phospholipid liposome having a cationic peptide bound to its surface and having a double membrane structure; And
(b) a complex comprising (i) a nucleotide molecule as a cargo, and (ii) a cationic peptide as a cell penetration peptide, the complex encapsulated inside the phospholipid liposome )
The gene carrier of claim 1, wherein the cationic peptide is indirectly bound to the surface of the phospholipid liposome through a hydrophilic polymer bound to the surface of the phospholipid liposome.
The peptide of claim 1, wherein the cationic peptide consists of human immunodeficiency virus (HIV) -Tat, 6-40 amino acid residues and at least three of the amino acid residues are lysine, arginine or lysine and arginine, KALA and protamine. Gene carrier, characterized in that selected from the group consisting of.
The method of claim 2, wherein the hydrophilic polymer is polyethylene glycol, polyvinylpyrrolidone, polyoxazoline, polypropylene glycol, copolymers of polyethylene glycol and polypropylene glycol, chitosan, dextran, cellulose, heparin, alginate or their A gene carrier, which is a monoesterified derivative.
The double-stranded ODN of claim 1, wherein the nucleotide molecule of the carrier is an antisense oligonucleotide, aptamer, siRNA, shRNA, miRNA, ribozyme, DNAzyme, or transcription factor binding sequence. ) Is a gene carrier.
According to claim 1, wherein the complex is characterized in that the ratio of the charge (negative charge: positive charge) of the nucleotide molecule as a cargo (cargo) and the cationic peptide as a cell penetration peptide is formed in a 1: 1-12 Gene carrier.
The gene carrier of claim 1, wherein the surface of the phospholipid liposome of the gene carrier further comprises a targeting ligand.
8. The gene carrier of claim 7, wherein the targeting ligand has a bipodal peptide binder (BPB) structure comprising: (i) a parallel covalent bond (parallel); ), A structure stabilizing region comprising antiparallel or parallel and antiparallel amino acid strands; And (ii) target binding region I and target binding region II, each of which is bound to both ends of the structure stabilization site and comprises randomly selected n and m amino acids, respectively.
Method for preparing a gene delivery system comprising the following steps:
forming a complex comprising (a) (i) a nucleotide molecule as a cargo and (ii) a cationic peptide as a cell penetration peptide;
(b) mixing the phospholipid and the phospholipid having a cationic peptide bound thereto to form a phospholipid film; And
(c) mixing and extruding the complex of step (a) and the phospholipid film of step (b) to obtain a liposome with the complex encapsulated.
10. The double-stranded ODN of claim 9 wherein the nucleotide molecule of the carrier is an antisense oligonucleotide, aptamer, siRNA, shRNA, miRNA, ribozyme, DNAzyme or transcription factor binding sequence. ).
10. The peptide, KALA and protamine of claim 9, wherein the cationic peptide consists of human immunodeficiency virus (HIV) -Tat, 6-40 amino acid residues and at least three of the amino acid residues are lysine, arginine or lysine and arginine. Method selected from the group consisting of.
10. The method of claim 9, wherein the step (b) further comprises mixing a phospholipid having a hydrophilic polymer bound to its surface.
Anticancer composition comprising:
(a) a phospholipid liposome having a cationic peptide bound to its surface and having a double membrane structure; And
(b) (i) a double-stranded ODN corresponding to the binding sequence of the NF-κB transcription factor as a cargo, and (ii) a cationic peptide as a cell penetration peptide. Complex (complex) containing, the complex is encapsulation (encapsulation) inside the phospholipid liposomes.
Anticancer sensitizing compositions comprising:
(a) a phospholipid liposome having a cationic peptide bound to its surface and having a double membrane structure; And
(b) (i) a double-stranded ODN corresponding to the binding sequence of the NF-κB transcription factor as a cargo, and (ii) a cationic peptide as a cell penetration peptide. Complex (complex) containing, the complex is encapsulation (encapsulation) inside the phospholipid liposomes.
The composition of claim 13 or 14, wherein the cationic peptide is indirectly bound to the surface of the phospholipid liposome through a hydrophilic polymer bound to the surface of the phospholipid liposome.
15. The composition of claim 13 or 14, wherein the surface of the phospholipid liposome further comprises a targeting ligand.
17. The composition of claim 16, wherein the targeting ligand has a bipodal peptide binder (BPB) structure comprising: (i) a parallel formed non-covalent bond between strands; A structure stabilizing region comprising antiparallel or parallel and antiparallel amino acid strands; And (ii) target binding region I and target binding region II, each of which is bound to both ends of the structure stabilization site and comprises randomly selected n and m amino acids, respectively.

Images