Artificial viral capsid

Abstract

The present invention provides an artificial viral capsid modified with a compound comprising a fluorine atom. The present invention relates to an artificial virus capsid formed by self-assembly of a plurality of subunits, the subunits containing a beta-cyclopeptide of tomato boslet dwarf virus, a group derived from a fluorine-containing compound, and a divalent linking group linking the beta-cyclopeptide and the group derived from the fluorine-containing compound, and the divalent linking group is linked to the C terminal of the beta-cyclopeptide.

Description

TECHNICAL FIELD

[0001] The present application relates to an artificial virus capsid modified by a compound containing a fluorine atom.

[0002] This application is based on Japanese Patent Application No. 2023-076053 filed on May 2, 2023, the content of which is incorporated herein by reference in its entirety. BACKGROUND

[0003] A spherical artificial virus capsid can be created by self-assembly of a β-annulus peptide from a protein of a positive dodecahedron inner skeleton of a capsid that forms a tomato bushy stunt virus in vitro (Non-Patent Documents 1 to 4). The β-annulus peptide is a peptide of 24 amino acids in length as the smallest unit corresponding to a part of a capsid protein composed of 388 amino acids. The β-annulus peptide spontaneously self-assembles in water to form a spherical and hollow nanocapsule, i.e., an artificial virus capsid, of about 30 to 50 nm in diameter. In this nanocapsule, the β-annulus peptide has the N-terminus toward the inside of the hollow capsule and the C-terminus toward the outside of the hollow capsule. It has been confirmed that various substances can be encapsulated in this artificial virus capsid (in the form of a free molecule or in the form of binding or interacting with the N-terminus of the β-annulus peptide). Such encapsulated substances are also referred to as guests, and depending on the kind of the guest, the diameter of the artificial virus capsid can be slightly expanded. In addition, it has also been confirmed that by linking molecules such as a gold nanoparticle, a single-stranded DNA of 20 nucleotides in length, a coiled coil-forming peptide, human serum albumin, ribonuclease S, and the like near the C-terminus of the β-annulus peptide, an artificial virus capsid modified on the outer surface with these molecules can be formed. In addition, it has also been reported that a modified artificial virus capsid obtained by self-assembly after linking an aptamer of a nucleic acid having a length of 40 or more nucleotides to the C-terminus side of the β-annulus peptide can effectively perform specific delivery (e.g., introduction into the inside of a specific target cell) via the aptamer portion (Patent Document 1).

[0004] On the other hand, it is known that a compound having a polyfluoro structure is stable in a living body and has low toxicity, and is excellent in uptake into a cell and escape from an endosome. It has also been studied to introduce a polyfluoro structure into an oligonucleotide or a peptide nucleic acid as a moiety having a cell membrane permeability (Patent Documents 2 and 3) using this property.

[0005] In addition, introduction of a polyfluoro structure into a peptide has also been performed. For example, it has been reported that a fluorine-containing amino acid obtained by introducing a polyfluoroalkyl group into a side chain is excellent in cell membrane permeability as a fluorine-containing peptide having the fluorine-containing amino acid as a constituent residue (Patent Document 4).

[0006] PRIOR ART DOCUMENTS

[0007] PATENT DOCUMENTS

[0008] Patent Document 1: Japanese Patent Application Publication No. 2022-173834

[0009] Patent Literature 2: International Publication No. 2012 / 130941

[0010] Patent Literature 3: International Publication No. 2021 / 060506

[0011] Patent Literature 4: International Publication No. 2023 / 048236

[0012] Non-Patent Literature

[0013] Non-Patent Literature 1: Chem. Commun., 2018, vol. 54, p. 8944.

[0014] Non-Patent Literature 2: Bioconjugate Chem. 2019, vol. 30, p. 1636-1641.

[0015] Non-Patent Literature 3: J. Org. Chem. 2020, vol. 85, p. 1668-1673.

[0016] Non-Patent Literature 4: J Pept Sci. 2017, vol. 23 (7-8), p. 636-643.

[0017] Non-Patent Literature 5: Kostrzewa-Nowak et al., British Journal of Cancer, 2005, vol. 93, p. 89-97. SUMMARY

[0018] An object of the present application is to provide an artificial virus capsid having excellent cell membrane permeability.

[0019] The present inventors have found that the cell membrane permeability of an artificial virus capsid formed by self-assembly of a β -loop peptide of Tomato bushy stunt virus (TBSV) can be improved by modifying the β -loop peptide with a peptide containing an amino acid residue having a fluorine atom introduced in a side chain, thereby completing the present application.

[0020] That is, the present application is as follows.

[0021] [1] An artificial virus capsid formed by self-assembly of a plurality of subunits,

[0022] the subunit containing a β -loop peptide of Tomato bushy stunt virus, a group derived from a fluorine-containing compound, and a divalent linking group linking the β -loop peptide and the group derived from the fluorine-containing compound,

[0023] The above bivalent linking group is attached to the C-terminal end of the above β-cyclic peptide.

[0024] [2] The artificial virus coat of the above [1], wherein the fluorine-containing compound is a fluorine-containing peptide.

[0025] [3] The artificial virus coat of the above [2], wherein the side chain of at least one of the amino acid residues constituting the fluorine-containing peptide is a group represented by the following general formula (1),

[0026]

[0027] [In the formula, Z 1 is a divalent, trivalent or tetravalent linking group other than an alkylene group; Rf is a C 1-30 alkyl group substituted with at least two fluorine atoms (the C 1-30 alkyl group can have 1 to 5 etheric oxygen atoms between carbon atoms when the number of carbon atoms is two or more), -SF5, or -SF4-CR 101 R 102 -CR 103 R 104 Cl (R 101 , R 102 , R 103 and R 104 each independently are a hydrogen atom, a fluorine atom or a chlorine atom, but two or more of R 101 , R 102 , R 103 and R 104 are fluorine atoms); n3 is 1, 2 or 3; n4 is 0 or 1; and the black dot indicates a bonding site].

[0028] [4] The artificial virus coat of the above [3], wherein Rf is a group represented by the following general formula (f-1) or (f-2),

[0029]

[0030] [In the formula, Rf P represents a perhalogenated C 1-10 alkyl group containing at least two or more fluorine atoms (the perhalogenated C 1-10 alkyl group can have etheric oxygen atoms between carbon atoms when the number of carbon atoms is two or more), n1 is an integer of 0 to 10, n2 is an integer of 0 to 9, and the black dot indicates a bonding site].

[0031] [5] The artificial virus coat of the above [3] or [4], wherein the amino acid residue whose side chain is represented by the above general formula (1) is an amino acid residue in which 1 to 3 Rf are directly or indirectly linked to the side chain of a natural amino acid.

[0032] [6] The artificial virus capsid according to any one of the above [1] to [5], wherein the above bivalent linking group is a bismaleimide group having a maleimide group at both terminals.

[0033] [7] The artificial virus capsid according to any one of the above [1] to [6], wherein the above β -ring peptide has a cysteine residue at the C-terminal end or in the vicinity thereof, and the above bivalent linking group is linked to the thiol group from the cysteine residue.

[0034] [8] A pharmaceutical composition comprising the artificial virus capsid according to any one of the above [1] to [7].

[0035] [9] A carrier composition for drug delivery comprising the artificial virus capsid according to any one of the above [1] to [7].

[0036] The artificial virus capsid of the present application is modified with a group derived from a fluorine-containing compound, and thus has excellent cell membrane permeability. Therefore, it is expected that the artificial virus capsid is utilized in the medical field as a carrier for delivery of a physiologically active substance or a drug (DDS carrier). BRIEF DESCRIPTION OF DRAWINGS

[0037] Figure 1 is a graph showing the results of reverse phase HPLC (A) and MALDI-TOF-MS (B) in the purification of the β-Annulus-Cys peptide in Example 1. Figure 1 Figure 1

[0038] Figure 2 is a graph showing the results of reverse phase HPLC (A) and MALDI-TOF-MS (B) in the purification of the TMR-β-Annulus-Cys peptide in Example 1. Figure 2 Figure 2

[0039] Figure 3 is a graph showing the results of reverse phase HPLC (A) and MALDI-TOF-MS (B) in the purification of the β-Annulus-PEG2-maleimide in Example 1. Figure 3 Figure 3

[0040] Figure 4 is a graph showing the results of reverse phase HPLC (A) and MALDI-TOF-MS (B) in the purification of the TMR-β-Annulus-PEG2-maleimide in Example 1. Figure 4 Figure 4

[0041] ​​​​​​​​Figure 5 is a graph showing the results of the determination of the particle size distribution (number function distribution) of the TMR-labeled fluorine-containing peptide-modified capsid in Example 1. C8F17 is a graph showing the results of the determination of the particle size distribution (number function distribution) of the TMR-labeled fluorine-containing peptide-modified capsid in Example 1. Figure 5 is a graph showing the results of the determination of the particle size distribution (number function distribution) of the TMR-labeled fluorine-containing peptide-modified capsid in Example 1. Figure 5 is a graph showing the results of the determination of the particle size distribution (number function distribution) of the TMR-labeled fluorine-containing peptide-modified capsid in Example 1.

[0042] Figure 6 is a graph showing the results of the determination of the particle size distribution (number function distribution) of the TMR-labeled fluorine-containing peptide-modified capsid in Example 1. C8F17 is a graph showing the results of the determination of the particle size distribution (number function distribution) of the TMR-labeled fluorine-containing peptide-modified capsid in Example 1. Figure 6 is a graph showing the results of the determination of the particle size distribution (number function distribution) of the TMR-labeled fluorine-containing peptide-modified capsid in Example 1. Figure 6 is a graph showing the results of the determination of the particle size distribution (number function distribution) of the TMR-labeled fluorine-containing peptide-modified capsid in Example 1.

