Membrane permeation promoter, membrane permeation promoting method, and intracellular delivery method

Abstract

Provided is a membrane permeation promoter that promotes permeation of a conjugate of a target substance and a transmembrane peptide into a lipid bilayer membrane, the membrane permeation promoter containing a flavonoid. Also provided are a membrane permeation promoting method and intracellular delivery method using the membrane permeation promoter, and an intracellular delivery kit comprising the membrane permeation promoter.

Description

Membrane permeation enhancer, membrane permeation enhancement method, and intracellular delivery method

[0001] The present invention relates to a membrane permeation enhancer that enhances the membrane permeability of a complex between a target substance and a membrane-permeating peptide, a membrane permeation enhancement method and an intracellular delivery method using the membrane permeation enhancer, and an intracellular delivery kit that includes the membrane permeation enhancer.

[0002] Biological membranes, such as cell membranes, serve to separate the inside of the membrane from the outside world so that normal biological functions can be performed inside the membrane. This membrane does not simply separate the inside from the outside world; for example, in the case of a cell membrane, it is equipped with devices for exchanging substances, information, and energy with the outside of the cell and for carrying out various metabolic reactions. Specifically, the cell membrane contains transporters and receptors, which selectively take in necessary substances and excrete various metabolic products through their functions.

[0003] In recent years, cell penetrating peptides (CPPs) that can penetrate biological membranes without the intervention of these transporters or receptors have become known, and it is expected that these membrane-penetrating peptides will be combined with target substances (cargo) such as drugs to be applied to drug delivery, where the peptides penetrate biological membranes.

[0004] However, in order to control the membrane permeability of a complex between a target substance and a membrane-penetrating peptide (hereinafter also referred to as a "membrane-penetrating peptide complex"), there are many factors that need to be considered, such as the concentration, application time, and the structure of the membrane-penetrating peptide itself, and there is a problem in that it is not easy to change the complex to suit the target substance being applied.

[0005] In view of these problems, the present inventors have proposed a method for enhancing the membrane permeability of a membrane-permeable peptide complex using a membrane permeation enhancer containing a surfactant having a steroid skeleton, such as cholate or taurocholate (see Patent Document 1).

[0006] International Publication No. 2023 / 027006

[0007] Although the membrane permeation enhancer described in Patent Document 1 has excellent biocompatibility, it is still difficult to say that its effect of enhancing the membrane permeability of the membrane-permeating peptide complex is sufficient.

[0008] The present invention has been proposed in view of the above, and aims to provide a novel membrane permeation enhancer that is highly biocompatible and can further enhance the membrane permeability of a membrane-permeating peptide complex, a membrane permeation enhancement method and an intracellular delivery method using the membrane permeation enhancer, and an intracellular delivery kit that includes the membrane permeation enhancer.

[0009] Specific means for solving the above problems include the following embodiments. <1> A membrane permeation enhancer that enhances the permeation of a complex of a target substance and a membrane-permeating peptide through a lipid bilayer membrane, the membrane permeation enhancer containing a flavonoid. <2> The membrane permeation enhancer according to <1>, wherein the flavonoid is a catechin. <3> The membrane permeation enhancer according to <1> or <2>, wherein the lipid bilayer membrane is a cell membrane. <4> A membrane permeation enhancement method for enhancing the permeation of a complex of a target substance and a membrane-permeating peptide through a lipid bilayer membrane, the method comprising contacting the complex and the membrane permeation enhancer according to any one of <1> to <3> with a lipid bilayer membrane. <5> A method for intracellular delivery of a target substance into a cell, the method comprising contacting a complex of the target substance and a membrane-permeating peptide with a cell in the presence of the membrane permeation enhancer according to <1> or <2>. <6> The intracellular delivery method according to <5>, wherein the target substance is a low-molecular-weight compound having a molecular weight of 2000 or less, a protein, a nucleic acid, an antibody or a functional fragment thereof, an organelle, or a substance obtained by binding a labeling substance to any of these. <7> The intracellular delivery method according to <5> or <6>, wherein the target substance is a therapeutic substance or a test substance. <8> The intracellular delivery method according to any one of <5> to <7>, wherein the target substance is delivered into a cell in vitro. <9> An intracellular delivery kit for delivering a target substance into a cell, comprising a complex of the target substance and a membrane-permeating peptide, and the membrane permeation enhancer according to <1> or <2>.

[0010] According to the present invention, it is possible to provide a novel membrane permeation enhancer that is highly biocompatible and can further enhance the membrane permeability of a membrane-permeating peptide complex, a membrane permeation enhancement method and an intracellular delivery method that use the membrane permeation enhancer, and an intracellular delivery kit that includes the membrane permeation enhancer.

