Cytoplasm delivery agent and use thereof

Abstract

To provide a cytoplasm delivery agent containing peptide and a cationic molecule used for delivering a target substance to a cytoplasm, and use thereof.SOLUTION: A cytoplasm delivery agent contains peptide including an amino acid sequence represented by the following formula (1) or a salt thereof, and a cationic molecule. X1X2X3X4 formula (1) X1 denotes an artificial amino acid residue or an aspartic acid residue in which artificial amino acid represented by the following general formula (A-1) or the following general formula (A-2) is bonded by peptide bond, X2, X3 and X4 denote alanine, leucine, isoleucine, valine or phenylalanine. In the formula, R11 and R21 denote a hydrocarbon group of carbon atom number 3 to 10 which may have a substituent group. R12 and R22 denote a protective group or a hydrogen atom.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to cytoplasmic delivery agents and uses thereof. [Background technology]

[0002] Liposomes with functional molecules introduced onto their outer surface have been developed to deliver target substances such as small molecule drugs, peptides, proteins, and sugars to the cytoplasm. For example, Patent Document 1 describes a method for delivering target substances to the cytoplasm using GALA peptide, an artificial peptide that mimics a viral membrane fusion protein.

[0003] When GALA peptide and liposomes containing cationic molecules and target substances are contacted with cells, the GALA peptide and liposomes are taken up by the cells by endocytosis. The GALA peptide taken up into the endosome changes its conformation due to a decrease in pH, destabilizing the endosomal membrane. This promotes the destruction of endosomes and the delivery of target substances to the cytoplasm (e.g., Non-Patent Documents 1 to 3). [Prior art documents] [Patent documents]

[0004] [Patent Document 1] International Publication No. 2012 / 124688 [Non-patent literature]

[0005] [Non-Patent Document 1] Luke van der Koog et al, Liposomes and Extracellular Vesicles as Drug Delivery Systems: A Comparison of Composition, Pharmacokinetics, and Functionalization, Advanced Healthcare Materials, 2022 Mar;11(5):e2100639. doi: 10.1002 / adhm.202100639. [Non-patent document 2] Nakase I and Futaki S, Combined treatment with a pH-sensitive fusogenic peptide and cationic lipids achieves enhanced cytosolic delivery of exosomes, Scientific Reports, 2015 May 26;5:10112. doi: 10.1038 / srep10112. [Non-patent document 3] Kobayashi et al., Cytosolic targeting of macromolecules using a pH-dependent fusogenic peptide in combination with cationic liposomes, Bioconjugate Chemmistry. 2009 May 20;20(5):953-9. doi: 10.1021 / bc800530v. Summary of the Invention [Problem to be solved by the invention]

[0006] In order to deliver larger amounts of target substances to the cytoplasm, the development of peptides other than GALA peptides is required. Therefore, an object of the present invention is to provide a cytoplasmic delivery agent containing a peptide and a cationic molecule, which is used to deliver a target substance to the cytoplasm, and a method for delivering a target substance to the cytoplasm of a target cell using the same. [Means for solving the problem]

[0007] The present invention includes the following aspects. [1] A cytoplasmic delivery agent for delivering a target substance to the cytoplasm of a target cell, the cytoplasmic delivery agent comprising a peptide having an amino acid sequence represented by the following formula (1) or a salt thereof, and a cationic molecule, and having cytoplasmic delivery activity: X 1 X 2 X 3 X 4 Formula (1) X 1 represents an artificial amino acid residue in which an artificial amino acid represented by the following general formula (A-1) or (A-2) is bound by a peptide bond, or an aspartic acid residue; X 2 , X 3 and X 4 each independently represents alanine, leucine, isoleucine, valine, or phenylalanine.

[0008] [ka] [R 11 and R 21 R each independently represents a hydrocarbon group having 3 to 10 carbon atoms which may have a substituent. 12 and R 22 each independently represents a protecting group or a hydrogen atom.

[0009] [2] In the general formula (A-1) and the general formula (A-2), R 11 and R 21 and each independently represent an optionally substituted aliphatic hydrocarbon group. [3] In the general formula (A-1) and the general formula (A-2), R 11 and R21 and each independently represent a chain aliphatic hydrocarbon group which may have a substituent. [4] In the general formula (A-1) and the general formula (A-2), R 11 and R 21 and each independently represent a chain aliphatic hydrocarbon group having 3 to 7 carbon atoms, which may have a substituent.

[0010] [5]X 1 The cytoplasmic delivery agent according to [1], wherein the artificial amino acid represented by the following formula (A-1-01) or the following formula (A-2-01) is an artificial amino acid residue bound by a peptide bond.

[0011] [ka] [R 12 and R 22 each independently represents a protecting group or a hydrogen atom.

[0012] [6] The cytoplasmic delivery agent according to [1], wherein the peptide contains repeats of the amino acid sequence represented by formula (1), and the number of repeats is 3 to 20. [7] The cytoplasmic delivery agent according to [1], wherein the peptide contains repeats of the amino acid sequence represented by formula (1), and the number of repeats is 4 to 10. [8]X 2 is alanine and X 3 is leucine and X 4 The cytoplasmic delivery agent according to [1], wherein R is alanine.

[0013] [9] The cytoplasmic delivery agent according to [1], wherein the peptide is selected from the group consisting of the following (a), (b), and (c): WXAALAXALAXALAXHLAXALAXALXALAA (SEQ ID NO: 1) (a) a peptide comprising the amino acid sequence set forth in SEQ ID NO: 1 and having cytoplasmic delivery activity; (b) a peptide comprising an amino acid sequence in which one or more amino acids are deleted, inserted, substituted, and / or added in the amino acid sequence set forth in SEQ ID NO: 1, and having cytoplasmic delivery activity; (c) a peptide comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 1 and having cytoplasmic delivery activity; Each of the multiple Xs independently represents an artificial amino acid residue in which an artificial amino acid represented by the following general formula (A-1) or the following general formula (A-2) is bound via a peptide bond, or an aspartic acid residue: The multiple Xs may be the same or different.

[0014] [ka] [R 11 and R 21 R each independently represents a hydrocarbon group having 3 to 10 carbon atoms which may have a substituent. 12 and R 22 each independently represents a protecting group or a hydrogen atom.

[0015]

[10] A method for delivering a target substance to the cytoplasm of a target cell, the method comprising the step of contacting the cytoplasmic delivery agent according to any one of [1] to [9] and the target substance with the target cell.

[0016]

[11] Use of a cytoplasmic delivery agent for delivering a target substance to a cytoplasm, wherein the cytoplasmic delivery agent contains a peptide having an amino acid sequence represented by the following formula (1) or a salt thereof, and a cationic molecule, and has cytoplasmic delivery activity: X 1 X 2 X 3 X 4 Formula (1) X 1 represents an artificial amino acid residue in which an artificial amino acid represented by the following general formula (A-1) or the following general formula (A-2) is bound via a peptide bond, or an aspartic acid residue, X 2 , X3 and X 4 each independently represents alanine, leucine, isoleucine, valine, or phenylalanine.

[0017] [ka] [R 11 and R 21 R each independently represents a hydrocarbon group having 3 to 10 carbon atoms which may have a substituent. 12 and R 22 each independently represents a protecting group or a hydrogen atom.

[0018]

[12] Use of a peptide or a salt thereof and a cationic molecule for producing a cytoplasmic delivery agent, wherein the cytoplasmic delivery agent contains a peptide or a salt thereof comprising an amino acid sequence represented by the following formula (1) and a cationic molecule, and has cytoplasmic delivery activity: X 1 X 2 X 3 X 4 Formula (1) X 1 represents an artificial amino acid residue in which an artificial amino acid represented by the following general formula (A-1) or the following general formula (A-2) is bound via a peptide bond, or an aspartic acid residue, X 2 , X 3 and X 4 each independently represents alanine, leucine, isoleucine, valine, or phenylalanine.