[0043] Figure 7 is a graph showing the results of the determination of the particle size distribution (number function distribution) of the TMR-labeled fluorine-containing peptide-modified capsid in Example 1. C8F17 is a graph showing the results of the determination of the particle size distribution (number function distribution) of the TMR-labeled fluorine-containing peptide-modified capsid in Example 1. Figure 7 is a graph showing the results of the determination of the particle size distribution (number function distribution) of the TMR-labeled fluorine-containing peptide-modified capsid in Example 1. Figure 7 is a graph showing the results of the determination of the particle size distribution (number function distribution) of the TMR-labeled fluorine-containing peptide-modified capsid in Example 1.

[0044] Figure 8 is a graph showing the results of the determination of the particle size distribution (number function distribution) of the TMR-labeled fluorine-containing peptide-modified capsid in Example 1. Figure 8 is a graph showing the results of the determination of the particle size distribution (number function distribution) of the TMR-labeled fluorine-containing peptide-modified capsid in Example 1. C8F17 is a graph showing the results of the determination of the particle size distribution (number function distribution) of the TMR-labeled fluorine-containing peptide-modified capsid in Example 1. Figure 8 is a graph showing the results of the determination of the particle size distribution (number function distribution) of the TMR-labeled fluorine-containing peptide-modified capsid in Example 1. C8F17 is a graph showing the results of the determination of the particle size distribution (number function distribution) of the TMR-labeled fluorine-containing peptide-modified capsid in Example 1.

[0045] Figure 9 is a graph showing the results of the determination of the particle size distribution (number function distribution) of the TMR-labeled fluorine-containing peptide-modified capsid in Example 1.

[0046] Figure 10 is a graph showing the results of the determination of the particle size distribution (number function distribution) of the TMR-labeled fluorine-containing peptide-modified capsid in Example 1.

[0047] Figure 11 is a graph showing the results of the determination of the particle size distribution (number function distribution) of the TMR-labeled fluorine-containing peptide-modified capsid in Example 1.

[0048] Figure 12is a graph showing the results of measurement of TMR fluorescence intensity of cells into which a TMR-labeled fluorine-containing peptide-modified capsid (left column), a TMR-labeled fluorine-containing peptide (middle column), and a TMR-labeled capsid (right column) were introduced in Example 1.

[0049] Figure 13 is a graph showing the results of flow cytometry measurement of untreated cells (A), cells into which a TMR-labeled fluorine-containing peptide-modified capsid (B), a TMR-labeled fluorine-containing peptide (C), and a TMR-labeled capsid (D) were introduced in Example 1 (left: scatter plot, right: histogram).

[0050] Figure 14 is a graph showing the results of measurement of the proportion (%) of TMR-uptaking cells of untreated cells, cells into which a TMR-labeled fluorine-containing peptide-modified capsid, a TMR-labeled fluorine-containing peptide, and a TMR-labeled capsid were introduced in Example 1 (A) and the central value of TMR fluorescence intensity (B). Figure 14 Figure 14

[0051] Figure 15 is a graph showing the results of measurement of the particle size distribution (number function distribution) of Dox-encapsulating fluorine-containing peptide-modified capsids in Example 2.

[0052] Figure 16 is a TEM image of Dox-encapsulating fluorine-containing peptide-modified capsids in Example 2.

[0053] Figure 17 is a graph showing the results of measurement of the UV-vis spectrum and Dox binding rate (%) of Dox-encapsulating fluorine-containing peptide-modified capsids and Dox-encapsulating fluorine-containing peptide-unmodified capsids in Example 2.

[0054] Figure 18 is a graph showing the results of measurement of the cell survival rate (%) of cells into which Dox-encapsulating fluorine-containing peptide-modified capsids were introduced in Example 2.

[0055] Figure 19 is a TMR fluorescence image of cells cultured in a medium containing only a TMR-labeled fluorine-containing peptide-modified capsid (Control), cells cultured in a medium containing a TMR-labeled fluorine-containing peptide and EIPA (80 μM EIPA), cells cultured in a medium containing a TMR-labeled fluorine-containing peptide and Pitstop2 (20 μM Pitstop2), and cells cultured in a medium containing a TMR-labeled capsid and Genistein (160 μM Genistein) in Example 3.

[0056] Figure 20 ​​is a graph showing the results of TMR fluorescence intensity of cells cultured in a medium containing only a TMR-labeled capsid of a modified artificial virus with a fluorine-containing peptide (Control), a medium containing a TMR-labeled capsid of a modified artificial virus with a fluorine-containing peptide and EIPA (EIPA), a medium containing a TMR-labeled capsid of a modified artificial virus with a fluorine-containing peptide and Pitstop2 (Pitstop2), and a medium containing a TMR-labeled capsid of a modified artificial virus with a fluorine-containing peptide and genistein (Genistein) in Example 3.

[0057] Figure 21 is a graph showing the results of β-Annulus-CAD C6F13 F peptide in Example 4. Figure 21 (A) and ESI-MS Figure 21 (B) in Example 4.

[0058] Figure 22 is a graph showing the results of Hoechst 33342 fluorescence images, mCherry fluorescence images, merged images of mCherry fluorescence images and Hoechst 33342 fluorescence images, and transmitted light images of cells into which mCherry mRNA was introduced using a transfection reagent (left column), cells into which mCherry mRNA was introduced using a CAD C6F13 F peptide-modified artificial virus capsid (middle column), and cells into which mCherry mRNA was introduced using an unmodified artificial virus capsid (right column) in Example 4.

[0059] Figure 23 is a graph showing the results of mCherry fluorescence intensity of cells into which mCherry mRNA was introduced using a transfection reagent (left column), cells into which mCherry mRNA was introduced using a CAD C6F13 F peptide-modified artificial virus capsid (middle column), and cells into which mCherry mRNA was introduced using an unmodified artificial virus capsid (right column) in Example 4. DETAILED DESCRIPTION

[0060] In the present invention and the present application specification, "C p1-p2 " (p1 and p2 are positive integers satisfying p1 < p2) means a group having a carbon number of p1 to p2.

[0061] In the present invention and the present application specification, "C 1-30 alkyl" is an alkyl group having a carbon number of 1 to 30, and can be linear or branched. "C 2-30 alkyl" is an alkyl group having a carbon number of 2 to 30, and can be linear or branched. As the C 1-30Examples of the "C1-10 alkyl group" include a methyl group, an ethyl group, a propyl group, an isopropyl group, a butyl group, an isobutyl group, a sec-butyl group, a t-butyl group, a pentyl group, an isopentyl group, a neopentyl group, a t-pentyl group, a hexyl group, a heptyl group, an octyl group, a nonyl group, a decyl group, an undecyl group, a dodecyl group, a tridecyl group, a tetradecyl group, a pentadecyl group, a hexadecyl group, a heptadecyl group, an octadecyl group, a nonadecyl group, an icosyl group, a hendecyl group, a docosyl group, a tricosyl group, a tetracosyl group, a pentacosyl group, a hexacosyl group, a heptacosyl group, an octacosyl group, a nonacosyl group, a triacontyl group, and the like.

[0062] In the present application and the present specification, the "C 1-10 alkyl group" is a C1-6 alkyl group, and can be either a straight chain or a branched chain. Examples of the "C 2-10 alkyl group" include a methyl group, an ethyl group, a propyl group, an isopropyl group, a butyl group, an isobutyl group, a sec-butyl group, a t-butyl group, a pentyl group, an isopentyl group, a neopentyl group, a t-pentyl group, a hexyl group, and the like. 1-10

[0063] In the present application and the present specification, the "C 1-6 alkyl group" is a C1-6 alkyl group, and can be either a straight chain or a branched chain. Examples of the "C 1-6 alkyl group" include a methyl group, an ethyl group, a propyl group, an isopropyl group, a butyl group, an isobutyl group, a sec-butyl group, a t-butyl group, a pentyl group, an isopentyl group, a neopentyl group, a t-pentyl group, a hexyl group, and the like.

[0064] In the present application and the present specification, the "C 6-14 aryl group" is a C6-14 aromatic hydrocarbon group, and is particularly preferably a C 6-12 aryl group. Examples of the "C 6-14 aryl group" include a phenyl group, a naphthyl group, an anthryl group, a 9-fluorenyl group, and the like, and a phenyl group is particularly preferred.

[0065] In the present application and the present specification, the "C 6-14 aryl group" is a C6-14 aromatic hydrocarbon group, and is particularly preferably a C 6-14 aryl group. Examples of the "C 1-6 aryl group" include a phenyl group, a naphthyl group, an anthryl group, a 9-fluorenyl group, and the like, and a phenyl group is particularly preferred. 1-6 alkyl group, a C 6-14 ​Examples of the "aryl group" include phenyl, naphthyl, anthryl, 4-nitrophenyl, 4-methoxyphenyl, 2,4-dimethoxyphenyl, 3,4-dimethoxyphenyl, 4-methylphenyl, 2,6-dimethylphenyl, 3-chlorophenyl, 1,3-benzodioxole-5-yl, and the like.