[0011] 1 is a diagram showing the fluorescence intensity per red blood cell when (-)-catechin is added to a red blood cell solution together with a membrane-permeable peptide conjugate (FITC-R8). 2 is a diagram showing the fluorescence intensity per red blood cell when (-)-epicatechin is added to a red blood cell solution together with a membrane-permeable peptide conjugate (FITC-R4). 3 is a diagram showing the fluorescence intensity per red blood cell when (-)-epicatechin is added to a red blood cell solution together with a membrane-permeable peptide conjugate (FITC-R8). 4 is a diagram showing the fluorescence intensity per red blood cell when (-)-epicatechin gallate is added to a red blood cell solution together with a membrane-permeable peptide conjugate (FITC-R4). 5 is a diagram showing the fluorescence intensity per red blood cell when (-)-epicatechin gallate is added to a red blood cell solution together with a membrane-permeable peptide conjugate (FITC-R8). 1 is a diagram showing the fluorescence intensity per red blood cell when (-)-epigallocatechin gallate is added together with a membrane-permeable peptide complex (FITC-R4) to a red blood cell solution. 2 is a diagram showing the fluorescence intensity per red blood cell when (-)-epigallocatechin gallate is added together with a membrane-permeable peptide complex (FITC-R8) to a red blood cell solution. 3 is a diagram showing the ratio of the fluorescence intensity (membrane permeation enhancement factor) when (-)-epicatechin is added together with a membrane-permeable peptide complex (FITC-R4) to the fluorescence intensity when only the membrane-permeable peptide complex (FITC-R4) is added to a GUV solution. 4 is a diagram showing the ratio of the fluorescence intensity (membrane permeation enhancement factor) when (-)-epigallocatechin gallate is added together with a membrane-permeable peptide complex (FITC-R8) to the fluorescence intensity when only the membrane-permeable peptide complex (FITC-R8) is added to a GUV solution. 1 is a diagram showing the fluorescence intensity per red blood cell when (-)-epigallocatechin gallate is added to a red blood cell solution together with a membrane-permeable peptide conjugate (FITC-Ap1). 2 is a diagram showing the fluorescence intensity per red blood cell when (-)-epigallocatechin gallate is added to a red blood cell solution together with a membrane-permeable peptide conjugate (FITC-Ap3). 3 is a diagram showing the fluorescence intensity per red blood cell when (-)-epigallocatechin gallate is added to a red blood cell solution together with a membrane-permeable peptide conjugate (FITC-Pf14_8).1 shows the fluorescence intensity per red blood cell when (-)-epigallocatechin gallate was added together with a membrane-permeable peptide conjugate (FITC-Pf14_10) to a red blood cell solution. 2 shows the fluorescence intensity per red blood cell when (-)-epigallocatechin gallate was added together with a membrane-permeable peptide conjugate (FITC-Pf14_11) to a red blood cell solution. 3 shows the ratio (membrane permeation enhancement factor) of the fluorescence intensity when (-)-epigallocatechin gallate was added together with a membrane-permeable peptide conjugate (FITC-Ap1 or FITC-Ap3) to the fluorescence intensity when only the membrane-permeable peptide conjugate (FITC-Ap1 or FITC-Ap3) was added to a GUV solution.

[0012] Specific embodiments to which the present invention is applied are described below. However, the present invention is not limited to the following embodiments. In this specification, numerical ranges indicated using "to" indicate ranges that include the numerical values ​​before and after "to" as the minimum and maximum values, respectively. In describing amino acid sequences, the left side is the N-terminus, and amino acid residues are represented using single-letter symbols well known in the art (for example, "R" for an arginine residue).

[0013] <Membrane Permeation Enhancer> The membrane permeation enhancer according to this embodiment is a membrane permeation enhancer that enhances the permeation of a complex of a target substance and a membrane-permeating peptide (membrane-permeating peptide complex) through a lipid bilayer membrane (hereinafter also simply referred to as "membrane"), and contains a flavonoid. The membrane permeation enhancer according to this embodiment can enhance the permeation of a membrane-permeating peptide through a lipid bilayer membrane by temporarily changing the structure of the lipid bilayer membrane without destroying the structure of the lipid bilayer membrane. In particular, flavonoids have excellent biocompatibility and can further enhance the membrane permeability of the membrane-permeating peptide complex compared to conventional membrane permeation enhancers such as surfactants having a steroid skeleton as described in Patent Document 1.

[0014] Hereinafter, each element related to the membrane permeation enhancer according to this embodiment will be described.

[0015] [Lipid bilayer membrane] The lipid bilayer membrane may be a naturally occurring membrane or an artificial membrane. Examples of naturally occurring membranes include biological membranes such as cell membranes (for example, membranes constituting blood cells such as red blood cells), nuclear membranes, and membranes surrounding organelles. Examples of artificial membranes include liposomes.

[0016] The lipid bilayer membrane may contain other membrane components such as proteins and sugar chains in addition to lipids.

[0017] [Membrane-penetrating peptide complex] The membrane-penetrating peptide complex is a complex of a target substance and a membrane-penetrating peptide.

[0018] (Membrane-penetrating peptide) A specific example of a membrane-penetrating peptide is polyarginine in which 3 to 13 consecutive arginines are linked (see, for example, Japanese Patent No. 6320469). Examples of such polyarginines include R8 (SEQ ID NO: 1: RRRRRRRRR) and R4 (SEQ ID NO: 2: RRRR).