[0019] [ka] [R 11 and R 21 R each independently represents a hydrocarbon group having 3 to 10 carbon atoms which may have a substituent. 12 and R 22 each independently represents a protecting group or a hydrogen atom. [Effects of the Invention]

[0020] According to the present invention, it is possible to provide a cytoplasmic delivery agent containing a peptide and a cationic molecule, which is used to deliver a target substance to the cytoplasm, and a method for delivering a target substance to the cytoplasm of a target cell using the same. [Brief explanation of the drawings]

[0021] [Figure 1A] 1 shows confocal laser scanning microscope images of FITC-labeled peptides in cells contacted with the cytoplasmic delivery agents of Test Examples 1 to 3 in Experimental Example 1. FIG. [Figure 1B] FIG. 1 shows the results of flow cytometry of cells contacted with the cytoplasmic delivery agents of Test Examples 4 to 6 in Experimental Example 1. [Figure 2A] 1 shows confocal laser scanning microscope images of FITC-avidin in cells contacted with the cytoplasmic delivery agents of Test Examples 7 to 9 in Experimental Example 2. FIG. [Figure 2B] FIG. 10 shows the results of flow cytometry of cells contacted with the cytoplasmic delivery agents of Test Examples 10 to 12 in Experimental Example 2. [Figure 3A] 1 shows confocal laser scanning microscope images of IgG-Alexa488 in cells contacted with the cytoplasmic delivery agents of Test Examples 13 to 15 in Experimental Example 3. FIG. [Figure 3B] FIG. 10 shows the results of flow cytometry of cells contacted with the cytoplasmic delivery agents of Test Examples 16 to 18 in Experimental Example 3. [Figure 4] FIG. 10 shows the results of measuring the survival rate of cells contacted with the cytoplasmic delivery agents of Test Examples 19 to 27 in Experimental Example 4. DETAILED DESCRIPTION OF THE INVENTION

[0022] The cytoplasmic delivery agent according to this embodiment is used to deliver a target substance to the cytoplasm of a target cell. The cytoplasmic delivery agent according to this embodiment contains a peptide comprising the amino acid sequence represented by the following formula (1) or a salt thereof, and a cationic molecule. X 1 X 2 X 3 X 4 (1) X 1 represents an artificial amino acid residue in which an artificial amino acid represented by the following general formula (A-1) or (A-2) is bound via a peptide bond, or an aspartic acid residue. X 2 , X 3 and X 4 each independently represents alanine, leucine, isoleucine, valine, or phenylalanine.

[0023] [ka] [R 11 and R 21 R each independently represents a hydrocarbon group having 3 to 10 carbon atoms which may have a substituent. 12 and R 22 each independently represents a protecting group or a hydrogen atom.

[0024] <Cells into which the target substance is introduced (target cells)> The biological species of the cells into which the target substance is introduced (sometimes referred to as target cells) is not particularly limited as long as the effects of the present invention are achieved, and may be any of eukaryotes, eubacteria, and archaea, with eukaryotes being preferred. The eukaryotes may be either multicellular or unicellular organisms. The multicellular organisms may be either animals or plants, with animals being preferred.

[0025] Examples of the animals include vertebrates such as mammals, reptiles, birds, amphibians, and fish, as well as insects. Among these, mammals are preferred. Examples of the mammals include rodents such as mice, rats, hamsters, and guinea pigs; lagomorphs such as rabbits; ungulates such as pigs, cows, goats, horses, and sheep; carnivores such as dogs and cats; and primates such as humans, monkeys, rhesus monkeys, cynomolgus monkeys, marmosets, orangutans, and chimpanzees.

[0026] When the target cells are animal or plant cells, the target cells may be either somatic cells or germ cells, and are preferably somatic cells.

[0027] The cells of interest may be in vitro cells (cultured cells), in vivo cells (cells inside a living organism), or ex vivo cells (cells outside a living organism). Furthermore, the target cells may be primary cultured cells or established cell lines. Established cell lines are generally immortalized cells, and may be cancer cells.

[0028] (Target substance) The target substance is not particularly limited, and examples thereof include drugs, nucleic acids, peptides, proteins, sugars, and complexes thereof. The target substance may be the same as a naturally occurring substance, an artificial substance that does not occur in nature, or a naturally occurring substance that has been partially artificially modified.

[0029] (Cytoplasmic delivery activity) As used herein, the cytoplasmic delivery activity of a cytoplasmic delivery agent means the activity of delivering a target substance to the cytoplasm of a target cell.

[0030] As used herein, the phrase "a cytoplasmic delivery agent has cytoplasmic delivery activity" has the following meaning. When a target cell is contacted with a target substance, a peptide or a salt thereof comprising the amino acid sequence represented by formula (1), and a cationic molecule, the amount of the target substance delivered to the cytoplasm of the target cell is quantified (this amount is designated as α). In the same manner as described above, except that a peptide containing the amino acid sequence represented by formula (1) is not used, a target substance and a cationic molecule are contacted with the target cells, and the amount of the target substance delivered to the cytoplasm of the target cells is quantified (this amount is designated as β).

[0031] The phrase "a cytoplasmic delivery agent has cytoplasmic delivery activity" means that the ratio of α to β, ie, the ratio expressed as α / β, is greater than 1. The ratio represented by α / β may be, for example, 1.5 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 15 or more, 20 or more, 30 or more, 40 or more, or 50 or more.

[0032] A method for quantifying the amount of a target substance delivered to the cytoplasm may be, for example, a method in which the target substance is labeled in advance and the labeled target substance delivered to the cytoplasm is detected using a fluorescence microscope, a confocal laser microscope, an electron microscope, flow cytometry, etc. Examples of labeled target substances include target substances bound to tags or fluorescent substances. Alternatively, the amount of the target substance delivered to the cytoplasm may be quantified using an antibody that recognizes the target substance.

[0033] (peptide) The cytoplasmic delivery agent according to this embodiment contains a peptide comprising an amino acid sequence represented by the following formula (1) or a salt thereof. X 1 X 2 X 3 X 4 Formula (1) X in the formula (1) 1 represents an artificial amino acid residue in which an artificial amino acid represented by the following general formula (A-1) or (A-2) is bound via a peptide bond, or an aspartic acid residue.

[0034] [ka] [R 11 and R 21 R each independently represents a hydrocarbon group having 3 to 10 carbon atoms which may have a substituent. 12 and R 22 each independently represents a protecting group or a hydrogen atom.

[0035] R 11 and R 21 The hydrocarbon group having 3 to 10 carbon atoms in R is preferably an aliphatic hydrocarbon group. 11 and R 21 is an aliphatic hydrocarbon group, R 11 and R 21 may be chain-like or cyclic.

[0036] R 11 and R 21 If is a chain, R 11 and R 21 may be a straight chain or a branched chain, and is preferably a straight chain. As the straight-chain aliphatic hydrocarbon group, a straight-chain alkylene group is preferred, and specific examples thereof include a trimethylene group [-(CH2)3-], a tetramethylene group [-(CH2)4-], and a pentamethylene group [-(CH2)5-]. The branched aliphatic hydrocarbon group is preferably a branched alkylene group, and specific examples thereof include alkyl alkylene groups such as alkylmethylene groups such as -CH(CHCH)-, -C(CH)-, -C(CH)(CHCH)-, -C(CH)(CHCHCH)-, and -C(CHCH)-; alkylethylene groups such as -CH(CH)CH-, -CH(CH)CH(CH)-, -C(CH)CH-, -CH(CHCH)CH-, and -C(CHCH)-CH-; alkyl trimethylene groups such as -CH(CH)CHCH- and -CHCH(CH)CH-; and alkyl tetramethylene groups such as -CH(CH)CHCHCHCH- and -CHCH(CH)CHCH-. The alkyl group in the alkylalkylene group is preferably a linear alkyl group having 1 to 5 carbon atoms.

[0037] R 11 and R 21 When is cyclic, the hydrocarbon group may be a monocyclic group or a polycyclic group. The monocyclic aliphatic hydrocarbon group is preferably a group in which two hydrogen atoms have been removed from a monocycloalkane. The monocycloalkane preferably has 3 to 6 carbon atoms, and specific examples include cyclopentane and cyclohexane. The aliphatic hydrocarbon group that is a polycyclic group is preferably a group in which one hydrogen atom has been removed from a polycycloalkane.

[0038] R 11 and R 21 Examples of the substituent that the hydrocarbon group in P1 , -R P2 -OR P1 , -R P2 -CO-R P1 , -R P2 -CO-OR P1 , -R P2 -O-CO-R P1 , -R P2 -OH, -R P2 -CN or -R P2 -COOH and the like. RP1 is a monovalent chain saturated hydrocarbon group having 1 to 3 carbon atoms, or a monovalent alicyclic saturated hydrocarbon group having 1 to 3 carbon atoms. R P2 is a single bond or a divalent chain saturated hydrocarbon group having 1 to 3 carbon atoms. R P1 and R P2 Some or all of the hydrogen atoms in the chain saturated hydrocarbon group and the alicyclic saturated hydrocarbon group may be substituted with halogen atoms.