[0066] In the present application and the present specification, the "C 6-14 In the present application and the present specification, the "C 1-6 In the present application and the present specification, the "C 1-6 In the present application and the present specification, the "C 6-14 In the present application and the present specification, the "C 6-14 In the present application and the present specification, the "C 1-6 In the present application and the present specification, the "C 6-14 In the present application and the present specification, the "C 6-14 In the present application and the present specification, the "C 1-6 In the present application and the present specification, the "C 1-6 In the present application and the present specification, the "C 1-4 In the present application and the present specification, the "C 6-14 In the present application and the present specification, the "C 1-6 Examples of the "C

[0067] In the present application and the present specification, the "halogen atom" means a fluorine atom, a chlorine atom, a bromine atom, or an iodine atom. The "halogen atom other than a fluorine atom" means a chlorine atom, a bromine atom, or an iodine atom. As examples of the "halogen atom other than a fluorine atom", a chlorine atom or a bromine atom is preferred, and a chlorine atom is particularly preferred.

[0068] In the present application and the present specification, the "C 1-6 In the present application and the present specification, the "C 1-6 In the present application and the present specification, the "C 1-6 The "C 1-6 Examples of the "C

[0069] In the present application and the present specification, the "ether-bonded oxygen atom" means an oxygen atom which connects carbon atoms, and does not include an oxygen atom which is connected to another oxygen atom in series. The "C

[0070] In the following, the "compound n" means a compound represented by the formula (n).

[0071] The artificial virus capsid of the present application is an artificial virus capsid formed by self-assembly of a plurality of subunits containing a β-cyclodextrin of TBSV, a group derived from a fluorine-containing compound, and a divalent linking group linking the β-cyclodextrin and the group derived from a fluorine-containing compound. In this artificial virus capsid, the divalent linking group is linked to the C-terminal end of the β-cyclodextrin, and thus the group derived from a fluorine-containing compound is displayed on the outside of the hollow capsule of the artificial virus capsid.

[0072] <β-cyclodextrin>

[0073] The β-cyclodextrin of TBSV (hereinafter also simply referred to as "β-cyclodextrin") is well known and is reviewed in Non-Patent Literature 1. The β-cyclodextrin is composed of an amino acid sequence represented by INHVGGTGGAIMAPVAVTRQLVGS (SEQ ID NO: 1). In the amino acid sequence of SEQ ID NO: 1, the 1st to 5th regions, the 10th to 13th regions, and the 17th to 22nd regions are portions necessary for forming an artificial virus capsid by self-assembly (hereinafter sometimes referred to as "consensus sequence of β-cyclodextrin"), and are the smallest units capable of forming an artificial virus capsid by self-assembly in water.

[0074] The β-cyclodextrin used in the present application and the present application specification is not particularly limited as long as it is a peptide containing the consensus sequence of β-cyclodextrin and capable of forming an artificial virus capsid by self-assembly in water. For example, it can be a peptide composed of only the amino acid sequence represented by SEQ ID NO: 1, or it can be a peptide in which, within the limits that do not hinder the formation of an artificial virus capsid, regions other than the consensus sequence of β-cyclodextrin in the amino acid sequence represented by SEQ ID NO: 1 can be replaced with other amino acid residues, can be deleted, or other amino acid residues or amino acid sequences can be added. In addition, further amino acid sequences can be added to the C-terminal end and / or the N-terminal end of SEQ ID NO: 1.

[0075] It has been demonstrated in past studies that even if further amino acid sequences are added to the C-terminal end and / or the N-terminal end of SEQ ID NO: 1, an artificial virus capsid can be formed. In the present application and the present application specification, as the β-cyclodextrin, it can be a peptide composed of an amino acid sequence in which, for example, 1 to 20 residues, 1 to 10 residues, or 1 to 5 residues of amino acids are added to the N-terminal end and / or the C-terminal end of the amino acid sequence represented by SEQ ID NO: 1.

[0076] As the amino acid sequence added to the amino acid sequence represented by SEQ ID NO: 1, it is not particularly limited as long as it is a sequence that does not hinder the formation of an artificial virus capsid based on self-assembly, and for example, it can be a linker sequence for linking to other substances that modify the β-cyclodextrin. It is within the skill of a person skilled in the art to synthesize a peptide in a manner that the N-terminal end and / or the C-terminal end of the amino acid sequence represented by SEQ ID NO: 1 has a functional group useful for binding to a linker or the linker itself.

[0077] In the present application and the present specification, "subunit" refers to a unit molecule that forms an artificial virus capsid by self-assembly. Each subunit contains at least a β-cyclodipeptide. However, the plurality of subunits that self-assemble into one artificial virus capsid are not limited to being completely identical to each other, and for example, a configuration in which a subunit modified with a fluorine-containing compound and a subunit not having a fluorine-containing compound are mixed is also contemplated. An artificial virus capsid formed by self-assembly of a plurality of subunits composed of subunits modified with mutually different fluorine-containing compounds is also contemplated. Self-assembly, as described in Non-Patent Literatures 1 to 4, indicates a phenomenon in which a plurality of subunits containing a β-cyclodipeptide spontaneously combine in water or an aqueous solution to form an artificial virus capsid in a substantially spherical shape, and a structure thus formed. The diameter of each artificial virus capsid formed by self-assembly (measured by DLS (dynamic light scattering method)) can vary depending on the presence or absence and the kind of a guest, and is typically 30 to 100 nm. It is inferred that one artificial virus capsid contains about 60 subunits. The pH of the aqueous solution at the time of self-assembly is typically 5 to 9, and for example, can be 6 to 8, or 6.5 to 7.5. The concentration of the subunit at the time of self-assembly can be, for example, 1 to 50 μM, 3 to 40 μM, or 5 to 30 μM.

[0078] The β-cyclodipeptide used in the present application and the present specification preferably has a cysteine residue at the C terminus or in the vicinity thereof, specifically, at 1 to 5 amino acid residues from the C terminus, and more preferably at 2 to 5 amino acid residues from the C terminus. Note that "1 amino acid residue from the C terminus" refers to the amino acid residue at the C terminus. As the β-cyclodipeptide having a cysteine residue at the C terminus or in the vicinity thereof, for example, a peptide composed of an amino acid sequence in which the 2nd amino acid residue (G) from the C terminus of the amino acid sequence of SEQ ID NO: 1 is replaced with a cysteine residue (SEQ ID NO: 2), a peptide composed of an amino acid sequence in which the amino acid residue (S) at the C terminus of the amino acid sequence of SEQ ID NO: 1 is replaced with a cysteine residue (SEQ ID NO: 3), or a peptide composed of an amino acid sequence in which a peptide composed of 1 to 4 amino acids is added to the C terminus side of these amino acid sequences. In addition, it can be a peptide composed of an amino acid sequence in which an amino acid sequence composed of 1 to 5 amino acids including a cysteine residue is added to the C terminus side of the amino acid sequence of SEQ ID NO: 1. As the amino acid sequence having a cysteine residue added to the C terminus side of the amino acid sequence of SEQ ID NO: 1, from the viewpoint of suppressing the influence on the formation of an artificial virus capsid, an amino acid sequence of 1 to 5 amino acids in which all except the cysteine residue are glycine residues is preferable, and an amino acid sequence of 2 to 5 amino acids in which the 2nd from the C terminus is a cysteine residue and the others are all glycine residues is more preferable.

[0079] The β -turn peptide used in the present application and the present application specification can have a cysteine residue at the N-terminus or in the vicinity thereof, specifically, 1 to 5 amino acid residues from the N-terminus, preferably 1 amino acid residue from the N-terminus. By having a cysteine residue at the N-terminus or in the vicinity thereof, it becomes easy to modify the N-terminus of the β -turn peptide with other substances. As the β -turn peptide having a cysteine residue at the N-terminus or in the vicinity thereof, for example, a peptide consisting of an amino acid sequence in which an amino acid sequence consisting of 1 to 5 amino acids including a cysteine residue is added to the N-terminal side of the amino acid sequence of SEQ ID NO: 1 can be given. As the amino acid sequence having a cysteine residue added to the N-terminal side of the amino acid sequence of SEQ ID NO: 1, from the viewpoint of suppressing the influence on the formation of artificial virus capsids, an amino acid sequence of 1 to 5 amino acids all of which are glycine residues except for the cysteine residue is preferred, and an amino acid sequence of 2 to 5 amino acids of which the first from the N-terminus is a cysteine residue and the others are all glycine residues is more preferred.

[0080] <group derived from a fluorine-containing compound>

[0081] The "group derived from a fluorine-containing compound" used in the present application and the present application specification means a group in which one hydrogen atom of a fluorine-containing compound having at least one fluorine atom is removed to become a bonding site. From the viewpoint of more sufficiently obtaining the effect of improving the cell membrane permeability based on fluorine, the "group derived from a fluorine-containing compound" used in the present application is preferably a group having a group represented by the following general formula (1). In general formula (1), a black dot represents a bonding site.

[0082]

[0083] In general formula (1), Rf is a C 1-30 alkyl group, -SF5, or -SF4-CR 101 R 102 -CR 103 R 104 Cl.

[0084] Rf is a C 1-30 alkyl group substituted with at least two fluorine atoms, the C 1-30 alkyl group can have 1 to 5 ether-bonding oxygen atoms between carbon atoms when the carbon atoms are two or more (C 2-30 alkyl group), can have 1 to 5 ether-bonding oxygen atoms between carbon atoms.

[0085] One or more hydrogen atoms bonded to a carbon atom of Rf can be further substituted with a halogen atom other than fluorine. Here, as the C 1-30 alkyl group of Rf, a C 1-20 alkyl group is preferred, and a C 1-10 alkyl group is more preferred, and a C2-10 alkyl, more preferably C 2-8 alkyl. The C 1-30 alkyl is C 2-30 alkyl, it can have 1 to 5 ether-bonding oxygen atoms between carbon atoms. In Rf, the number of hydrogen atoms substituted with fluorine atoms is not particularly limited as long as it is 2 or more, for example, it is preferably 3 or more, more preferably 6 or more, and further preferably 7 or more.