[0019] Other specific examples of membrane-permeable peptides include Tat Peptide (48-60) (SEQ ID NO: 3: GRKKRRQRRRPPQ); Penetratin (SEQ ID NO: 4: RQIKIWFQNRRMKWKK); (Lys) ８(SEQ ID NO:5: KKKKKKKK); HIV-1 Rev(34-50) (SEQ ID NO:6: TRQRRNRRRRWRERQR); FHV Coat(35-49) (SEQ ID NO:7: RRRRNRTRRNRRRVR); BMV Gag(7-25) (SEQ ID NO:8: KMTRAQRRAAARRNRWTAR); HTLV-II Rex(4-16) (SEQ ID NO:9: TRRQRTRRARRNR); CCMV Gag(7-25) (SEQ ID NO:10: KLTRAQRRAAARKNKRNTR); P22 N(14-30) (SEQ ID NO: 11: NAKTRRHERRRKLAIER); λN(1-22) (SEQ ID NO: 12: MDAQTRRRERRAEKQAQWKAAN); φ21N(12-29) (SEQ ID NO: 13: TAKTRYKARRAELIAERR); yeast PRP6(129-144) (SEQ ID NO: 14: TRRNKRNRIQEQLNRK); human U2AF(142-153) (SEQ ID NO: 15: SQMTRQARRLYV); CTP512 (SEQ ID NO: 16: YGRRARRRRRRRR); Buforin II (SEQ ID NO: 17: TRSSRAGLQFPVGRVHRLLRK); Transportan (SEQ ID NO: 18: GWTLNSAGYLLGKINLKALAALAKKIL); MAP (SEQ ID NO: 19: KLALKLALKALKAALKLA); Pep-1 (SEQ ID NO: 20: KETWWETWWTEWSQPKKKRKV; MPG (SEQ ID NO: 21: GALFLGFFLGAAGSTTMGAWSQPKKKRKV); PenetraMax (SEQ ID NO: 22: KWFKIQMQIRRWKNKR); LL-37 (SEQ ID NO: 23: LLGDFERKSKEKIGKEFKRIVQRIKDFL RNLVPRTESC); Pep-7 (SEQ ID NO: 24: SDLWEMMMVSLACQY); K-FGF (SEQ ID NO: 25: AAVALLPAVLLALLAP); Ku70 (SEQ ID NO: 26: VPMLK); Ku70 (SEQ ID NO: 27: PMLKE); prion (SEQ ID NO: 28: MANLGYWLLALFVTMWTDVGLCKKRPKP); pVEC (SEQ ID NO: 29: LLIILRRRIRKQAHAHSK); SynB1 (SEQ ID NO: 30: RGGRLSYSRRRFSTSTGR); HN-1 (SEQ ID NO: 31: TSPLNIHNGQKL); and amidated products thereof.

[0020] Furthermore, examples of membrane-permeable peptides include peptides newly discovered from malaria parasite proteins. Specific examples include Ap1 (SEQ ID NO: 32: KTNEKLCDKKEKKRLPSK), Ap3 (SEQ ID NO: 33: KWKKRLAKEIRDKPIKK), Pf14_8 (SEQ ID NO: 34: SKKKKKKENEKEKERKRKEKE), Pf14_10 (SEQ ID NO: 35: KERKRKEKEQEKERKNKENKKKE), and Pf14_11 (SEQ ID NO: 36: TKNKIKTWK).

[0021] (Target Substance) Examples of target substances that can be permeated through a lipid bilayer membrane include low molecular weight compounds with a molecular weight of 2000 g / mol or less (preferably a molecular weight of 1500 g / mol or less), proteins, nucleic acids, antibodies or functional fragments thereof, organelles, and substances obtained by binding these with labeling substances (fluorophores, affinity labeling agents, etc.). Examples of proteins include enzymes, receptors, fluorescent proteins, etc. Examples of nucleic acids include DNA, RNA, hybrid nucleic acids, aptamers, etc. The isotype of the antibody is not particularly limited and may be any of IgG, IgM, IgA, IgD, IgE, etc. Furthermore, the antibody may be either a polyclonal antibody or a monoclonal antibody. Examples of functional fragments of antibodies include Fab, Fab', F(ab') ２ , Fv, scFv, etc. Examples of the organelle include mitochondria, etc.

[0022] The target substance may be covalently bound to the membrane-permeable peptide directly or via a linker. The binding position of the target substance may be either the C-terminal side or the N-terminal side of the membrane-permeable peptide. When the target substance is a protein, the complex of the target substance and the membrane-permeable peptide may be a fusion protein.

[0023] [Flavonoids] Flavonoids are classified according to their basic structure into flavones (apigenin, chrysin, luteolin, etc.), flavans (flavan, etc.), flavanones (naringenin, hesperetin, etc.), flavonols (galangin, quercetin, kaempferol, myricetin, etc.), flavanols (catechin, theaflavin, etc.), flavanonols (dihydroquercetin, etc.), isoflavones (daidzein, genistein, etc.), chalcones (carthamin, phloretin, etc.), anthocyanidins (cyanidin, delphinidin, pelargonidin, etc.), etc. Flavonoids may be derived from natural products or may be synthetic products that do not exist in nature. In addition, flavonoids may be used alone, or two or more may be used in combination.

[0024] Among flavonoids, flavanols are preferred, and catechins are more preferred, from the viewpoint of the effect of promoting the membrane permeability of the membrane-permeating peptide complex.

[0025] Here, catechins refer to polyoxy derivatives of 3-oxyflavan. Catechins include not only free catechins such as catechin, epicatechin, gallocatechin, and epigallocatechin, but also esterified catechins and methylated catechins. Catechins may be in the (+) form, the (-) form, or the racemic form.

[0026] Examples of ester-type catechins include compounds obtained by dehydration condensation of the hydroxy group of free catechins with the carboxy group of organic acids such as trihydroxybenzoic acid, dihydroxybenzoic acid, monohydroxybenzoic acid, etc. Specific examples of ester-type catechins include catechin gallate, epicatechin gallate, gallocatechin gallate, and epigallocatechin gallate.