[0039] R 11 and R 21 The number of carbon atoms in is preferably 3 to 8, more preferably 4 to 7, still more preferably 4 to 6, and particularly preferably 5.

[0040] R 12 and R 22 The protecting group in R is not particularly limited, but can be formed using, for example, t-butyl ester. 12 -O- is (CH3)3C-O-.

[0041] X 1 is an artificial amino acid residue, X 1 Preferably, the artificial amino acid represented by the general formula (A-1) is an artificial amino acid residue bound by a peptide bond.

[0042] Or X 1 Preferably, the artificial amino acid represented by the general formula (A-2) is an artificial amino acid residue linked by a peptide bond. In this case, the peptide containing the amino acid sequence represented by the formula (1) is less susceptible to degradation by enzymes such as proteases, and therefore the cytoplasmic delivery activity is more likely to be enhanced.

[0043] X 1 is an artificial amino acid residue, the artificial amino acid is preferably an artificial amino acid represented by the following formula (A-1-01) or the following formula (A-2-01), and more preferably an artificial amino acid represented by the following formula (A-1-01).

[0044] [ka] [R 12 and R 22 each independently represents a protecting group or a hydrogen atom.

[0045] The peptide preferably contains repeats of the amino acid sequence shown in formula (1). The number of repetitions may be, for example, 2 to 20, 3 or more, 4 or more, or 5 or more. The number of repetitions is preferably 3 to 20, and more preferably 5 to 10.

[0046] In formula (1), X 2 , X 3 and X 4 are preferably each independently alanine, leucine or isoleucine. In formula (1), X 2 is alanine and X 3 is leucine and X 4 is preferably alanine.

[0047] The peptide comprising the amino acid sequence represented by formula (1) may be a fusion peptide in which another protein or peptide tag is fused. For example, another protein or peptide tag may be bound to either or both of the N-terminus and C-terminus of a peptide comprising the amino acid sequence represented by formula (1) (e.g., one of the peptides exemplified above). Examples of other proteins or peptides include marker enzyme proteins such as alkaline phosphatase or partial peptides thereof; fluorescent proteins such as GFP or partial peptides thereof; and tag peptides such as His tags and FLAG tags.

[0048] The peptide is preferably a peptide selected from the group consisting of the following (a), (b), and (c): WXAALAXALAXALAXHLAXALAXALXALAA (SEQ ID NO: 1) (a) a peptide comprising the amino acid sequence set forth in SEQ ID NO: 1 and having cytoplasmic delivery activity; (b) a peptide comprising an amino acid sequence in which one or more amino acids are deleted, inserted, substituted, and / or added in the amino acid sequence set forth in SEQ ID NO: 1, and having cytoplasmic delivery activity; (c) a peptide comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 1 and having cytoplasmic delivery activity; Each of the multiple Xs independently represents an artificial amino acid residue in which an artificial amino acid represented by the following general formula (A-1) or the following general formula (A-2) is bound via a peptide bond, or an aspartic acid residue: The multiple Xs may be the same or different.

[0049] [ka] [R 11 and R 21 R each independently represents a hydrocarbon group having 3 to 10 carbon atoms which may have a substituent. 12 and R 22 each independently represents a protecting group or a hydrogen atom.

[0050] Examples of the artificial amino acid represented by the general formula (A-1) or (A-2) include those mentioned above.

[0051] The peptide (a) may have 10 to 1000 residues, 15 to 700 residues, 20 to 500 residues, 30 to 300 residues, 30 to 100 residues, 30 to 70 residues, 30 to 50 residues, or 30 to 40 amino acid residues.

[0052] The "plurality" in the peptide (b) is not particularly limited, and examples include 2 to 20, 2 to 15, 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, 2 to 3, or 2, etc.

[0053] The peptide (c) contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 1 (hereinafter, this sequence is referred to as "XALA homologous sequence"). The XALA homologous sequence in the amino acid sequence of peptide (c) has an amino acid sequence identity of 90% or more, preferably 93% or more, more preferably 95% or more, even more preferably 97% or more, and particularly preferably 98% or more, to the amino acid sequence represented by sequence number 1.

[0054] Here, the sequence identity of an amino acid sequence is a value indicating the percentage of identity between a target amino acid sequence (target amino acid sequence) and a reference amino acid sequence (reference amino acid sequence). The reference amino acid sequence is the amino acid sequence represented by SEQ ID NO: 1. The target amino acid sequence is the XALA-homologous sequence in peptide (c). The sequence identity of a subject amino acid sequence to a reference amino acid sequence can be determined, for example, as follows: First, the reference amino acid sequence and the subject amino acid sequence are aligned. Here, gaps may be included in each amino acid sequence to maximize sequence identity. Next, the number of matching amino acids in the reference amino acid sequence and the subject amino acid sequence is calculated, and the sequence identity can be determined according to the following formula (F1). Sequence identity (%) = number of matching amino acids / total number of amino acids in the target amino acid sequence × 100 …(F1)

[0055] When the peptide (c) is a peptide in which one or more amino acids have been added to an XALA homologous sequence, the amino acids may be added to either the N-terminus or C-terminus of the XALA homologous sequence, or to both the N-terminus and C-terminus.

[0056] The peptide (c) is not particularly limited as long as the peptide has cytoplasmic delivery activity, and may be, for example, 5,000 residues or less, 3,000 residues or less, 2,000 residues or less, 1,000 residues or less, 500 residues or less, 300 residues or less, 100 residues or less, 60 residues or less, or 50 residues or less.

[0057] When the peptide (c) is a peptide in which one or more amino acids are added to an XALA homologous sequence, the number of amino acids added to the XALA homologous sequence is not particularly limited as long as it has cytoplasmic delivery activity, but may be 1 to 5,000 residues, 1 to 3,000 residues, 1 to 2,000 residues, 1 to 1,000 residues, 1 to 500 residues, 1 to 300 residues, 1 to 100 residues, or 1 to 40 residues.

[0058] The salt of the peptide containing the amino acid sequence represented by formula (1) that can be contained in the cytoplasmic delivery agent of this embodiment is not particularly limited as long as it is a physiologically acceptable salt, and may be an acid addition salt or a basic salt. Examples of acid addition salts include salts with inorganic acids such as hydrochloric acid, phosphoric acid, hydrobromic acid, and sulfuric acid; and salts with organic acids such as acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid, tartaric acid, citric acid, malic acid, benzoic acid, methanesulfonic acid, and benzenesulfonic acid. Examples of basic salts include salts with inorganic bases such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, and magnesium hydroxide; and salts with organic bases such as caffeine, piperidine, trimethylamine, and pyridine.

[0059] <Peptide concentration> When the cytoplasmic delivery agent of this embodiment is brought into contact with a target cell, the concentration of the peptide comprising the amino acid sequence represented by formula (1) or its salt is not particularly limited as long as the effects of the present invention are achieved, and may be, for example, 10 pM to 1 mM, preferably 100 pM to 100 μM, and more preferably 1 nM to 10 μM.

[0060] <Method of producing peptides> The peptide containing the amino acid sequence represented by formula (1) may be produced by a method commonly used in the art, for example, an organic chemical synthesis method such as the Fmoc method (fluorenylmethyloxycarbonyl method) or the tBoc method (t-butoxycarbonyl method), or may be produced using a commercially available device generally called a peptide synthesizer.

[0061] The peptide containing the amino acid sequence represented by the formula (1) may be produced as a recombinant protein by applying various known gene recombination techniques to a nucleic acid encoding the peptide.

[0062] The peptide containing the amino acid sequence shown in formula (1) may be produced in a form in which a tag peptide such as a Flag tag, a polyhistidine tag, a c-Myc tag, an HA tag, an AU1 tag, a GST tag, or an MBP tag is attached to its N-terminus and / or C-terminus, or may be produced in the form of a fusion protein with other proteins such as a fluorescent protein, glutathione transferase, or alkaline phosphatase. These tag peptides and other proteins are preferably removed before contacting the peptide with target cells. The peptide containing the amino acid sequence represented by formula (1) may be labeled with a fluorescent substance, a luminescent substance, biotin, or other appropriate labeling agent.

[0063] <Cationic molecules> The cationic molecule is not particularly limited as long as it exhibits the effects of the present invention, and examples thereof include cationic lipids and cationic polymers.