[0086] As examples of Rf, mention can be made of trifluoromethyl, pentafluoroethyl, heptafluoropropyl, nonafluorobutyl, perfluoropentyl, perfluorohexyl, perfluoroheptyl, perfluorooctyl, perfluorononyl, perfluorodecyl, difluoromethyl, 1,1-difluoroethyl, 2,2-difluoroethyl, 1,1,2,2-tetrafluoroethyl, 1,1,2,2,3,3-hexafluoropropyl, 1,1,2,3,3,3-hexafluoropropyl, 1,1,2,2,3,3-hexafluorohexyl, 1,1,2,2,3,3-hexafluorooctyl, 1,1,2,2,3,3-hexafluorodecyl, 1,1,2,2,3,3-hexafluorooctadecyl, 1,1,2,2,3,3-hexafluorohexacosyl, and the like.

[0087] When Rf is a group having 2 carbon atoms, as Rf, a group in which at least 4 or more of the hydrogen atoms bonded to the carbon atoms are substituted with fluorine atoms, such as pentafluoroethyl, is preferable compared with 1,1,1-trifluoroethyl (CF3-CH2-). In addition, when Rf is a group having 3 carbon atoms, as Rf, a straight-chain group is preferable, and in the case of a branched-chain group, 1,1,1,3,3,3-hexafluoropropan-2-yl ((CF3)2-CH-), (CF3)2-CF- group are preferable. When Rf is a group having 4 carbon atoms, as Rf, it can be a straight-chain group or a branched-chain group. In the case of a branched-chain group, a group in which the hydrogen atoms bonded to the carbon atoms constituting the alkylene moiety are substituted with fluorine atoms, or a completely fluorinated group is preferable.

[0088] As Rf, a group represented by the following general formula (f-1) or (f-2) is preferable. In general formulae (f-1) and (f-2), the black dot indicates the bonding site.

[0089]

[0090] In general formulae (f-1) and (f-2), Rf P represents a completely halogenated C 1-10 alkyl. Rf P is a C 1-10 alkyl in which all the hydrogen atoms are substituted with halogen atoms, and at least 2 or more of these halogen atoms are fluorine atoms. Rf P When the number of carbon atoms is 2 or more, that is, when the completely halogenated C2-10 In the case of an alkyl group, 1 to 5 oxygen atoms having ether linkage can be present between carbon atoms. In General Formula (f-2), 2 Rf P may be the same kind of group, or different kinds of groups.

[0091] In General Formula (f-1) or (f-2) below, n1 is an integer of 0 to 10, and n2 is an integer of 0 to 9. When n1 and n2 are 0, each represents a single bond. That is, when n1 is 0, the group represented by General Formula (f-1) is Rf P , and when n2 is 0, the group represented by General Formula (f-2) is (Rf P )2-CH-.

[0092] When Rf is a group represented by General Formula (f-1), Rf is preferably a group in which Rf P is a trifluoromethyl group, a pentafluoroethyl group, a heptafluoropropyl group, a nonafluorobutyl group, a perfluoropentyl group, a perfluorohexyl group, a perfluoroheptyl group, a perfluorooctyl group, a perfluorononyl group, or a perfluorodecyl group, n1 is an integer of 0 to 4, more preferably a group in which Rf P is a trifluoromethyl group, a pentafluoroethyl group, a heptafluoropropyl group, a nonafluorobutyl group, a perfluoropentyl group, a perfluorohexyl group, a perfluoroheptyl group, a perfluorooctyl group, a perfluorononyl group, or a perfluorodecyl group, n1 is an integer of 0 to 2, and further preferably a group in which Rf P is a trifluoromethyl group, a pentafluoroethyl group, a heptafluoropropyl group, a nonafluorobutyl group, a perfluoropentyl group, or a perfluorohexyl group, n1 is an integer of 0 to 2 (where a group in which n1 is 1 and Rf P is a trifluoromethyl group is excluded).

[0093] When Rf is a group represented by General Formula (f-2), Rf is preferably a group in which Rf P is a trifluoromethyl group, a pentafluoroethyl group, a heptafluoropropyl group, a nonafluorobutyl group, a perfluoropentyl group, a perfluorohexyl group, a perfluoroheptyl group, a perfluorooctyl group, a perfluorononyl group, or a perfluorodecyl group, n2 is an integer of 0 to 4, more preferably a group in which Rf P is a trifluoromethyl group, a pentafluoroethyl group, a heptafluoropropyl group, a nonafluorobutyl group, a perfluoropentyl group, a perfluorohexyl group, a perfluoroheptyl group, a perfluorooctyl group, a perfluorononyl group, or a perfluorodecyl group, n2 is an integer of 0 to 2, and further preferably a group in which Rf P is a trifluoromethyl group, a pentafluoroethyl group, a heptafluoropropyl group, a nonafluorobutyl group, a perfluoropentyl group, or a perfluorohexyl group, n2 is an integer of 0 to 2 (where a group in which n2 is 0 or 1 and Rf P is a trifluoromethyl group is excluded).

[0094] As examples of Rf, a difluoromethyl group, a 1,1-difluoroethyl group, a 2,2-difluoroethyl group, a 1,1,2,2-tetrafluoroethyl group, a 1,1,2,2,3,3-hexafluoropropyl group, a 1,1,2,3,3,3-hexafluoropropyl group, and the like can be given.

[0095] Rf is -SF4-CR 101 R 102 -CR 103 R 104 When R 101 , R 102 , R 103 and R 104 are each independently a hydrogen atom, a fluorine atom or a chlorine atom, among them, R 101 , R 102 , R 103 and R 104 are two or more fluorine atoms. As the -SF4-CR 101 R 102 -CR 103 R 104 group represented by R 1 , specifically, there can be mentioned -SF4-CF2-CF2Cl, -SF4-CF2-CFCl2, -SF4-CF2-CHF-Cl, -SF4-CF2-CCl3, -SF4-CF2-CHCl2, -SF4-CF2-CH2Cl, -SF4-CFCl-CFCl2, -SF4-CFCl-CHF-Cl, -SF4-CHF-CHF-Cl.

[0096] In General Formula (1), Z 1 is a divalent, trivalent or tetravalent linking group other than an alkylene group, n3 is 1, 2 or 3, and n4 is 0 or 1. When n4 is 0, -(Z 1 )n4- represents a single bond. As Z 1 , there is no particular limitation as long as it is a divalent to tetravalent group other than an alkylene group. For example, as Z 1 , there can be mentioned an alkylene group, an oxygen atom (-0-), a sulfur atom (-S-), -NH-, -N(CH3)-, -N(C2H5)-, -N(C3H7)-, a trivalent nitrogen atom, -C(=0)-, -S(=0)2-, a group obtained by removing 2 to 4 hydrogen atoms from a cycloalkane, a group obtained by removing 2 to 4 hydrogen atoms from an aromatic ring (an aryl group), a group obtained by removing 2 to 4 hydrogen atoms from a heterocycle (a heteroaryl group), or a combination thereof. As the aryl group and the heteroaryl group, the above-mentioned listed groups can be used. Among them, a group consisting only of an alkylene group, and a group in which the linking portion to Rf is an alkylene group are excluded.

[0097] Z 1Z is a linking group having 1 to 3 valences, and the group represented by General Formula (1) becomes a group having 1 to 3 Rf groups. Z 1 Z is a linking group having 2 valences, and the group represented by General Formula (1) becomes a group having 1 Rf group. Z 1 When Z has a nitrogen atom having 3 valences, the group represented by General Formula (1) can be a group having 2 Rf groups by bonding 2 Rf groups directly or via another 2-valent linking group to the nitrogen atom.

[0098] Z is a linking group having 1 to 3 valences, and the group represented by General Formula (1) becomes a group having 1 to 3 Rf groups. Z 1 Z can be a linking group having a group (ring group) obtained by removing a hydrogen atom from a ring, or a linking group not having a ring group. Z 1 When Z has a ring group, the group represented by General Formula (1) can be a group having 2 or 3 Rf groups. As the ring group, a group obtained by removing 2 to 4 hydrogen atoms from a cycloalkane, an aromatic ring, or a heterocycle can be mentioned. As the heterocycle, a ring in which 1 to 3 carbon atoms of an aromatic ring are substituted with one or more atoms selected from a nitrogen atom, an oxygen atom, and a sulfur atom is preferable. In addition, the ring group can be a group obtained by removing a hydrogen atom from a monocyclic ring, or a group obtained by removing a hydrogen atom from a fused ring. As Z in General Formula (1) 1 Z contains a ring group, a group obtained by removing 2 to 4 hydrogen atoms from cyclohexane, benzene, imidazole, or indole is preferable. For example, Z 1 When Z is a linking group having a ring group obtained by removing 2 to 4 hydrogen atoms from benzene, the ring group is 1,4-phenylene, 1,3-phenylene, 1,5-phenylene, or 1,3,5-substituted phenylene, and 1,4-phenylene or 1,3,5-substituted phenylene is preferable.