[0027] Methylated catechins refer to compounds in which some of the hydroxy groups of free catechins or ester catechins have been methoxylated. Specific examples of methylated catechins include catechin-3-O-(3-O-methyl)gallate, catechin-3-O-(4-O-methyl)gallate, epicatechin-3-O-(3-O-methyl)gallate, epicatechin-3-O-(4-O-methyl)gallate, epicatechin-3-O-(3,4-O-dimethyl)gallate, and epicatechin-3-O-(3,5-O-dimethyl)gallate. gallocatechin-3-O-(3-O-methyl)gallate, gallocatechin-3-O-(4-O-methyl)gallate, epigallocatechin-3-O-(3-O-methyl)gallate, epigallocatechin-3-O-(4-O-methyl)gallate, epigallocatechin-3-O-(3,4-O-dimethyl)gallate, and epigallocatechin-3-O-(3,5-O-dimethyl)gallate.

[0028] Among these catechins, at least one selected from the group consisting of catechin, epicatechin, gallocatechin, epigallocatechin, catechin gallate, epicatechin gallate, gallocatechin gallate, and epigallocatechin gallate is preferred.

[0029] [Additives] The membrane permeation enhancer according to this embodiment may contain various additives in addition to flavonoids.

[0030] [Target of application of membrane permeation enhancer] The target of application of the membrane permeation enhancer according to this embodiment is not particularly limited, and examples thereof include mammals (mouse, rat, hamster, rabbit, cat, dog, cow, sheep, monkey, human, etc.) and animal cells.

[0031] <Membrane Permeation Promotion Method> The membrane permeation promotion method according to this embodiment is a method for promoting the permeation of a membrane-permeating peptide complex into a lipid bilayer membrane, and includes contacting the membrane-permeating peptide complex and the membrane permeation promoter according to this embodiment with a lipid bilayer membrane.

[0032] An example of the method for promoting membrane permeation according to this embodiment is a method in which the membrane-permeating peptide complex, the membrane permeation enhancer according to this embodiment, and, if necessary, an additive are mixed and administered to a living body as a formulation, and the formulation is brought into contact with target cells in the living body, thereby promoting the permeation of the membrane-permeating peptide complex into target cells.

[0033] Another example of the membrane permeation enhancing method according to this embodiment is a method of enhancing permeation of a membrane-permeating peptide complex into a target cell by contacting collected target cells (e.g., blood cells such as red blood cells) with the membrane-permeating peptide complex and the membrane permeation enhancer according to this embodiment in a solution. The concentration of the flavonoid in the solution is, for example, preferably 1 to 1,000 μM, more preferably 10 to 350 μM, and even more preferably 15 to 200 μM.

[0034] <Intracellular delivery method> The intracellular delivery method according to this embodiment is an intracellular delivery method for delivering a target substance into a cell, and includes contacting a complex of the target substance and a membrane-permeable peptide (membrane-permeable peptide complex) with a cell in the presence of the membrane permeation enhancer according to this embodiment.

[0035] Examples of target substances to be delivered into cells include low molecular weight compounds with a molecular weight of 2000 g / mol or less (preferably 1500 g / mol or less), proteins, nucleic acids, antibodies or functional fragments thereof, organelles, and substances obtained by binding these with labeling substances (fluorophores, affinity labeling agents, etc.). Examples of proteins include enzymes, receptors, fluorescent proteins, etc. Examples of nucleic acids include DNA, RNA, hybrid nucleic acids, aptamers, etc. The isotype of the antibody is not particularly limited and may be any of IgG, IgM, IgA, IgD, IgE, etc. Furthermore, the antibody may be either a polyclonal antibody or a monoclonal antibody. Examples of functional fragments of antibodies include Fab, Fab', F(ab') ２ , Fv, scFv, etc. Examples of the organelle include mitochondria, etc.

[0036] The target substance may be covalently bound to the membrane-permeable peptide directly or via a linker. The binding position of the target substance may be either the C-terminal side or the N-terminal side of the membrane-permeable peptide. When the target substance is a protein, the complex of the target substance and the membrane-permeable peptide may be a fusion protein.

[0037] The target substance may be for therapeutic purposes or for testing purposes, in other words, the target substance may be a therapeutic substance or a testing substance.

[0038] Examples of therapeutic substances include drugs such as small molecule drugs, nucleic acid drugs, and polymer drugs. By conjugating such drugs with membrane-permeable peptides and contacting them with cells together with the membrane permeation enhancer according to this embodiment to allow them to permeate into the cells, the therapeutic effect can be further improved. Another example of a therapeutic substance is a substance introduced into transplant cells for cell medicine. For example, by conjugating mitochondria with membrane-permeable peptides and contacting them with transplant cells together with the membrane permeation enhancer according to this embodiment to allow them to permeate into the cells, the therapeutic effect of cell medicine can be further improved.

[0039] Examples of test substances include antibodies or functional fragments thereof, lectins, nucleic acids, and substances obtained by binding a labeling substance to any of these. Specific examples of test substances include antibodies or functional fragments thereof against malaria-derived proteins (malaria severity markers) present in the red blood cells of malaria-infected patients, cancer markers, etc. Such antibodies or functional fragments thereof are conjugated with membrane-permeable peptides, and then contacted with cells together with the membrane permeation enhancer according to this embodiment to allow them to permeate into the cells, thereby further improving the accuracy of tests for intracellular molecules.

[0040] The delivery of the target substance into cells may be carried out in vivo or in vitro.

[0041] <Intracellular delivery kit> The intracellular delivery kit according to this embodiment is an intracellular delivery kit for delivering a target substance into a cell, and includes a complex of the target substance and a membrane-permeable peptide (membrane-permeable peptide complex) and a membrane permeation enhancer according to this embodiment. The intracellular delivery kit according to this embodiment can be suitably used in the intracellular delivery method described above.