[0064] Examples of cationic lipids include 1,2-dioleyloxy-3-(trimethylammonium)propane (DOPTAP), N,N-dioctadecylamidoglycylspermine (DOGS), dimethyldioctadecylammonium bromide (DDAB), N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), 2,3-dioleyloxy-N-[2(spermine-carboxamido)ethyl]-N,N-dimethyl-1-propanaminium trifluoroacetate (DOSPA), N-[1-(2,3-dimyristyloxy)propyl]-N,N-dimethyl-N-(2-hydroxyethyl)ammonium bromide (DMRIE), or esters of dipalmitoylphosphatidic acid (DPPA) and hydroxyethylenediamine, and esters of distearoylphosphatidic acid (DSPA) and hydroxyethylenediamine.

[0065] The commercially available reagent containing a cationic lipid is not particularly limited as long as it exhibits the effects of the present invention, and examples thereof include LIPOFECTAMINE (registered trademark), LIPOFECTAMINE 2000 (registered trademark), LIPOFECTAMINE 3000 (registered trademark), and LIPOFECTAMINE LTX (registered trademark).

[0066] Examples of cationic polymers include poly-L-lysine (PLL), chitosan, polyethyleneimine (PEI), polydimethylaminoethyl methacrylate (PD-MAEMA), and polyamidoamine (PAMAM).

[0067] The cationic molecule is preferably a cationic lipid.

[0068] <Concentration of cationic molecules> The concentration of the cationic molecule when the cytoplasmic delivery agent according to this embodiment is brought into contact with the target cells is not particularly limited as long as the effects of the present invention are achieved. For example, the concentration may be 0.01 to 20% by mass, preferably 0.1 to 5% by mass, and more preferably 0.5 to 3% by mass, relative to the total mass (100% by mass) of the cytoplasmic delivery agent to be brought into contact with the target cells.

[0069] The cytoplasmic delivery agent according to this embodiment may be used in combination with a known transfection reagent. Examples of transfection reagents include reagents for transfecting nucleic acids and reagents for transfecting proteins.

[0070] The cytoplasmic delivery agent according to this embodiment may be used in combination with a buffer, etc. Examples of buffers include, but are not limited to, PBS, physiological saline, and Tris buffer.

[0071] As described above, the cytoplasmic delivery agent of this embodiment contains a peptide having the amino acid sequence shown in formula (1) or a salt thereof, and a cationic molecule, and is thereby capable of delivering a target substance to the cytoplasm. After the target substance is taken up into the endosome, the peptide containing the amino acid sequence represented by formula (1) promotes the destruction of the endosomal membrane, thereby preventing the target substance from being degraded in the lysosome, and as a result, the target substance is delivered to the cytoplasm with high efficiency. X 1 However, when the artificial amino acid represented by the general formula (A-1) or the following general formula (A-2) is an artificial amino acid residue linked by a peptide bond, the hydrophobicity of the peptide is increased compared to when a GALA peptide is used, and the interaction between the peptide and the cell membrane is enhanced, thereby enabling a higher cytoplasmic delivery activity. 1 When the residue is an aspartic acid residue, it is possible to deliver the target substance to the cytoplasm with the same efficiency as when the GALA peptide is used.

[0072] (Method for delivering a target substance into the cytoplasm of a target cell) The method for delivering a target substance to the cytoplasm of a target cell according to this embodiment includes the step of contacting the target cell with the cytoplasm delivery agent according to the embodiment and the target substance.

[0073] Examples of the target substance and the target cells include those described above in the above embodiments. The concentration of the target substance to be contacted with the target cells is not particularly limited as long as the effects of the present invention are achieved, and can be appropriately determined by a person skilled in the art.

[0074] According to the method of this embodiment, it is possible to deliver a target substance to the cytoplasm by using the cytoplasmic delivery agent of the embodiment.

[0075] (Other embodiments) In one embodiment, the present invention provides the use of a cytoplasmic delivery agent to deliver a substance of interest to the cytoplasm of a target cell. The cytoplasmic delivery agent contains a peptide comprising the amino acid sequence shown in the following formula (1) or a salt thereof, and a cationic molecule, and has cytoplasmic delivery activity. X 1 X 2 X 3 X 4 Formula (1) X 1 represents an artificial amino acid residue in which an artificial amino acid represented by the following general formula (A-1) or the following general formula (A-2) is bound via a peptide bond, or an aspartic acid residue, X 2 , X 3 and X 4 each independently represents alanine, leucine, isoleucine, valine, or phenylalanine.

[0076] [ka] [R 11 and R 21R each independently represents a hydrocarbon group having 3 to 10 carbon atoms which may have a substituent. 12 and R 22 each independently represents a protecting group or a hydrogen atom.

[0077] Examples of the target substance, target cell, and cytoplasmic delivery agent include those similar to those described above in the previous embodiment.

[0078] In one embodiment, the present invention provides use of a peptide or a salt thereof and a cationic molecule for producing a cytoplasmic delivery agent. Cytoplasmic delivery agents are used to deliver target substances into the cytoplasm of target cells. The cytoplasmic delivery agent has cytoplasmic delivery activity. The cytoplasmic delivery agent contains a peptide comprising the amino acid sequence shown in the following formula (1) or a salt thereof, and a cationic molecule. X 1 X 2 X 3 X 4 Formula (1) X 1 represents an artificial amino acid residue in which an artificial amino acid represented by the following general formula (A-1) or the following general formula (A-2) is bound by a peptide bond, or represents aspartic acid, X 2 , X 3 and X 4 each independently represents alanine, leucine, isoleucine, valine, or phenylalanine.

[0079] [ka] [R 11 and R 21 R each independently represents a hydrocarbon group having 3 to 10 carbon atoms which may have a substituent. 12 and R 22 each independently represents a protecting group or a hydrogen atom.

[0080] Examples of the target substance, target cell, and cytoplasmic delivery agent include those similar to those described above in the previous embodiment. [Example]

[0081] The present invention will be described below with reference to examples, but the present invention is not limited to the following examples.

[0082] (Synthesis of GALA, DALA, and AsuALA peptides) GALA, DALA, and AsuALA peptides were synthesized as follows.

[0083] The GALA peptide is a peptide consisting of the amino acid sequence shown in SEQ ID NO:2. WEAALAEALAEALAEHLAEALAEALEALAA (SEQ ID NO: 2)

[0084] The DALA peptide is a peptide consisting of the amino acid sequence shown in SEQ ID NO:3. WDAALADALADALADHLADALADALDALAA (SEQ ID NO: 3)

[0085] The AsuALA peptide is a peptide consisting of the amino acid sequence shown in SEQ ID NO:4. WXAALAXALAXALAXHLAXALAXALXALAA (SEQ ID NO: 4) In SEQ ID NO: 4, X represents an artificial amino acid residue to which the artificial amino acid represented by the following formula (A-1-1) is bound via a peptide bond.

[0086] [ka]

[0087] All peptides were synthesized using the Fmoc solid-phase method. The amino acids used were Fmoc amino acids (Peptide Institute) and Fmoc-Asu(OtBu)-OH (GL Biochem). Fmoc-Asu(OtBu)-OH is (S)-Fmoc-2-amino-octanedioic acid-8-tert-butyl ester, CAS Registry Number 276869-41-1. By using Fmoc-Asu(OtBu)-OH, a peptide having an artificial amino acid residue in which the artificial amino acid represented by the formula (A-1-1) is bound by a peptide bond was synthesized.

[0088] The peptide chain was elongated by repeating the condensation reaction (1-hydroxybenzotriazole (HOBt) (Peptide Institute) / 2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) (Peptide Institute) / N,N-diisopropylethylamine (DIEA) (Sigma-Aldrich)) onto a peptide synthesis resin (TGS-RAM, Shimadzu Corporation) and the Fmoc deprotection reaction (30% piperidine (Nacalai Tesque)) (PSSM-8 Automatic Peptide Synthesizer, Shimadzu Corporation).

[0089] After completion of the elongation reaction of the target peptide chain on the resin, the peptide was deprotected and cleaved from the resin using trifluoroacetic acid (TFA) (Watanabe Chemical Industries)-ethanedithiol (EDT) (Nacalai Tesque) (95:5) at 20°C for 3 hours. The crude peptide cleaved from the resin was purified using reversed-phase high-performance liquid chromatography (Chromaster, Hitachi High-Tech Science) using solvents: 0.1% TFA in HO and 0.1% TFA in CHCN (Nacalai Tesque). After lyophilization, the molecular weight of the target peptide was confirmed using a matrix-assisted laser desorption / ionization time-of-flight mass spectrometer (Microflex, Bruker Daltonics). The measurement results are shown below.