[0099] Specifically, as Z in General Formula (1) 1 -C(=O)-, -C(=O)-O-, -O-C(=O)-, -NH-C(=O)-O-, -O-C(=O)-NH-, -C(=O)-NH-, -NH-C(=O)-, -S-S-, -S(=O)2-NH-, -NH-S(=O)2-, -S(=O)2-NH-S(=O)2-, -C(=O)-NH-Ph- (-Ph- is 1,4-phenylene, 1,3-phenylene, 1,5-phenylene, or 1,3,5-substituted phenylene). In addition, a linking group in which any of these groups is combined with C 1-6 -C(=O)-, -C(=O)-O-, -O-C(=O)-, -NH-C(=O)-O-, -O-C(=O)-NH-, -C(=O)-NH-, -NH-C(=O)-, -S-S-, -S(=O)2-NH-, -NH-S(=O)2-, -S(=O)2-NH-S(=O)2-, -C(=O)-NH-Ph- (-Ph- is 1,4-phenylene, 1,3-phenylene, 1,5-phenylene, or 1,3,5-substituted phenylene). In addition, a linking group in which any of these groups is combined with C 1 -C(=O)-, -C(=O)-O-, -O-C(=O)-, -NH-C(=O)-O-, -O-C(=O)-NH-, -C(=O)-NH-, -NH-C(=O)-, -S-S-, -S(=O)2-NH-, -NH-S(=O)2-, -S(=O)2-NH-S(=O)2-, -C(=O)-NH-Ph- (-Ph- is 1,4-phenylene, 1,3-phenylene, 1,5-phenylene, or 1,3,5-substituted phenylene). In addition, a linking group in which any of these groups is combined with C

[0100] Z in general formula (1) 1 Preferably, the linking group is represented by the following general formula (2). In general formula (2), black dots represent bonding sites.

[0101]

[0102] In general formula (2), Z 2 It is a divalent, trivalent, or tetravalent linking group other than an alkylene group. As Z 2 There are no particular limitations as long as the group is a divalent to tetravalent group other than an alkylene group. For example, as Z... 2 Examples of aryl groups include alkylene groups, oxygen atoms (-O-), sulfur atoms (-S-), -NH-, -N(CH3)-, -N(C2H5)-, -N(C3H7)-, trivalent nitrogen atoms, -C(=O)-, -S(=O)2-, groups obtained by removing 2 to 4 hydrogen atoms from a cycloalkanes, groups obtained by removing 2 to 4 hydrogen atoms from an aromatic ring (aryl), groups obtained by removing 2 to 4 hydrogen atoms from a heterocyclic ring (heteroaryl), or combinations thereof. As aryl and heteroaryl groups, the groups listed above can be used. However, groups consisting solely of alkylene groups and groups whose linking portion to Rf is alkylene groups are excluded.

[0103] Z in general formula (2) 2 Specifically, it can be used with Z 1 The same groups listed in the formula (2) are also included. 2 Preferred are -C(=O)-, -C(=O)-O-, -OC(=O)-, -NH-C(=O)-O-, -OC(=O)-NH-, -C(=O)-NH-, -NH-C(=O)-, -SS-, -S(=O)2-NH-, -NH-S(=O)2-, -S(=O)2-NH-S(=O)2-, -C(=O)-NH-Ph- (-Ph- is 1,4-phenylene, 1,3-phenylene, 1,5-phenylene or 1,3,5-substituted phenyl).

[0104] In general formula (2), Rh is a hydrogen atom or C 1-6 Alkyl group. Rh is C10. 1-6 When alkyl, C is preferred as Rh. 1-3 Alkyl, more preferably methyl or ethyl.

[0105] As a group represented by general formula (1), for example, Z can be cited. 1 Rf is a group represented by general formula (2), and Rf is a group represented by general formula (f-1) or (f-2). Preferably, Z in general formula (2) is preferred. 2The following are possible values: -C(=O)-, -C(=O)-O-, -OC(=O)-, -NH-C(=O)-O-, -OC(=O)-NH-, -C(=O)-NH-, -NH-C(=O)-, -SS-, -S(=O)2-NH-, -NH-S(=O)2-, -S(=O)2-NH-S(=O)2-, -C(=O)-NH-Ph- (-Ph- is 1,4-phenylene, 1,3-phenylene, 1,5-phenylene, or 1,3,5-substituted phenyl), Rh is a hydrogen atom or C 1-6 Alkyl groups, Rf is a group represented by the general formula (f-1) or (f-2).

[0106] The fluorinated compound-derived group in the subunit of the artificial virus capsid of the present invention is not particularly limited as long as it is a group having at least one fluorine atom and does not hinder the formation of a β-cyclic peptide-based self-assembled artificial virus capsid when linked to a β-cyclic peptide. Examples of fluorinated compounds containing such a group include those derived from compounds in which fluorine atoms or fluorinated groups are introduced into peptides, nucleic acids, low-molecular-weight compounds, and chimeric molecules consisting of two or more of these. Preferably, compounds in which at least one hydrogen atom of the peptide, nucleic acid, or low-molecular-weight compound is replaced by a group represented by the above general formula (1).

[0107] The groups derived from fluorinated compounds that form the capsid of the artificial virus of the present invention are preferably fluorinated amino acid residues or fluorinated peptides. As fluorinated amino acids, amino acids with groups represented by the above general formula (1) bonded to an α-carbon are preferred. As fluorinated peptides, peptides in which at least one side chain of the amino acid residue constituting the peptide is a group represented by the above general formula (1) or a group containing such a group are preferred.

[0108] The fluorinated peptide, which is present in the artificial virus capsid of the present invention, preferably has one or more groups having a side chain represented by the above general formula (1) [-(Z 1 A peptide consisting of amino acid residues of [n4-(Rf)n3] or amino acid residues directly or indirectly linked to the side chain of a natural amino acid. Specifically, as a fluorinated peptide comprising the artificial viral capsid of the present invention, a peptide having at least one of the following amino acid residues is preferred: a peptide in which one or two hydrogen atoms of the amino group of the side chain of an arginine residue, asparagine residue, glutamine residue, or lysine residue are connected with -Rf or -Z. 3 -(Rf)n5(Z 3 The linking group is divalent, trivalent, or tetravalent, excluding alkylene groups. This excludes groups whose linking portion to Rf is alkylene. Additionally, n5 is 2, 3, or 4. (This refers to amino acid residues obtained by substitution; the hydrogen atom of the imino group on the side chain of an arginine residue is replaced with -Rf or -Z.) 3- an amino acid residue substituted with (Rf)n5; the hydrogen atom of the carboxyl group of the side chain of an asparagine acid residue or a glutamic acid residue is substituted with -Rf or -Z 3 - an amino acid residue substituted with (Rf)n5; the hydrogen atom of the thiol group of the side chain of a cysteine residue or a methionine residue is substituted with -Rf or -Z 3 - an amino acid residue substituted with (Rf)n5; the hydrogen atom of the hydroxyl group of the side chain of a serine residue, a threonine residue or a tryptophan residue is substituted with -Rf or -Z 3 - an amino acid residue substituted with (Rf)n5; 1 to 3 hydrogen atoms of the benzene ring of the side chain of a tyrosine residue or a phenylalanine residue is substituted with -Rf or -Z 3 - an amino acid residue substituted with (Rf)n5; 1 to 3 hydrogen atoms of the imidazole ring of the side chain of a histidine residue is substituted with -Rf or -Z 3 - an amino acid residue substituted with (Rf)n5; 1 to 3 hydrogen atoms of the indole ring of the side chain of a tryptophan residue is substituted with -Rf or -Z 3 - an amino acid residue substituted with (Rf)n5.

[0109] as Z 3 There is no particular limitation as long as it is a 2- to 4-valent group other than an alkylene group. For example, as Z 3 alkylene group, an oxygen atom (-0-), a sulfur atom (-S-), -NH-, -N(CH3)-, -N(C2H5)-, -N(C3H7)-, a 3-valent nitrogen atom, -C(=0)-, -S(=0)2-, a group obtained by removing 2 to 4 hydrogen atoms from a cycloalkane, a group obtained by removing 2 to 4 hydrogen atoms from an aromatic ring (an aryl group), a group obtained by removing 2 to 4 hydrogen atoms from a heterocycle (a heteroaryl group), or a combination thereof. As the aryl group and the heteroaryl group, the above-mentioned listed groups can be used. Among them, a group consisting of only an alkylene group, and a group in which the linking portion to Rf is an alkylene group are excluded. Specifically, as Z 3 a group other than an alkylene group can be used. 1 or Z 2 the same linking group.

[0110] the hydrogen atom of the amino group, the imino group, the carboxyl group, the hydroxyl group, the thiol group, the benzene ring, the imidazole ring, the indole ring, and the like of the side chain of a natural amino acid is substituted with -Rf or -Z 3 Substitution of - (Rf)n5may be performed by a general synthetic reaction such as an ester reaction.

[0111] As the fluorine-containing peptide possessed by the artificial virus capsid of the present application, a peptide containing at least one amino acid residue having - (Z 1 )n4- (Rf)n3in the side chain can be mentioned. Among the amino acid residues constituting the peptide, only at least one side chain has - (Z1 ) n4- (Rf) n3, all of the side chains of the amino acid residues can have - (Z 1 ) n4- (Rf) n3. Two or more of the side chains have - (Z 1 ) n4- (Rf) n3in one molecule of the peptide, these equal number of - (Z 1 ) n4- (Rf) n3may be the same kind as each other, or can be different kinds. In addition, in the peptide, the side chain having - (Z 1 ) n4- (Rf) n3may be at the N terminus, or at the C terminus, or other than the termini.

[0112] The fluorine-containing peptide possessed by the artificial virus coat of the present application is preferably a peptide composed of 3 or more amino acids. As the amino acid residues constituting the fluorine-containing peptide possessed by the artificial virus coat of the present application, 2 to 20, more preferably 3 to 20, further preferably 3 to 10, and more further preferably 3 to 6 are preferable.