[0042] The present invention will be explained in more detail below with reference to examples, but the present invention is not limited to these examples.

[0043] Preparation Example 1: Stored sheep red blood cells (15 mL) were added to a plastic tube and centrifuged (3000 rpm, 5 minutes), after which the supernatant was removed. Next, 0.9% saline (12 mL) was added, and the tube was centrifuged again (3000 rpm, 5 minutes), after which the supernatant was removed. This procedure was repeated three times, after which 0.9% saline (12 mL) was added and gently stirred to obtain a 20% red blood cell solution. Next, 0.9% saline (49 mL) was added to the obtained 20% red blood cell solution (1 mL) and gently stirred to obtain a 0.4% red blood cell solution.

[0044] In Test Example 1, (-)-catechin was used as the flavonoid and FITC-labeled arginine octamer (FITC-R8) was used as a model molecule of the membrane-permeable peptide complex to conduct a membrane permeation experiment on red blood cells. FITC-labeled arginine octamer (FITC-R8) is a compound represented by the following formula (1) in which x is 8:

[0045]

[0046] A 0.4% erythrocyte solution (100 μL), an 80 mM PBS solution (140 μL), a 100 μM membrane-permeable peptide complex (FITC-R8) (160 μL), and a 54 μM, 75 μM, or 689 μM (-)-catechin solution (100 μL) were added to a vial and incubated at 25°C for 60 min. The final concentration of (-)-catechin after mixing with the erythrocyte solution was 10.8 μM, 15 μM, or 138 μM. After incubation, the mixture was centrifuged at 4°C, 10,500 rpm, and 5 min to remove the supernatant. Then, 80 mM PBS solution (500 μL) and 0.05% trypsin solution (160 μL) were added and incubated at 37°C for 10 min. The mixture was then centrifuged twice (4°C, 10,500 rpm, 5 min) to remove the supernatant, and 80 mM PBS solution (500 μL) was added. The resulting sample was then analyzed by flow cytometry to determine the fluorescence intensity per red blood cell. As controls, a sample containing no (-)-catechin solution but with 240 μL of PBS solution added, and a sample containing no (-)-catechin solution or membrane-permeable peptide complex but with 400 μL of PBS solution added were prepared. These were similarly analyzed by flow cytometry to determine the fluorescence intensity per red blood cell.

[0047] The fluorescence intensity per red blood cell is shown in Figure 1. As shown in Figure 1, when (-)-catechin was added together with the membrane-permeable peptide conjugate (FITC-R8), the uptake of the membrane-permeable peptide conjugate into red blood cells was promoted in a (-)-catechin concentration-dependent manner. Specifically, when (-)-catechin was added together with FITC-R8 at a final concentration of 138 μM, the fluorescence intensity per red blood cell increased 1.31-fold compared to the control without (-)-catechin.

[0048] In Test Example 2, (-)-epicatechin was used as the flavonoid, and FITC-labeled arginine tetramer (FITC-R4) and FITC-labeled arginine octamer (FITC-R8) were used as model molecules of the membrane-permeable peptide complex to conduct a membrane permeation experiment for red blood cells. FITC-labeled arginine tetramer (FITC-R4) is a compound represented by the above formula (1) in which x is 4.

[0049] A 0.4% erythrocyte solution (100 μL), an 80 mM PBS solution (140 μL), a 100 μM membrane-permeable peptide conjugate (FITC-R4 or FITC-R8) (160 μL), and a 54 μM, 75 μM, or 1.7 mM (-)-epicatechin solution (100 μL) were added to a vial and incubated at 25°C for 60 min. The final (-)-epicatechin concentrations after mixing with the erythrocyte solution were 10.8 μM, 15 μM, or 340 μM. After incubation, the mixture was centrifuged at 4°C, 10,500 rpm, and 5 min to remove the supernatant. Then, 80 mM PBS solution (500 μL) and 0.05% trypsin solution (160 μL) were added and incubated at 37°C for 10 min. The mixture was then centrifuged twice (4°C, 10,500 rpm, 5 min) to remove the supernatant, and 80 mM PBS solution (500 μL) was added. The resulting sample was then analyzed by flow cytometry to determine the fluorescence intensity per red blood cell. As controls, a sample containing no (-)-epicatechin solution but with 240 μL of PBS solution added was prepared, and a sample containing no (-)-epicatechin solution or membrane-permeable peptide complex but with 400 μL of PBS solution added was prepared. These controls were similarly analyzed by flow cytometry to determine the fluorescence intensity per red blood cell.

[0050] The fluorescence intensity per red blood cell when FITC-R4 was added as the membrane-permeable peptide conjugate is shown in Figure 2A, and the fluorescence intensity per red blood cell when FITC-R8 was added as the membrane-permeable peptide conjugate is shown in Figure 2B. As shown in Figures 2A and 2B, when (-)-epicatechin was added together with the membrane-permeable peptide conjugate (FITC-R4 or FITC-R8), the uptake of the membrane-permeable peptide conjugate into red blood cells was promoted in a (-)-epicatechin concentration-dependent manner. Specifically, when (-)-epicatechin was added together with FITC-R4 at a final concentration of 340 μM, the fluorescence intensity per red blood cell increased 33.81-fold compared to the control without (-)-epicatechin. Furthermore, when (-)-epicatechin was added at a final concentration of 340 μM together with FITC-R8, the fluorescence intensity per red blood cell increased 25.70 times compared to the control without (-)-epicatechin.