[0090] GALA peptide (Trp-Glu-Ala-Ala-Leu-Ala-Glu-Ala-Leu-Ala-Glu-Ala-Leu-Ala-Glu-His-Leu-Ala-Glu-Ala-Leu-Ala-Glu-Ala-Leu-Glu-Ala-Leu-Ala-Ala-amide) MALDI-TOF-MS: 3031.8 [calcd for (M+H) + : 3032.4] Column: COSMOSIL 5C18-AR-II Gradient: 5%(0min)-5%(5min)-95%(35min) B in A (A = H2O / 0.1% TFA, B = CH3CN / 0.1% TFA) Flow rate: 1.0 ml / min Detection: 220 nm Yield from the starting resin: 28%

[0091] DALA peptide (Trp-Asp-Ala-Ala-Leu-Ala-Asp-Ala-Leu-Ala-Asp-Ala-Leu-Ala-Asp-His-Leu-Ala-Asp-Ala-Leu-Ala-Asp-Ala-Leu-Asp-Ala-Leu-Ala-Ala-amide) MALDI-TOF-MS: 2933.4 [calcd for (M+H) + : 2934.2] Column: COSMOSIL 5C18-AR-II Gradient: 5%(0min)-5%(5min)-95%(35min) B in A (A = H2O / 0.1% TFA, B = CH3CN / 0.1% TFA) Flow rate: 1.0 ml / min Detection: 220 nm Yielding from the starting resin: 23%

[0092] AsuALA Peptide (Trp-Asu-Ala-Ala-Leu-Ala-Asu-Ala-Leu-Ala-Asu-Ala-Leu-Ala-Asu-His-Leu-Ala-Asu-Ala-Leu-Ala-Asu-Ala-Leu-Asu-Ala-Leu-Ala-Ala-amide) MALDI-TOF-MS: 3326.0 [calcd for (M+H) + : 3327.0] Column: COSMOSIL 5C4-AR-300 Gradient: 5%(0min)-5%(5min)-95%(35min)-95%(40min) B in A (A = H2O / 0.1% TFA, B = CH3CN / 0.1% TFA) Flow rate: 1.0 ml / min Detection: 220 nm Yielding from the starting resin: 35%

[0093] (Synthesis of FITC-labeled peptide) The following FITC-labeled peptides were synthesized by the following procedure.

[0094] FITC-GABA-GALA peptide FITC-NH-(CH)-CO-WEAALAEALAEALAEHLAEALAEALEALAA (SEQ ID NO: 5)

[0095] FITC-GABA-DALA peptide FITC-NH—(CH)-CO-WDAALADALADALADHLADALADALDALAA (SEQ ID NO: 6)

[0096] FITC-GABA-AsuALA peptide FITC-NH-(CH2)3-CO-WXAALAXALAXALAXHLAXALAXALXALAA (SEQ ID NO: 7) In SEQ ID NO: 7, X represents an artificial amino acid residue to which the artificial amino acid represented by the following formula (A-1-1) is bound via a peptide bond.

[0097] [ka]

[0098] The above-mentioned automatically synthesized GALA peptide (sequence number 2), DALA peptide (sequence number 3), and AsuALA peptide (sequence number 4) were manually synthesized with Fmoc-γ-Abu-OH(GABA) (Merck) as a linker and FITC, Isomer I (Tokyo Chemical Industry) as a fluorophore to obtain FITC-GABA-GALA, FITC-GABA-DALA, and FITC-GABA-AsuALA peptides, respectively.

[0099] For manual synthesis, 5 equivalents of GABA, HOBt, HBTU, and DIEA were dissolved in 500 μL of DMF and then added to 1 equivalent of resin. The mixture was incubated at 50°C for 2 hours in DMF. The mixture was then washed with DMF (1 mL, 5 times). A Ninhydrin test using Kaiser Test Reagents (Ninhydrin / EtOH, Phenol / EtOH, KCN / Pyridine) (Kokusan Chemical) was performed to confirm GABA condensation to the N-terminus of the peptide chain. 500 μL of 30% piperidine was added and the mixture was stirred at room temperature for 20 minutes to carry out the Fmoc removal reaction. The mixture was washed with DMF (1 mL, 5 times). A Ninhydrin test using Kaiser Test Reagents (Ninhydrin / EtOH, Phenol / EtOH, KCN / Pyridine) (Kokusan Chemical) was performed to confirm the removal of the Fmoc group from the N-terminus.

[0100] Five equivalents of FITC, Isomer I, and DIEA were dissolved in 500 μL of DMF, added to 1 equivalent of resin, and reacted in DMF at 50°C for 2 hours. After washing with DMF (1 mL, 5 times), a Ninhydrin test was performed using Kaiser Test Reagents (Ninhydrin / EtOH, Phenol / EtOH, KCN / Pyridine) (Kokusan Chemical Co., Ltd.), confirming that FITC had been condensed to the N-terminus of the peptide chain. After the FITC condensation reaction on the resin was completed, the peptide was deprotected and cleaved from the resin using trifluoroacetic acid (TFA) (Watanabe Chemical Industries)-ethanedithiol (EDT) (Nacalai Tesque) (95:5) at 20°C for 3 hours. The crude peptide cleaved from the resin was purified using reversed-phase high-performance liquid chromatography (Chromaster, Hitachi High-Tech Science) using solvents: 0.1% TFA in HO and 0.1% TFA in CHCN (Nacalai Tesque). After lyophilization, the molecular weight of the target peptide was confirmed using a matrix-assisted laser desorption / ionization time-of-flight mass spectrometer (Microflex, Bruker Daltonics). The measurement results are shown below.

[0101] FITC-GABA-GALA peptide (FITC-NH-(CH2)3-CO-Trp-Glu-Ala-Ala-Leu-Ala-Glu-Ala-Leu-Ala-Glu-Ala-Leu-Ala-Glu- His-Leu-Ala-Glu-Ala-Leu-Ala-Glu-Ala-Leu-Glu-Ala-Leu-Ala-Ala-amide) MALDI-TOF-MS: 3506.3 [calcd for (M+H) + : 3506.9] Column: COSMOSIL 5C18-AR-II Gradient: 5%(0min)-5%(5min)-95%(35min) B in A (A = H2O / 0.1% TFA, B = CH3CN / 0.1% TFA) Flow rate: 1.0 ml / min Detection: 220 nm Yielding from the starting resin: 16%

[0102] FITC-GABA-DALAペプチド (FITC-NH-(CH2)3-CO-Trp-Asp-Ala-Ala-Leu-Ala-Asp-Ala-Leu-Ala-Asp-Ala-Leu-Ala-Asp-His-Leu-Ala-Asp-Ala-Leu-Ala-Asp-Ala-Leu-Asp-Ala-Leu-Ala-Ala-amide) MALDI-TOF-MS: 3409.6 [calcd for (M+H) + : 3408.7] Column: COSMOSIL 5C18-AR-II Gradient: 5%(0min)-5%(5min)-95%(35min) B in A (A = H2O / 0.1% TFA, B = CH3CN / 0.1% TFA) Flow rate: 1.0 ml / min Detection: 220 nm Yielding from the starting resin: 15%

[0103] FITC-GABA-AsuALAペプチド (FITC-NH-(CH2)3-CO-Trp-Asu-Ala-Ala-Leu-Ala-Asu-Ala-Leu-Ala-Asu-Ala-Leu-Ala-Asu-His-Leu-Ala-Asu-Ala-Leu-Ala-Asu-Ala-Leu-Asu-Ala-Leu-Ala-Ala-amide) MALDI-TOF-MS: 3801.2 [calcd for (M+H) + : 3801.4] Column: COSMOSIL 5C4-AR-300 Gradient: 5%(0min)-5%(5min)-95%(35min)-95%(40min) B in A (A = H2O / 0.1% TFA, B = CH3CN / 0.1% TFA) Flow rate: 1.0 ml / min Detection: 220 nm Yielding from the starting resin: 28%

[0104] (Synthesis and purification of biotin-labeled peptides) The following biotin-labeled peptides were synthesized by the following procedure.

[0105] Biotin-GALA peptide Biotin-WEAALAEALAEALAEHLAEALAEALEALAA (SEQ ID NO: 8)

[0106] Biotin-DALA peptide Biotin-WDAALADALADALADHLADALADALDALAA (SEQ ID NO: 9)

[0107] Biotin-AsuALA Biotin-WXAALAXALAXALAXHLAXALAXALXALAA (SEQ ID NO: 10) In SEQ ID NO: 10, X represents an artificial amino acid residue to which the artificial amino acid represented by the following formula (A-1-1) is bound via a peptide bond.