[0113] In the fluorine-containing peptide possessed by the artificial virus coat of the present application, as the amino acid residue other than - (Z 1 ) n4- (Rf) n3, there is no particular limitation, and it can be an amino acid residue of an α-amino acid, an amino acid residue of a β-amino acid, an amino acid residue of a γ-amino acid, or an amino acid residue of a δ-amino acid. In addition, it can be an amino acid residue of an L-amino acid, or an amino acid residue of a D-amino acid. As the amino acid residue other than - (Z 1 ) n4- (Rf) n3included in the fluorine-containing peptide possessed by the artificial virus coat of the present application, it is preferably an amino acid residue of a protein-constituting amino acid, or a modified amino acid in which the side chain of the D form thereof is modified.

[0114] As the protein-constituting amino acid, glycine, alanine, valine, leucine, isoleucine, serine, threonine, phenylalanine, tyrosine, tryptophan, asparagine, glutamine, proline, aspartic acid, glutamic acid, lysine, arginine, histidine, and the like can be given. In addition, as the modified amino acid in which the protein-constituting amino acid is modified, for example, an amino acid in which the hydrogen atom of the amino group of the side chain of lysine, arginine, or histidine is substituted with a protecting group for an amino group or Pbf (N-ω- (2, 2, 4, 6, 7-pentamethylbenzofuran-5-sulfonyl) group; an amino acid in which the hydrogen atom of the carboxyl group of the side chain of aspartic acid or glutamic acid is substituted with a protecting group for a carboxyl group or a t-butyl alkyl group; an amino acid in which the hydrogen atom of the thiol group of cysteine is substituted with a benzyl group can be given.

[0115] The fluorinated peptide comprising the artificial virus capsid of the present invention is preferably a peptide consisting of 3 to 20, preferably 3 to 10, and more preferably 3 to 6 amino acid residues, wherein the N-terminus is a cysteine ​​residue and the second residue from the opposite end of the end bonded to the divalent linker is a fluorinated amino acid residue. Particularly preferred are peptides comprising an N-terminus of a cysteine ​​residue and a second residue from the C-terminus of a natural amino acid whose side chain hydrogen atom is replaced with -Rf or -Z. 3 A peptide consisting of 3 to 20, preferably 3 to 10, more preferably 3 to 6 amino acid residues, of which -(Rf)n5-substituted amino acid residues are present; more preferably, the peptide consists of an N-terminus cysteine ​​residue and a second C-terminus consisting of a hydrogen atom of the side chain of a natural amino acid replaced with -Rf or -Z. 3 A peptide consisting of 3 to 20, preferably 3 to 10, more preferably 3 to 6 amino acid residues, wherein the second to third amino acid residues from the N-terminus are independently alanine or glycine residues; and further preferably, a peptide consisting of a cysteine ​​residue at the N-terminus and a hydrogen atom of the side chain of the natural amino acid with a hydrogen atom replaced by -Rf or -Z. 3 A peptide consisting of 3 to 20, preferably 3 to 10, more preferably 3 to 6 amino acid residues, wherein the second to third amino acid residues from the N-terminus are independently alanine or glycine, and the C-terminus is phenylalanine. The hydrogen atoms of the side chain of this natural amino acid are represented by -Rf or -Z. 3 The amino acid residue substituted with -(Rf)n5 is preferably any of the following amino acid residues: one or two hydrogen atoms of the amino group of the side chain of an arginine residue, asparagine residue, glutamine residue, or lysine residue are substituted with -Rf or -Z. 3 -(Rf)n5(Z 3 The linking group is divalent, trivalent, or tetravalent, excluding alkylene groups. This excludes groups whose linking portion to Rf is alkylene. Additionally, n5 is 2, 3, or 4. (This refers to amino acid residues obtained by substitution; the hydrogen atom of the imino group on the side chain of an arginine residue is replaced with -Rf or -Z.) 3 Amino acid residues formed by substituting -(Rf)n5; hydrogen atoms of the carboxyl groups of the side chains of aspartic acid or glutamic acid residues are replaced with -Rf or -Z. 3 Amino acid residues formed by substituting -(Rf)n5; hydrogen atoms of the thiol groups in the side chains of cysteine ​​or methionine residues replaced with -Rf or -Z. 3 Amino acid residues formed by substituting -(Rf)n5; hydrogen atoms of the hydroxyl groups on the side chains of serine, threonine, or tryptophan residues replaced with -Rf or -Z. 3- an amino acid residue substituted with (Rf)n5; 1 to 3 hydrogen atoms of the benzene ring of the side chain of a tyrosine residue or a phenylalanine residue is substituted with -Rf or -Z 3 - an amino acid residue substituted with (Rf)n5; 1 to 3 hydrogen atoms of the imidazole ring of the side chain of a histidine residue is substituted with -Rf or -Z 3 - an amino acid residue substituted with (Rf)n5; 1 to 3 hydrogen atoms of the indole ring of the side chain of a tryptophan residue is substituted with -Rf or -Z 3 - an amino acid residue substituted with (Rf)n5.

[0116] In the artificial virus capsid of the present application, when the bivalent linking group described above is linked to the N terminus of the fluorine-containing peptide, the C terminus of the fluorine-containing peptide can be protected with a protecting group. As the protecting group of the C terminus, there is no particular limitation as long as it is a protecting group of a carboxyl group, and for example, a protecting group of a carboxyl group used in peptide synthesis can be used. As the protecting group of the carboxyl group, specifically, a protecting group selected from the group represented by General Formula (p-1), 2-(9,10-dioxo)anthrylmethyl, benzyloxymethyl, and benzoylmethyl. In General Formula (p-1), R 3 is a C 6-14 aryl group, R 4 and R 5 are each independently a hydrogen atom or a C 6-14 aryl group which can be substituted. In addition, a black dot indicates a bonding site.

[0117]

[0118] As the protecting group of the carboxyl group, benzyl, diphenylmethyl, triphenylmethyl, 4-nitrobenzyl, 4-methoxybenzyl, 2,4-dimethoxybenzyl, 3,4-dimethoxybenzyl, 4-methylbenzyl, 2,6-dimethylbenzyl, 3-chlorobenzyl, 9-anthrylmethyl, piperonyl, 2-(9,10-dioxo)anthrylmethyl, benzyloxymethyl, benzoylmethyl, and the like can be given. From the aspect that deprotection can be performed under mild conditions, the protecting group of the carboxyl group at the C terminus is preferably benzyl, triphenylmethyl, and more preferably benzyl.

[0119] In the artificial virus capsid of the present invention, when the aforementioned divalent linker is attached to the N-terminus of a fluorinated peptide, the N-terminus of the fluorinated peptide can be protected by an amino protecting group. There is no particular limitation on the protecting group at the N-terminus, as long as it is an amino protecting group; for example, an amino protecting group used in peptide synthesis can be used. Examples of amino protecting groups include tert-butoxycarbonyl (Boc), 9-fluorenylmethoxycarbonyl (Fmoc), benzyloxycarbonyl (Cbz), allyloxycarbonyl (Alloc), and 2,2,2-trichloroethoxycarbonyl (Troc), etc., which are carbamate-based protecting groups. From the perspective of enabling deprotection under mild conditions, tert-butoxycarbonyl (Boc) or 9-fluorenylmethoxycarbonyl (Fmoc) is preferred.

[0120] The artificial virus capsid of the present invention has a fluorinated peptide, except that at least one side chain is -(Z). 1 Besides amino acids of type n4-(Rf)n3 used as raw materials, peptide synthesis can be carried out using conventional methods. For example, it can be carried out using solid-phase peptide synthesis. This fluorinated peptide can have side chains with -(Z) 1 Using amino acids of type n4-(Rf)n3 as raw materials, they can be easily synthesized using an automated peptide synthesizer. As fluorinated amino acids, those with a side chain having -(Z) 1 Amino acids of n4-(Rf)n3.

[0121] Peptides can be manufactured by sequentially condensing an amino acid, formed by binding its C-terminus to a solid, with an amino acid protecting its amino group, thereby detaching the peptide from the solid phase. The amino acid raw material preferably uses amino acids whose amino groups are protected by a Boc or Fmoc group. The side chain functional groups of the amino acid raw material preferably use side chain functional groups protected by a protecting group. Examples of protecting groups for side chain functional groups include Boc, triphenylmethyl, benzyl, and 2,2,5,7,8-pentamethylchromane-6-sulfonyl (Pmc).

[0122] Examples of condensing agents that form peptide bonds include N,N-dicyclohexylcarbodiimide (DCC), 1-ethyl-3-(3'-dimethylaminopropyl)carbodiimide (WSC), and benzotriazol-1-yloxy-tris(dimethylamino). Hexafluorophosphate (BOP), benzotriazole-1-yloxytripyrrolidinyl Hexafluorophosphate (pyBOP), 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethylurea hexafluorophosphate (HBTU), 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethylurea tetrafluoroborate, 1-cyano-2-ethoxy-2-oxoethyleneaminooxy)dimethylaminomorpholinylcarbon hexafluorophosphate (COMU) and the like. In addition, N-hydroxybenzotriazole (HOBt), (hydroxyimino)cyanoacetic acid ethyl ester (oxyma) can also be used in combination with the above-mentioned condensing agent at a preferred ratio.