[0051] <Test Example 3> In Test Example 3, flow cytometry analysis was performed in the same manner as in Test Example 2, except that (-)-epicatechin gallate was added instead of (-)-epicatechin to a final concentration of 2.2 μM, 10.8 μM, or 15 μM, and the fluorescence intensity per red blood cell was determined.

[0052] The fluorescence intensity per red blood cell when FITC-R4 was added as the membrane-penetrating peptide conjugate is shown in Figure 3A, and the fluorescence intensity per red blood cell when FITC-R8 was added as the membrane-penetrating peptide conjugate is shown in Figure 3B. As shown in Figures 3A and 3B, when (-)-epicatechin gallate was added together with the membrane-penetrating peptide conjugate (FITC-R4 or FITC-R8), the uptake of the membrane-penetrating peptide conjugate into red blood cells was promoted in a concentration-dependent manner of (-)-epicatechin gallate. Specifically, when (-)-epicatechin gallate was added together with FITC-R4 at a final concentration of 15 μM, the fluorescence intensity per red blood cell increased 2.80-fold compared to the control without (-)-epicatechin gallate. Furthermore, when (-)-epicatechin gallate was added at a final concentration of 15 μM together with FITC-R8, the fluorescence intensity per red blood cell increased 14.52 times compared to the control without (-)-epicatechin gallate.

[0053] <Test Example 4> In Test Example 4, flow cytometry analysis was performed in the same manner as in Test Example 2, except that (-)-epigallocatechin gallate was added to a final concentration of 10.8 μM, 15 μM, or 22 μM instead of (-)-epicatechin, and the fluorescence intensity per red blood cell was determined.

[0054] The fluorescence intensity per red blood cell when FITC-R4 was added as the membrane-penetrating peptide conjugate is shown in Figure 4A, and the fluorescence intensity per red blood cell when FITC-R8 was added as the membrane-penetrating peptide conjugate is shown in Figure 4B. As shown in Figures 4A and 4B, when (-)-epigallocatechin gallate was added together with the membrane-penetrating peptide conjugate (FITC-R4 or FITC-R8), the uptake of the membrane-penetrating peptide conjugate into red blood cells was promoted in a concentration-dependent manner of (-)-epigallocatechin gallate. Specifically, when (-)-epigallocatechin gallate was added together with FITC-R4 at a final concentration of 15 μM, the fluorescence intensity per red blood cell increased 5.31-fold compared to the control without (-)-epigallocatechin gallate. Furthermore, when (-)-epigallocatechin gallate was added at a final concentration of 22 μM together with FITC-R8, the fluorescence intensity per red blood cell increased 45.96 times compared to the control without (-)-epigallocatechin gallate.

[0055] Preparation Example 2: 1-Stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC) and methanol (1200 μL) were added to a vial and stirred for 1 minute to dissolve the SOPC. After dissolution, the vial was filled with nitrogen gas to form a thin film, and then dried under reduced pressure for 24 hours. Next, a 3 M potassium chloride solution was added, and the solution was stirred for 1 minute to form multilamellar vesicles (MLVs), which were then ultrasonicated for 30 minutes to form unilamellar vesicles (ULVs). After six freeze-thaw cycles, the solution was dialyzed for 3 days and aged for 5 days to obtain a 20 mM giant unilamellar vesicle (GUV) suspension.

[0056] Test Example 5 In Test Example 5, (-)-epicatechin was used as the flavonoid, and FITC-labeled arginine tetramer (FITC-R4) was used as a model molecule of the membrane-permeable peptide complex, and a membrane permeation experiment for GUV was carried out.

[0057] A 20 mM GUV suspension (200 μL), a 20% PBS solution (260 μL), a 200 μM membrane-permeable peptide complex (FITC-R4) (40 μL), and a 54 μM, 75 μM, or 110 μM (-)-epicatechin solution (100 μL) were added to a vial and incubated at 25°C for 60 min. The final concentration of (-)-epicatechin after mixing with the GUV suspension was 9 μM, 12.5 μM, or 18.3 μM. After incubation, the solution was centrifuged at 4°C, 10,500 rpm, and 5 min to remove the supernatant. A 0.05% trypsin solution (500 μL) was added and the mixture was incubated at 37°C for 10 min. The mixture was then centrifuged twice (4°C, 10,500 rpm, 5 min) to remove the supernatant, and then 20% PBS solution (500 μL) was added. The resulting sample was analyzed by flow cytometry to determine its fluorescence intensity. As a control, a sample was prepared in which no (-)-epicatechin solution was added, but 360 μL of PBS solution was added. This sample was similarly analyzed by flow cytometry to determine its fluorescence intensity.

[0058] The ratio of the fluorescence intensity when the (-)-epicatechin solution was added to the fluorescence intensity when the (-)-epicatechin solution was not added (membrane permeation enhancement factor) is shown in Figure 5. As shown in Figure 5, when (-)-epicatechin was added together with the membrane-permeating peptide conjugate (FITC-R4), the uptake of the membrane-permeating peptide conjugate into GUVs was promoted in a (-)-epicatechin concentration-dependent manner.

[0059] In addition, when a similar experiment was performed using a 600 mM sodium cholate solution or a 600 mM sodium taurocholate solution instead of the (-)-epicatechin solution, the membrane permeation promotion factor when the sodium cholate solution was used was 1.32-fold, and the membrane permeation promotion factor when the sodium taurocholate solution was used was 1.12-fold.

[0060] <Test Example 6> In Test Example 6, a membrane permeation experiment for GUV was carried out in the same manner as in Test Example 5, except that a (-)-epigallocatechin gallate solution was used instead of the (-)-epicatechin solution, and an FITC-labeled arginine octamer (FITC-R8) was used instead of the FITC-labeled arginine tetramer (FITC-R4).