[0108] [ka]

[0109] Biotin-NHS (Funakoshi) was manually added to the automatically synthesized GALA peptide (sequence number 2), DALA peptide (sequence number 3), and AsuALA peptide (sequence number 4), respectively, to obtain Biotin-GALA, Biotin-DALA, and Biotin-AsuALA peptides.

[0110] For manual synthesis, 5 equivalents of Biotin-NHS and DIEA were first dissolved in 500 μL of DMF, then added to 1 equivalent of resin and allowed to react overnight in DMF at 50°C. After washing with DMF (1 mL, 5 times), a Ninhydrin test was performed using Reagents for Kaiser Test (Ninhydrin / EtOH, Phenol / EtOH, KCN / Pyridine) (Kokusan Chemical Co., Ltd.), confirming that Biotin-NHS had been condensed to the N-terminus of the peptide chain.

[0111] After the biotin-NHS condensation reaction on the resin was completed, the peptide was deprotected and cleaved from the resin using trifluoroacetic acid (TFA) (Watanabe Chemical Industries)-ethanedithiol (EDT) (Nacalai Tesque) (95:5) at 20°C for 3 hours. The crude peptide cleaved from the resin was purified using reversed-phase high-performance liquid chromatography (Chromaster, Hitachi High-Tech Science) using solvents: 0.1% TFA in HO and 0.1% TFA in CHCN (Nacalai Tesque). After lyophilization, the molecular weight of the target peptide was confirmed using a matrix-assisted laser desorption / ionization time-of-flight mass spectrometer (Microflex, Bruker Daltonics). The measurement results are shown below.

[0112] Biotin-GALA peptide (Biotin-Trp-Glu-Ala-Ala-Leu-Ala-Glu-Ala-Leu-Ala-Glu-Ala-Leu-Ala-Glu-His-Leu-Ala-Glu-Ala-Leu-Ala-Glu-Ala-Leu-Glu-Ala-Leu-Ala-Ala-amide) MALDI-TOF-MS: 3257.8 [calcd for (M+H) + : 3257.7] Column: COSMOSIL 5C18-AR-II Gradient: 5%(0min)-5%(5min)-95%(35min) B in A (A = H2O / 0.1% TFA, B = CH3CN / 0.1% TFA) Flow rate: 1.0 ml / min Detection: 220 nm Yield from the starting resin: 24%

[0113] Biotin-DALA peptide (Biotin-Trp-Asp-Ala-Ala-Leu-Ala-Asp-Ala-Leu-Ala-Asp-Ala-Leu-Ala-Asp-His-Leu-Ala-Asp-Ala-Leu-Ala-Asp-Ala-Leu-Asp-Ala-Leu-Ala-Ala-amide) MALDI-TOF-MS: 3160.3 [calcd for (M+H) + : 3159.5] Column: COSMOSIL 5C18-AR-II Gradient: 5%(0min)-5%(5min)-95%(35min) B in A (A = H2O / 0.1% TFA, B = CH3CN / 0.1% TFA) Flow rate: 1.0 ml / min Detection: 220 nm Yield from the starting resin: 26%

[0114] Biotin-AsuALA peptide (Biotin-Trp-Asu-Ala-Ala-Leu-Ala-Ala-Ala-Leu-Ala-Asu-Ala-Leu-Ala-Asu-His-Leu-Ala-Asu-Ala-Leu-Ala-Asu-Ala-Leu-Asu-Ala-Leu-Ala-Ala-amide) MALDI-TOF-MS: 3552.8 [calcd for (M+H) + : 3552.3] Column: COSMOSIL 5C4-AR-300 Gradient: 5%(0min)-5%(5min)-95%(35min)-95%(45min) B in A (A = H2O / 0.1% TFA, B = CH3CN / 0.1% TFA) Flow rate: 1.0 ml / min Detection: 220 nm Yielding from the starting resin: 26%

[0115] In the following Experimental Examples 1 to 4, Lipofectamine LTX (Thermo Fisher Scientific) was used as a reagent containing a cationic lipid.

[0116] (Experimental Example 1) The cytoplasmic delivery activity of FITC-labeled peptides was measured for cytoplasmic delivery agents containing FITC-labeled peptides consisting of the amino acid sequences shown in SEQ ID NOs: 5 to 7 and cationic lipids.

[0117] Observation of intracellular internalization of FITC-labeled peptides using a confocal laser scanning microscope HeLa cells (2.4×10 4 The cells (200 μL) were subcultured onto a 35 mm glass-based dish (Iwaki) and cultured in α-MEM medium containing 10% FBS at 37°C and 5% CO 2 for 24 hours.

[0118] A 10 μL mixture of each FITC-labeled peptide of SEQ ID NOs: 5 to 7 (final concentration 200 nM), Lipofectamine LTX (final concentration 1% v / v), and PBS was left to stand at 25° C. for 20 minutes to form complexes. Next, α-MEM and FBS (final concentration 10%) were added to obtain 100 μL of Test Examples 1 to 3 containing the FITC-labeled peptides of SEQ ID NOs: 5 to 7, respectively.

[0119] Test Example 1 FITC-GABA-GALA peptide (SEQ ID NO: 5) Test Example 2 FITC-GABA-DALA peptide (SEQ ID NO: 6) Test Example 3 FITC-GABA-AsuALA peptide (SEQ ID NO: 7)

[0120] The culture medium was then removed, and the cells were washed with α-MEM containing 10% FBS (100 μL, 3 times). Test Examples 1 to 3 (100 μL / dish) were added and cultured for 4 hours at 37°C and 5% CO2. The cells were then observed using a confocal laser scanning microscope (FV1200, Olympus). The results are shown in Figure 1A.

[0121] As shown in FIG. 1A, it was confirmed that all of the FITC-labeled peptides consisting of SEQ ID NOs: 5 to 7 were taken up into cells.

[0122] <Evaluation of intracellular internalization of FITC-labeled peptides using a flow cytometer> HeLa cells (1.4×10 5 The cells (600 μL) were subcultured in a 24-well microplate (Iwaki) and cultured in α-MEM containing 10% FBS for 24 hours (37°C, 5% CO2).

[0123] A 60 μL mixture of each FITC-labeled peptide of SEQ ID NOs: 5 to 7 (final concentration 200 nM), Lipofectamine LTX (final concentration 1% v / v), and PBS was left to stand at 25° C. for 20 minutes to form complexes. Next, α-MEM and FBS (final concentration 10%) were added to give 600 μL of Test Examples 4 to 6 containing the FITC-labeled peptides of SEQ ID NOs: 5 to 7, respectively.

[0124] Test Example 4 FITC-GABA-GALA peptide (SEQ ID NO: 5) Test Example 5 FITC-GABA-DALA peptide (SEQ ID NO: 6) Test Example 6 FITC-GABA-AsuALA peptide (SEQ ID NO: 7)

[0125] The culture medium for the cultured cells was removed, and the cells were washed with medium (α-MEM containing 10% FBS) (200 μL, 3 times). Test Examples 4 to 6 (600 μL / well) were then added to the cultured cells, and the cells were cultured for 4 hours (37°C, 5% CO2).

[0126] As control 1, unmanipulated HeLa cells were prepared. As control 2, HeLa cells were prepared by adding only Lipofectamine LTX (final concentration 1% v / v) in the same manner as in Test Examples 4 to 6, except that the FITC-labeled peptide was not used.

[0127] After washing the cells with PBS (200 μL, 3 times), the cells were detached by treatment with 0.1 g / L trypsin-0.106 mmol EDTA (Nacalai, 200 μL, 37°C, 10 minutes, 5% CO2).

[0128] The suspension containing the detached cells was collected and centrifuged twice at 4°C, 1500 rpm (200g), and the supernatant was removed. Next, 400 μL of PBS was added to disperse the cells, and the mixture was centrifuged again at 4°C, 1500 rpm (200g). The supernatant was removed, and 400 μL of PBS was added. After this cell washing, the cell fluorescence intensity (excitation: 488 nm, emission: 525 nm) of 10,000 live cells was measured using a flow cytometer (Guava easyCyte, Merck Millipore). The results are shown in Figure 1B.

[0129] As shown in FIG. 1B, Test Example 5 containing the FITC-GABA-DALA peptide was confirmed to have cytoplasmic delivery activity at the same level as Test Example 4 containing the FITC-GABA-GALA peptide. Test Example 6 containing the FITC-GABA-AsuALA peptide was confirmed to have higher cytoplasmic delivery activity than Test Example 4 containing the FITC-GABA-GALA peptide.