[0123] The formation of the peptide bond can use a method of activating the carboxyl terminal end. As the activating agent, for example, N-hydroxysuccinimide, p-nitrophenyl ester, pentafluorophenyl ester, and the like can be mentioned. As the base used in the formation of the peptide bond, for example, triethylamine, diisopropylethylamine (DIPEA), and the like can be mentioned. As the solvent used in the peptide bond formation reaction, for example, chloroform, dichloromethane (DCM), dichloroethane (DCE), acetonitrile (MeCN), N,N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), N-methyl-2-pyrrolidone (NMP), and the like can be mentioned.

[0124] The Boc group and the Fmoc group as the protecting group of the amino terminal end amino group of the peptide or the amino acid can be removed by trifluoroacetic acid (TFA) or piperidine, respectively. The protecting group of the side chain functional group of the amino acid residue of the peptide can be removed by TFA, hydrogen fluoride (HF), trifluoromethanesulfonic acid, and the like, for example.

[0125] In addition, in the peptide solid phase synthesis method, as a method of detaching the peptide having the protecting group added to the side chain functional group of the peptide or the amino acid residue from the peptide solid phase synthesis resin, TFA can be used, for example. The detachment of the peptide from the peptide solid phase resin and the detachment of the protecting group of the side chain functional group of the amino acid residue can be simultaneously performed in the same reaction system, respectively. Or, they can be independently performed. As the peptide solid phase synthesis resin for the peptide solid phase synthesis, commercially available resins such as 4-hydroxymethyl-3-methoxyphenoxybutyric acid-diphenylmethylamine-polystyrene resin, p-benzyloxybenzyl alcohol-polystyrene resin, oxime resin, and the like can be used, for example.

[0126] The target peptide or the intermediate thereof can be isolated and purified by various methods such as ion chromatography, gel filtration chromatography, reverse phase chromatography, normal phase chromatography, recrystallization, extraction, fractional crystallization, and the like, for example. In addition, the peptide thus obtained can be converted into a salt by a conventional method.

[0127] The protecting group of the amino group or the carboxyl group of the fluorine-containing peptide produced can also be deprotected as needed. The deprotection can be performed by a conventional method according to the kind of the protecting group.

[0128] <Divalent Linking Group>

[0129] As the divalent linking group that links the β-cyclododecapeptide and the group derived from the fluorine-containing compound, there is no particular limitation as long as it is a divalent organic group. For example, as Z 1alkylene group, an oxygen atom (-0-), a sulfur atom (-S-), -NH-, -N(CH3)-, -N(C2H5)-, -N(C3H7)-, a trivalent nitrogen atom, -C(=0)-, -S(=0)2-, a group obtained by removing 2 to 4 hydrogen atoms from a cycloalkane, a group obtained by removing 2 to 4 hydrogen atoms from an aromatic ring (an aryl group), a group obtained by removing 2 to 4 hydrogen atoms from a heterocycle (a heteroaryl group), or a combination thereof. As the aryl group and the heteroaryl group, the above-listed groups can be used.

[0130] As the divalent linking group of the artificial virus coat of the present application, from the viewpoint of being able to easily bond with a cysteine residue or the like, a divalent group having at least one group capable of selectively and easily bonding with a thiol group is preferable. As the group capable of selectively and easily bonding with a thiol group, for example, a maleimide group, an iodoacetyl group, a bromoacetyl group, or the like can be given. As the divalent linking group, for example, a bismaleimide having a maleimide group at both ends such as a group represented by the following general formula (3) can be given.

[0131]

[0132] In general formula (3), the black dots indicate bonding sites. One of the two black dots bonds with the β-cyc peptide, and the other bonds with the group derived from the fluorine-containing compound. By a chemical reaction in which a thioether group is formed from a maleimide group and a thiol group, it is possible to easily bond with the thiol group of the cysteine residue in the β-cyc peptide or the thiol group in the group derived from the fluorine-containing compound. The reaction of the thiol group with the maleimide group can be performed using a general organic synthesis reaction.

[0133] In general formula (3), as Z 4 alkylene group, an oxygen atom (-0-), a sulfur atom (-S-), -NH-, -N(CH3)-, -N(C2H5)-, -N(C3H7)-, a trivalent nitrogen atom, -C(=0)-, -S(=0)2-, a group obtained by removing 2 to 4 hydrogen atoms from a cycloalkane, a group obtained by removing 2 to 4 hydrogen atoms from an aromatic ring (an aryl group), a group obtained by removing 2 to 4 hydrogen atoms from a heterocycle (a heteroaryl group), or a combination thereof. As the aryl group and the heteroaryl group, the above-listed groups can be used.

[0134] In general formula (3), as Z 4 , specifically, the same groups as those listed in Z 1 in general formula (3) can be used. In general formula (3), as Z 4, preferably an alkylene group having 2 to 6 carbon atoms, -C2H4-O-C2H4-, -C2H4-O-C2H4-O-C2H4-, -C2H4-O-C2H4-O-C2H4-O-C2H4-, -C2H4-O-C2H4-O-C2H4-O-C2H4-O-C2H4-, -Ph-, -Ph-CH2-Ph-, -C(=O)-, -C(=O)-O-, -O-C(=O)-, -NH-C(=O)-O-, -O-C(=O)-NH-, -C(=O)-NH-, -NH-C(=O)-, -S(=O)2-NH-, -NH-S(=O)2-, -S(=O)2-NH-S(=O)2-, -C(=O)-NH-Ph-, more preferably an alkylene group having 2 to 6 carbon atoms, -C2H4-O-C2H4-, -C2H4-O-C2H4-O-C2H4-, -C2H4-O-C2H4-O-C2H4-O-C2H4-, or -C2H4-O-C2H4-O-C2H4-O-C2H4-O-C2H4-. Note that -Ph- is 1,4-phenylene, 1,3-phenylene, 1,5-phenylene, or 1,3,5-substituted phenylene.

[0135] In the artificial virus coat of the present application, the divalent linking group bonded to the group derived from a fluorine-containing compound can also be bonded to the N-terminal side of the β -ring peptide, but is preferably bonded to the C-terminal side of the β -ring peptide. In the artificial virus coat of the β -ring peptide which is originally a hollow nanocapsule, the C-terminus of the β -ring peptide faces the outside of the nanocapsule. Therefore, by connecting the group derived from a fluorine-containing compound to the C-terminal side of the β -ring peptide, it is possible to present the group derived from a fluorine-containing compound on the surface of the artificial virus coat, and it is possible to more efficiently improve the cell membrane permeability.

[0136] The artificial virus coat of the present application can be bonded to other substances at the terminal of the β -ring peptide which is not bonded to the above-described divalent linking group. As the other substances, there is no particular limitation as long as it does not hinder the formation of the artificial virus coat based on the self-assembly of the β -ring peptide, and peptides, nucleic acids, low molecular compounds, and chimeric molecules composed of two or more of them can be cited.

[0137] Since the artificial virus capsid of the present application has a group derived from a fluorine-containing compound on the surface, the cell membrane permeability is excellent. Therefore, the artificial virus capsid is expected to be utilized in the medical field as a physiologically active substance. For example, the artificial virus capsid of the present application is useful as a DDS carrier for delivering a target substance into a cell. For example, in the artificial virus capsid of the present application, the artificial virus capsid is formed by self-assembly in a state in which a target substance for delivery into a cell is linked to the N-terminal side of a β-cyclodextrin. The artificial virus capsid thus obtained has the target substance encapsulated therein, and the surface is modified with a group derived from a fluorine-containing compound, and therefore the target substance can be efficiently taken into a cell.

[0138] In addition, in transfection, the cytotoxicity of the transfection reagent used also affects the cell introduction efficiency of the target substance. A reagent having strong cytotoxicity greatly reduces the survival rate of the cell after introduction, and as a result, the introduction efficiency also becomes low. In contrast, the artificial virus capsid of the present application has low cytotoxicity. Therefore, for various cells, the target substance can be efficiently taken into a cell without excessively reducing the cell survival rate.

[0139] The artificial virus capsid of the present application can also encapsulate a target substance for delivery into a cell as a guest. For example, when the subunit of the artificial virus capsid of the present application is self-assembled in an aqueous medium, by dissolving or dispersing the target substance in the aqueous medium in advance, an artificial virus capsid having the target substance encapsulated therein can be obtained. The aqueous medium can be water, a buffer such as phosphate buffered saline (PBS), or a solution containing a small amount of an organic solvent in them.

[0140] As the target substance for delivery into a cell encapsulated in the artificial virus capsid of the present application, there is no particular limitation as long as it is a substance that can be encapsulated in the artificial virus capsid formed from a β-cyclodextrin, and for example, a substance for cell labeling such as a fluorescent substance, a quantum dot, a physiologically active substance such as a pharmaceutical ingredient can be mentioned. As the pharmaceutical ingredient encapsulated in the artificial virus capsid of the present application, any one of a peptide, a nucleic acid, a low molecular compound, and a chimeric molecule composed of two or more of them can be mentioned. As the nucleic acid, for example, a nucleic acid that facilitates RNA interference such as siRNA, a nucleic acid for gene recombination, genome editing can be mentioned. That is, the artificial virus capsid of the present application, by encapsulating a target substance, can be used as an effective ingredient of a DDS carrier composition.

[0141] The DDS carrier composition can be appropriately combined with a pharmaceutically acceptable carrier, an excipient, an additive, and the like, in addition to the artificial virus capsid. One example of the appropriate carrier is water, which can be an aqueous solution containing a salt (electrolyte) at a concentration suitable for administration to a human body, a mixed solvent containing an organic solvent such as alcohol in water. As the administration route, oral, intravenous, intra-arterial, intramuscular, subcutaneous, transdermal, intraperitoneal, intraspinal, transrectal, transvaginal, ocular, and inhalation can be given, but are not limited to these.