[0061] The ratio of the fluorescence intensity when a (-)-epigallocatechin gallate solution was added to the fluorescence intensity when no (-)-epigallocatechin gallate solution was added (membrane permeation enhancement factor) is shown in Figure 6. As shown in Figure 6, when (-)-epigallocatechin gallate was added together with the membrane-permeating peptide complex (FITC-R8), the uptake of the membrane-permeating peptide complex into GUVs was promoted in a (-)-epigallocatechin gallate concentration-dependent manner.

[0062] In addition, when a similar experiment was performed using a 600 mM sodium cholate solution or a 600 mM sodium taurocholate solution instead of the (-)-epigallocatechin gallate solution, the membrane permeation promotion factor when the sodium cholate solution was used was 1.20-fold, and the membrane permeation promotion factor when the sodium taurocholate solution was used was 1.08-fold.

[0063] Test Example 7 In Test Example 7, (-)-epigallocatechin gallate was used as the flavonoid, and FITC-labeled Ap1 (FITC-Ap1) was used as a model molecule of the membrane-permeable peptide complex, and a membrane permeation experiment for red blood cells was carried out.

[0064] A 0.4% erythrocyte solution (100 μL), an 80 mM PBS solution (140 μL), a 100 μM membrane-permeable peptide conjugate (FITC-Ap1) (160 μL), and a 54 μM or 0.11 mM (-)-epigallocatechin gallate solution (100 μL) were added to a vial and incubated at 25°C for 60 min. The final concentration of (-)-epigallocatechin gallate after mixing with the erythrocyte solution was 10.8 μM or 22 μM. After incubation, the mixture was centrifuged at 4°C, 10,500 rpm, for 5 min to remove the supernatant. Then, 80 mM PBS solution (500 μL) and 0.05% trypsin solution (160 μL) were added and incubated at 37°C for 10 min. The mixture was then centrifuged twice (4°C, 10,500 rpm, 5 min) to remove the supernatant, and 80 mM PBS solution (500 μL) was added. The resulting sample was then analyzed by flow cytometry to determine the fluorescence intensity per red blood cell. Control samples included one containing no (-)-epigallocatechin gallate solution but 240 μL of PBS solution; one containing no (-)-epigallocatechin gallate solution or membrane-permeable peptide complex but 400 μL of PBS solution; and one containing sodium taurocholate to a final concentration of 370 μM instead of (-)-epigallocatechin gallate. These samples were similarly analyzed by flow cytometry to determine the fluorescence intensity per red blood cell.

[0065] The fluorescence intensity per red blood cell is shown in Figure 7. As shown in Figure 7, when (-)-epigallocatechin gallate was added together with the membrane-permeable peptide conjugate (FITC-Ap1), the uptake of the membrane-permeable peptide conjugate into red blood cells was promoted in a concentration-dependent manner of (-)-epigallocatechin gallate. Specifically, when (-)-epigallocatechin gallate was added together with FITC-Ap1 at a final concentration of 22 µM, the fluorescence intensity per red blood cell increased 4.03-fold compared to the control without (-)-epigallocatechin gallate. In contrast, when sodium taurocholate was added together with FITC-Ap1 at a final concentration of 370 µM, the increase in fluorescence intensity per red blood cell was only 1.08-fold.

[0066] <Test Example 8> In Test Example 8, flow cytometry analysis was performed in the same manner as in Test Example 7, except that FITC-labeled Ap3 (FITC-Ap3) was used instead of FITC-labeled Ap1 (FITC-Ap1), and the fluorescence intensity per red blood cell was determined.

[0067] The fluorescence intensity per red blood cell is shown in Figure 8. As shown in Figure 8, when (-)-epigallocatechin gallate was added together with the membrane-permeable peptide conjugate (FITC-Ap3), the uptake of the membrane-permeable peptide conjugate into red blood cells was promoted in a concentration-dependent manner of (-)-epigallocatechin gallate. Specifically, when (-)-epigallocatechin gallate was added together with FITC-Ap3 at a final concentration of 22 µM, the fluorescence intensity per red blood cell increased 8.14-fold compared to the control without (-)-epigallocatechin gallate. In contrast, when sodium taurocholate was added together with FITC-Ap3 at a final concentration of 370 µM, the increase in fluorescence intensity per red blood cell was only 1.18-fold.

[0068] <Test Example 9> In Test Example 9, flow cytometry analysis was performed in the same manner as in Test Example 7, except that FITC-labeled Pf14_8 (FITC-Pf14_8) was used instead of FITC-labeled Ap1 (FITC-Ap1), and the fluorescence intensity per red blood cell was determined.

[0069] The fluorescence intensity per red blood cell is shown in Figure 9. As shown in Figure 9, when (-)-epigallocatechin gallate was added together with the membrane-permeable peptide conjugate (FITC-Pf14_8), the uptake of the membrane-permeable peptide conjugate into red blood cells was promoted in a concentration-dependent manner depending on the (-)-epigallocatechin gallate concentration. Specifically, when (-)-epigallocatechin gallate was added together with FITC-Pf14_8 at a final concentration of 22 µM, the fluorescence intensity per red blood cell increased 4.89-fold compared to the control without (-)-epigallocatechin gallate. In contrast, when sodium taurocholate was added together with FITC-Pf14_8 at a final concentration of 370 µM, the increase in fluorescence intensity per red blood cell was 0.83-fold.