[0130] (Experimental Example 2) The activity of cytoplasmic delivery agents containing each biotin-labeled peptide consisting of the amino acid sequences shown in SEQ ID NOs: 8 to 10 and a cationic lipid to deliver FITC-avidin into the cytoplasm was measured.

[0131] <Observation of intracellular internalization of FITC-avidin using a confocal laser scanning microscope> HeLa cells (2.4×10 4 The cells (200 μL) were subcultured onto a 35 mm glass-based dish (Iwaki) and cultured in α-MEM medium containing 10% FBS at 37°C and 5% CO 2 for 24 hours.

[0132] FITC-avidin (Sigma-Aldrich), each biotin-labeled peptide, and PBS were mixed and incubated at 25°C for 10 minutes. Lipofectamine LTX was added to the mixture (10 μL), which was then incubated at 25°C for 20 minutes to allow complex formation. The final concentrations of the mixture were 0.50 μM FITC-avidin, 2 μM each biotin-labeled peptide, and 2% Lipofectamine LTX. Next, α-MEM, FBS (final concentration 10%), and 1% Penicillin-Streptomycin (Sigma-Aldrich) were added to obtain 100 μL of Test Examples 7 to 9, each containing the biotin-labeled peptides of SEQ ID NOs: 8 to 10.

[0133] Test Example 7 Biotin-GALA peptide (SEQ ID NO: 8) Test Example 8 Biotin-DALA peptide (SEQ ID NO: 9) Test Example 9 Biotin-AsuALA peptide (SEQ ID NO: 10)

[0134] As control 3, unmanipulated HeLa cells were prepared. As control 4, HeLa cells were prepared to which only FITC-avidin was added in the same manner as in Test Examples 7 to 9, except that the biotin-labeled peptide and Lipofectamine LTX were not used. As control 5, HeLa cells were prepared by adding only FITC-avidin and Lipofectamine LTX in the same manner as in Test Examples 7 to 9, except that no biotin-labeled peptide was used.

[0135] The culture medium was removed, and the cells were washed with α-MEM containing 10% FBS (100 μL, 3 times). Test samples 7 to 9 (100 μL / dish) were added and cultured for 6 hours at 37°C and 5% CO2. The cells were then observed using a confocal laser scanning microscope (FV1200, Olympus). The results are shown in Figure 2A.

[0136] As shown in FIG. 2A, Test Examples 7 to 9, which contain biotin-labeled peptides of SEQ ID NOs: 8 to 10, respectively, were confirmed to have the activity of delivering FITC-avidin into cells.

[0137] <Evaluation of FITC-avidin intracellular internalization using a flow cytometer> HeLa cells (1.4×10 5 The cells (600 μL) were subcultured in a 24-well microplate (Iwaki) and cultured in α-MEM medium containing 10% FBS for 24 hours (37°C, 5% CO ).

[0138] FITC-avidin, each biotin-labeled peptide, and PBS were mixed and incubated at 25°C for 10 minutes. Lipofectamine LTX was added to the mixture (60 μL), which was then incubated at 25°C for 20 minutes to allow complex formation. The final concentrations of the mixture were 0.50 μM FITC-avidin, 2 μM each biotin-labeled peptide, and 2% Lipofectamine LTX. Next, α-MEM, FBS (final concentration 10%), and 1% penicillin-streptomycin were added to obtain 600 μL of Test Examples 10 to 12, each containing the biotin-labeled peptides of SEQ ID NOs: 8 to 10.

[0139] Test Example 10 Biotin-GALA peptide (SEQ ID NO: 8) Test Example 11 Biotin-DALA peptide (SEQ ID NO: 9) Test Example 12 Biotin-AsuALA peptide (SEQ ID NO: 10)

[0140] The culture medium for the cultured cells was removed, and the cells were washed with medium (α-MEM containing 10% FBS) (200 μL, 3 times). Test Examples 10 to 12 (600 μL / well) were then added to the cultured cells, and the cells were cultured for 4 hours (37°C, 5% CO2).

[0141] As control 6, unmanipulated HeLa cells were prepared. As control 7, HeLa cells were prepared to which only FITC-avidin was added in the same manner as in Test Examples 10 to 12, except that neither the biotin-labeled peptide nor Lipofectamine LTX was used. As control 8, HeLa cells were prepared to which only FITC-avidin and Lipofectamine LTX were added in the same manner as in Test Examples 10 to 12, except that no biotin-labeled peptide was used.

[0142] After washing the cells with PBS (200 μL, 3 times), the cells were detached by treatment with 0.1 g / L trypsin-0.106 mmol EDTA (Nacalai, 200 μL, 37°C, 10 minutes, 5% CO2).

[0143] The suspension containing the detached cells was collected and centrifuged twice at 4°C, 1500 rpm (200g), and the supernatant was removed. Next, 400 μL of PBS was added to disperse the cells, and the mixture was centrifuged again at 4°C, 1500 rpm (200g). The supernatant was removed, and 400 μL of PBS was added. After this cell washing, the cell fluorescence intensity (excitation: 488 nm, emission: 525 nm) of 10,000 live cells was measured using a flow cytometer (Guava easyCyte, Merck Millipore). The results are shown in Figure 2B.

[0144] As shown in Figure 2B, Test Example 11, which contains the Biotin-DALA peptide, was confirmed to have the same or higher activity of delivering FITC-avidin into cells as Test Example 10, which contains the Biotin-GALA peptide. It was confirmed that Test Example 12 containing Biotin-AsuALA peptide had significantly higher activity in delivering FITC-avidin into cells than Test Example 10 containing Biotin-GALA peptide.

[0145] (Experimental Example 3) The activity of cytoplasmic delivery agents containing each peptide consisting of the amino acid sequences shown in SEQ ID NOs: 2 to 4 and a cationic lipid to deliver IgG-Alexa488 to the cytoplasm was measured.

[0146] <Observation of intracellular internalization of IgG-Alexa488 using a confocal laser scanning microscope> HeLa cells (2.4×10 4 The cells (200 μL) were subcultured onto a 35 mm glass-based dish (Iwaki) and cultured in α-MEM medium containing 10% FBS at 37°C and 5% CO 2 for 24 hours.

[0147] A 10 μL mixture of IgG-Alexa488 (Alexa Fluor 488 goat anti-mouse IgG antibody, ThermoFisher) (final concentration 10 μg / mL), each peptide (final concentration 1 μM), Lipofectamine LTX (final concentration 1% v / v), and PBS was incubated at 25°C for 20 minutes to allow complex formation. Next, α-MEM and FBS (final concentration 10%) were added to give 100 μL of Test Examples 13 to 15 containing peptides consisting of the amino acid sequences shown in SEQ ID NOs: 2 to 4.

[0148] Test Example 13 GALA peptide (SEQ ID NO: 2) Test Example 14 DALA peptide (SEQ ID NO: 3) Test Example 15 AsuALA peptide (SEQ ID NO: 4)

[0149] As a control 9, unmanipulated HeLa cells were prepared. As a control 10, HeLa cells were prepared to which only IgG-Alexa488 was added in the same manner as in Test Examples 13 to 15, except that the peptide and Lipofectamine LTX were not used. As a control 11, HeLa cells were prepared to which only IgG-Alexa488 and Lipofectamine LTX were added in the same manner as in Test Examples 13 to 15, except that no peptide was used.

[0150] The culture medium was then removed, and the cells were washed with α-MEM containing 10% FBS (100 μL, 3 times). Test Examples 13 to 15 (100 μL / dish) were added, and the cells were cultured for 24 hours at 37°C and 5% CO2. The cells were then observed using a confocal laser scanning microscope (FV1200, Olympus). The results are shown in Figure 3A.

[0151] As shown in FIG. 2A, Test Example 15, which contains a peptide consisting of the amino acid sequence shown in SEQ ID NO: 4, was confirmed to have the activity of delivering IgG-Alexa488 into cells with high efficiency.

[0152] <Evaluation of IgG-Alexa488 intracellular internalization using a flow cytometer> HeLa cells (1.4×10 5 The cells (600 μL) were subcultured in a 24-well microplate (Iwaki) and cultured in α-MEM containing 10% FBS for 24 hours (37°C, 5% CO2).

[0153] A 60 μL mixture of IgG-Alexa488 (final concentration 10 μg / mL), each peptide (final concentration 1 μM), Lipofectamine LTX (final concentration 1% v / v), and PBS was left to stand at 25°C for 20 minutes to form complexes. Next, α-MEM, FBS (final concentration 10%), and 1% Penicillin-Streptomycin (Sigma-Aldrich) were added to obtain 600 μL of Test Examples 16 to 18 containing peptides consisting of the amino acid sequences shown in SEQ ID NOs: 2 to 4.

[0154] Test Example 16 GALA peptide (SEQ ID NO: 2) Test Example 17 DALA peptide (SEQ ID NO: 3) Test Example 18 AsuALA peptide (SEQ ID NO: 4)

[0155] As a control 12, HeLa cells were prepared to which only IgG-Alexa488 was added in the same manner as in Test Examples 16 to 18, except that the peptide and Lipofectamine LTX were not used. As a control 13, HeLa cells were prepared to which only IgG-Alexa488 and Lipofectamine LTX were added in the same manner as in Test Examples 16 to 18, except that no peptide was used.

[0156] The culture medium for the cultured cells was removed, and the cells were washed with medium (α-MEM containing 10% FBS) (200 μL, 3 times). Next, Test Examples 16 to 18 (600 μL / well) were added to the cultured cells, and the cells were cultured for 24 hours (37°C, 5% CO2).

[0157] After washing the cells with PBS (200 μL, 3 times), the cells were detached by treatment with 0.1 g / L trypsin-0.106 mmol EDTA (Nacalai, 200 μL, 37°C, 10 minutes, 5% CO2).

[0158] The suspension containing the detached cells was collected and centrifuged twice at 4°C, 1500 rpm (200g), and the supernatant was removed. Next, 400 μL of PBS was added to disperse the cells, and the mixture was centrifuged again at 4°C, 1500 rpm (200g). The supernatant was removed, and 400 μL of PBS was added. After this cell washing, the cell fluorescence intensity (excitation: 488 nm, emission: 525 nm) of 10,000 live cells was measured using a flow cytometer (Guava easyCyte, Merck Millipore). The results are shown in Figure 3B.

[0159] As shown in Figure 3B, Test Example 18, which contains the AsuALA peptide, was found to have a higher activity in delivering IgG-Alexa488 to the cytoplasm than Test Examples 16 to 17, which contain the GALA peptide or DALA peptide, respectively.

[0160] (Experimental Example 4) The cytotoxicity of the cytoplasmic delivery agent was examined.

[0161] Human cervical cancer-derived HeLa cells (1.2 × 10 4 The cells (100 μL) were subcultured in a 96-well microplate (Iwaki) and cultured in α-MEM (Gibco) medium containing 10% Fetal Bovine Serum (FBS) (Gibco) at 37°C and 5% CO for 24 hours.

[0162] 5 μL of a mixture of each peptide (final concentrations: 20 nM, 200 nM, 1 μM), Lipofectamine LTX (final concentration: 1% v / v), and PBS was left to stand at 25° C. for 20 minutes to form complexes. Next, α-MEM and FBS (final concentration 10%) were added to give 50 μL of Test Examples 19 to 27 containing peptides consisting of the amino acid sequences shown in SEQ ID NOs: 2 to 4.

[0163] The peptide concentration in each test example below is the concentration at which the complex was formed. Test Example 19 GALA peptide (SEQ ID NO: 2) 1 μM Test Example 20 GALA peptide (SEQ ID NO: 2) 200 nM Test Example 21 GALA peptide (SEQ ID NO: 2) 20 nM Test Example 22 DALA peptide (SEQ ID NO: 3) 1 μM Test Example 23 DALA peptide (SEQ ID NO: 3) 200 nM Test Example 24 DALA peptide (SEQ ID NO: 3) 20 nM Test Example 25 AsuALA peptide (SEQ ID NO: 4) 1 μM Test Example 26 AsuALA peptide (SEQ ID NO: 4) 200 nM Test Example 27 AsuALA peptide (SEQ ID NO: 4) 20 nM

[0164] As a control 14, unmanipulated HeLa cells were prepared. As a control 15, HeLa cells were prepared to which only Lipofectamine LTX was added in the same manner as in Test Examples 19 to 27, except that no peptide was used.

[0165] After sample preparation, the culture medium was removed, and the cells were washed with α-MEM containing 10% FBS (100 μL, 3 times). Test Examples 19–27 (50 μL / well) were added and incubated for 4 hours at 37°C and 5% CO2. WST-1 (4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzenedisulfonate) reagent (10 μL / well) was then added and incubated at 37°C for 45 minutes. Cell viability was quantified by measuring absorbance at 450 nm (A450) and 620 nm (A620). The results are shown in Figure 4. In Figure 4, the percentage of cell viability for each test is expressed as a percentage of the cell viability of control 14 (100%).

[0166] As shown in FIG. 4, it was confirmed that the cytoplasmic delivery agents of Test Examples 19 to 27 did not have cytotoxicity. [Industrial Applicability]

[0167] The present invention can be suitably used for the purpose of transfecting proteins, nucleic acids, sugars, etc. into cells and delivering them into the cytoplasm of the cells.

Claims

1. A cytoplasmic delivery agent for delivering a target substance to the cytoplasm of a target cell, comprising: The present invention comprises a peptide or a salt thereof comprising an amino acid sequence represented by the following formula (1), and a cationic molecule, a cytoplasmic delivery agent having cytoplasmic delivery activity; X １ X ２ X ３ X ４ Formula (1) X １ represents an artificial amino acid residue in which an artificial amino acid represented by the following general formula (A-1) or (A-2) is bound via a peptide bond, or an aspartic acid residue, X ２ , X ３ and X ４ each independently represents alanine, leucine, isoleucine, valine, or phenylalanine. 【Chemical 1】 [R １１ and R ２１ R each independently represents a hydrocarbon group having 3 to 10 carbon atoms, which may have a substituent. １２ and R ２２ each independently represents a protecting group or a hydrogen atom.

2. In the general formula (A-1) and the general formula (A-2), R １１ and R ２１ and each independently represent an optionally substituted aliphatic hydrocarbon group.

3. In the general formula (A-1) and the general formula (A-2), R １１ and R ２１ and each independently represent a chain aliphatic hydrocarbon group which may have a substituent.

4. In the general formula (A-1) and the general formula (A-2), R １１ and R ２１ and each independently represent an optionally substituted chain aliphatic hydrocarbon group having 3 to 7 carbon atoms.

5. X １ The cytoplasmic delivery agent according to claim 1, wherein the artificial amino acid represented by the following formula (A-1-01) or the following formula (A-2-01) is an artificial amino acid residue bound by a peptide bond. 【Chemistry 2】 [R １２ and R ２２ each independently represents a protecting group or a hydrogen atom.

6. The peptide contains repeats of the amino acid sequence represented by formula (1), The cytoplasmic delivery agent according to claim 1 , wherein the number of repeats is 3 to 20.

7. The peptide contains repeats of the amino acid sequence represented by formula (1), The cytoplasmic delivery agent according to claim 1, wherein the number of repeats is 4 to 10.

8. X ２ is alanine, and X ３ is leucine, and X ４ The cytoplasmic delivery agent of claim 1 , wherein is alanine.

9. The cytoplasmic delivery agent according to claim 1 , wherein the peptide is a peptide selected from the group consisting of the following (a), (b), and (c): WXAALAXALAXALAXALAXHLAXALAXALAXALXALAA (SEQ ID NO: 1) (a) a peptide comprising the amino acid sequence set forth in SEQ ID NO: 1 and having cytoplasmic delivery activity; (b) a peptide comprising an amino acid sequence in which one or more amino acids are deleted, inserted, substituted, and / or added in the amino acid sequence set forth in SEQ ID NO: 1, and having cytoplasmic delivery activity; (c) a peptide comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 1 and having cytoplasmic delivery activity; Each of the multiple Xs independently represents an artificial amino acid residue in which an artificial amino acid represented by the following general formula (A-1) or the following general formula (A-2) is bound via a peptide bond, or an aspartic acid residue: The plural X's may be the same or different. 【Chemistry 3】 [R １１ and R ２１ R each independently represents a hydrocarbon group having 3 to 10 carbon atoms, which may have a substituent. １２ and R ２２ each independently represents a protecting group or a hydrogen atom.

10. A method for delivering a substance of interest to the cytoplasm of a target cell, comprising: A method comprising the step of contacting a cytoplasmic delivery agent according to any one of claims 1 to 9 and the target substance with a cell of a subject.

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