[0070] <Test Example 10> In Test Example 10, flow cytometry analysis was performed in the same manner as in Test Example 7, except that FITC-labeled Pf14_10 (FITC-Pf14_10) was used instead of FITC-labeled Ap1 (FITC-Ap1), and the fluorescence intensity per red blood cell was determined.

[0071] The fluorescence intensity per red blood cell is shown in Figure 10. As shown in Figure 10, when (-)-epigallocatechin gallate was added together with the membrane-permeable peptide conjugate (FITC-Pf14_10), the uptake of the membrane-permeable peptide conjugate into red blood cells was promoted in a concentration-dependent manner of (-)-epigallocatechin gallate. Specifically, when (-)-epigallocatechin gallate was added together with FITC-Pf14_10 at a final concentration of 22 µM, the fluorescence intensity per red blood cell increased 1.44-fold compared to the control without (-)-epigallocatechin gallate. In contrast, when sodium taurocholate was added together with FITC-Pf14_10 at a final concentration of 370 µM, the increase in fluorescence intensity per red blood cell was only 1.15-fold.

[0072] <Test Example 11> In Test Example 11, flow cytometry analysis was performed in the same manner as in Test Example 7, except that FITC-labeled Pf14_11 (FITC-Pf14_11) was used instead of FITC-labeled Ap1 (FITC-Ap1), and the fluorescence intensity per red blood cell was determined.

[0073] The fluorescence intensity per red blood cell is shown in Figure 11. As shown in Figure 11, when (-)-epigallocatechin gallate was added together with the membrane-permeable peptide conjugate (FITC-Pf14_11), the uptake of the membrane-permeable peptide conjugate into red blood cells was promoted in a concentration-dependent manner of (-)-epigallocatechin gallate. Specifically, when (-)-epigallocatechin gallate was added together with FITC-Pf14_11 at a final concentration of 22 µM, the fluorescence intensity per red blood cell increased 1.56-fold compared to the control without (-)-epigallocatechin gallate. In contrast, when sodium taurocholate was added together with FITC-Pf14_11 at a final concentration of 370 µM, the increase in fluorescence intensity per red blood cell was only 1.18-fold.

[0074] Test Example 12 In Test Example 12, a membrane permeation experiment for GUV was carried out using (-)-epigallocatechin gallate as a flavonoid and FITC-labeled Ap1 (FITC-Ap1) or FITC-labeled Ap3 (FITC-Ap3) as a model molecule of a membrane-permeable peptide complex.

[0075] A 20 mM GUV suspension (200 μL), 20% PBS solution (260 μL), 100 μM membrane-permeable peptide conjugate (FITC-Ap1 or FITC-Ap3) (40 μL), and 110 μM (-)-epigallocatechin gallate solution (100 μL) were added to a vial and incubated at 25°C for 60 min. The final concentration of (-)-epigallocatechin gallate after mixing with the GUV suspension was 18.3 μM. After incubation, the mixture was centrifuged at 4°C, 10,500 rpm for 10 min, and the supernatant was removed. A 0.05% trypsin solution (500 μL) was added and the mixture was incubated at 37°C for 10 min. The mixture was then centrifuged twice (4°C, 10,500 rpm, 10 min) to remove the supernatant, after which 20% PBS solution (500 μL) was added. The resulting sample was analyzed by flow cytometry to determine its fluorescence intensity. As a control, a sample was prepared in which no (-)-epigallocatechin gallate solution was added, but 360 μL of PBS solution was added. This sample was similarly analyzed by flow cytometry to determine its fluorescence intensity.

[0076] The ratio of the fluorescence intensity when the (-)-epigallocatechin gallate solution was added to the fluorescence intensity when the (-)-epigallocatechin gallate solution was not added (membrane permeation enhancement factor) is shown in Figure 12. As shown in Figure 12, when (-)-epigallocatechin gallate was added together with the membrane-permeating peptide conjugate (FITC-Ap1 or FITC-Ap3), the uptake of the membrane-permeating peptide conjugate into the GUV was promoted.

Claims

1. A membrane permeation enhancer that promotes the permeation of a complex of a target substance and a membrane-permeating peptide through a lipid bilayer membrane, the membrane permeation enhancer containing flavonoids.

2. The membrane permeation enhancer according to claim 1, wherein the flavonoids are catechins.

3. The membrane permeation enhancer according to claim 1, wherein the lipid bilayer membrane is a cell membrane.

4. A method for promoting membrane permeation by promoting the permeation of a complex of a target substance and a membrane-permeating peptide through a lipid bilayer membrane, the method comprising contacting the complex and a membrane permeation enhancer described in any one of claims 1 to 3 with a lipid bilayer membrane.

5. An intracellular delivery method for delivering a target substance into a cell, comprising contacting a complex of the target substance and a membrane-permeable peptide with a cell in the presence of a membrane permeation enhancer described in claim 1 or 2.

6. The intracellular delivery method according to claim 5, wherein the target substance is a low molecular weight compound having a molecular weight of 2000 or less, a protein, a nucleic acid, an antibody or a functional fragment thereof, an organelle, or a substance to which a labeling substance is bound.

7. The intracellular delivery method according to claim 5, wherein the target substance is a therapeutic substance or a test substance.

8. The intracellular delivery method according to claim 5, wherein the target substance is delivered into cells in vitro.

9. An intracellular delivery kit for delivering a target substance into a cell, comprising a complex of the target substance and a membrane-permeable peptide, and the membrane permeation enhancer described in claim 1 or 2.

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