[0142] The artificial virus capsid of the present application can be used for the treatment of a disease in which a therapeutic effect can be obtained by a pharmacodynamic ingredient by encapsulating the pharmacodynamic ingredient. That is, the artificial virus capsid of the present application can be used as an effective ingredient of a pharmaceutical composition. A pharmaceutical composition can be prepared by encapsulating a therapeutically effective amount of a pharmacodynamic ingredient in the inside of the artificial virus capsid of the DDS carrier composition. The subject to be administered with the artificial virus capsid of the present application or a composition containing the same is typically an animal individual such as a mammalian individual, particularly a human individual, and an in-vitro tissue or a cultured cell of these animals can also be the subject to be administered.

[0143] Examples

[0144] Hereinafter, the present application will be described by way of examples, but the present application is not limited to these examples.

[0145] The NMR device used in the NMR analysis was JNM-ECZ400S (400 MHz) manufactured by JEOL Ltd., and the solvent was CDCl3. 1 In the1H NMR, tetramethylsilane was set as 0 PPM. 19 In the19F NMR, C6F6was set as a reference value of -162 PPM.

[0146] [Measurement of particle diameter by dynamic light scattering method (DLS)]

[0147] The particle diameter of the artificial virus capsid in each sample was measured by a dynamic light scattering method. The measurement was performed at 25°C using a dynamic light scattering device DLS (Zetasizer Nano ZS manufactured by MALVERN) equipped with an incident helium-neon laser (633 nm) and a low-volume glass cuvette (ZEN2112 manufactured by Malvern). In the measurement, the sample diffusion intensity was displayed, the correlation time of the scattering intensity G (τ) was measured 10 times, and the averaged value was substituted into the following formula (1).

[0148]

[0149] [B: baseline, A: amplitude, q: scattering vector, τ: delay time, D: diffusion coefficient]

[0150] Hydrodynamic radius (R HThe Stokes-Einstein formula represented by the following equation (2) can be used to calculate the diffusion coefficient.

[0151]

[0152] [η: solvent viscosity, k B : Boltzmann constant, T: absolute temperature]

[0153] [Observation using a transmission electron microscope (TEM)]

[0154] The observation of the artificial virus capsid in each sample was performed using a TEM (JEM 1400 Plus, manufactured by JEOL Ltd.). The TEM grid (Thin Carbon film TEM grids, manufactured by ALLANCE Biosystems) was subjected to hydrophilization treatment by plasma treatment (25°C, 60 Hz, 500 VA, 40 s, JEOL HD Treatment). After adding the sample aqueous solution (5 μL) to the TEM grid on which the hydrophilization treatment was completed and leaving it for 1 minute, it was removed. Next, a staining agent (5 μL, EM stainer, manufactured by Nisshin EM Co., Ltd.) was added to the TEM grid, and after leaving it for 1 minute, it was removed. After staining, the grid was subjected to reduced pressure drying, and observation was performed using a TEM at an acceleration voltage of 80 kV.

[0155] [Cell culture]

[0156] HepG2 cells were cultured with Dulbecco's modified Eagle's medium (DMEM). The entire culture medium contained 10 v / v% fetal bovine serum (FBS), 100 μg / mL streptomycin, 100 units / mL penicillin, 1 mM sodium pyruvate, and 1 v / v% MEM non-essential amino acids. The cells were maintained at 37°C in a 5% CO2 incubator, and subculturing was performed every 3 to 4 days.

[0157] [Example 1]

[0158] An artificial virus capsid was formed by self-assembly of a subunit in which a β-cyclopeptide was linked to a fluorine-containing peptide via a bismaleimide group, and the cell membrane permeability was investigated.

[0159] (1) Synthesis of β-Annulus-Cys Peptide

[0160] Fmoc-Ser-Alko-PEG resin (87 mg, 0.24 mmol / g; manufactured by Watanabe Chemical Industries, Ltd.) was elongated to the target amino acid sequence (H-Ile-Asn(Trt)-His(Trt)-Val-Gly-Gly-Thr(tBu)-Gly-Gly-Ala-Ile-Met-Ala-Pro-Val-Ala-Val-Thr(tBu)-Arg(Pbf)-Gln(Trt)-Leu-Val-Cys(Trt)-Ser(tBu)-Alko-PEG resin; SEQ ID NO: 2) by the Fmoc solid-phase synthesis method. Deprotection (removal of Fmoc) was performed by adding piperidine / DMF solution (piperidine:DMF = 40:60 (volume ratio)) 2 mL in a column equipped with the amino acid-introduced resin. In the column equipped with the resin, 4 equivalents of each of Fmoc-group-bearing amino acid / DMF solution, HBTU / DMF solution, HOBt·H2O / DMF solution, and DIPEA / NMP solution were added, and a condensation reaction was performed at room temperature for 2 hours. After deprotection (removal of Fmoc) was performed by adding piperidine / DMF solution in the peptide-bearing resin obtained by the reaction, washing with NMP was performed, and then, reduced pressure drying was performed. Next, a cleavage cocktail (a mixture of TFA / 1,2-ethanedithiol / trisopropylsilane / water / phenylthioether = 2.58 / 0.09 / 0.03 / 0.15 / 0.15 (mL)) was added, and a treatment was performed at room temperature for 3 hours to cleave the peptide from the resin and to deprotect the protecting groups of the amino acid side chains. The solution in which the peptide was recovered was moved to a centrifuge tube, about 15 mL of methyl tert-butyl ether (MTBE) was added, and the operation of precipitating by centrifugation (3500 rpm, 10 minutes) was repeated 3 times, and the recovered precipitate was freeze-dried, thereby obtaining a crude peptide as a white solid. The crude peptide was purified by reverse phase HPLC using a C18 column (Inertsil WP300 C18 column; manufactured by GL Science) (linear gradient of MeCN / 0.1% TFA aqueous solution (100 minutes, from 5 / 95 to 100 / 0)), and the molecular weight of the target (m / z = 2351, matrix: a-CHCA) was identified by MALDI-TOF-MS, thereby obtaining a purified β-Annulus-Cys peptide (theoretical yield: 26.3 mg, yield: 18.1 mg, yield: 69%). The reverse phase HPLC chart is shown in (A) of Figure 1 , and the results of MALDI-TOF-MS are shown in (B) of Figure 1 .

[0161] (2) Synthesis of TMR-β-Annulus-Cys Peptide

[0162] The β-Annulus-Cys peptide labeled with the fluorescent dye tetramethylrhodamine (TMR) (TMR-β-Annulus-Cys peptide) was synthesized as follows. First, the target amino acid sequence (H-Ile-Asn(Trt)-His(Trt)-Val-Gly-Gly-Thr(tBu)-Gly-Gly-Ala-Ile-Met-Ala-Pro-Val-Ala-Val-Thr(tBu)-Arg(Pbf)-Gln(Trt)-Leu-Val-Cys(Trt)-Ser(tBu)-Alko-PEG resin) was elongated by the same synthesis as described in (1) above, and Fmoc removal was performed. After Fmoc removal, 4 equivalents of TMR-COOH, 4 equivalents of COMU, 8 equivalents of DIPEA, and 2 to 3 mL of NMP were added to the resin, and the mixture was dissolved and stirred for 3 hours under light shielding to allow the reaction to proceed. After the reaction, the solvent was removed, and the mixture was washed with NMP and DCM five times. Peptide cleavage from the resin, deprotection of the amino acid side chain protecting groups, and recovery of the peptide powder were performed by the same method as described in (1) above, and a crude peptide in the form of a red powder was obtained as a result. The crude peptide was purified by reverse phase HPLC in the same manner as described in (1) above, and the molecular weight of the target substance was identified by MALDI-TOF-MS (m / z = 2763, matrix: α-CHCA), and thus purified TMR-β-Annulus-Cys peptide was obtained (theoretical yield: 5 mg, yield: 1 mg, yield: 20%). The reverse phase HPLC chart is shown in FIG. 10(A), and the results of MALDI-TOF-MS are shown in FIG. 10(B). Figure 2 Figure 2

[0163] (3) Synthesis of β-Annulus-PEG2-maleimide

[0164] ​​β-Annulus-PEG2-maleimide was synthesized by bonding a thiol group in the cysteine residue of the 2 amino acid residues from the C-terminal of the β-Annulus-Cys peptide with bismaleimide-PEG2 (1,8-bismaleimido-diethyleneglycol) according to the following method. First, bismaleimide-PEG2 (1.5 mg) was dissolved in acetonitrile (125 μL). Next, after dissolving β-Annulus-Cys peptide (1.2 mg) in ion exchange water (375 μL), it was added to the bismaleimide-PEG2 / MeCN solution and allowed to react by shaking at 25°C for 24 hours (final concentration: 1 mM β-Annulus-Cys peptide, 10 mM bismaleimide-PEG2). This reaction solution was purified by reverse phase HPLC as in (1) above, and the molecular weight of the target substance was identified by MALDI-TOF-MS (m / z = 2659, matrix: α-CHCA), whereby purified β-Annulus-PEG2-maleimide was obtained (theoretical yield: 1.33 mg, yield: 0.60 mg, yield: 45.1%). The reverse phase HPLC chart is shown in (A) of FIG. 10, and the results of MALDI-TOF-MS are shown in (B) of FIG. 10. Figure 3 Figure 3

[0165] (4) Synthesis of TMR-β-Annulus-PEG2-maleimide

[0166] ​​TMR-β-Annulus-PEG2-maleimide was synthesized by the following method, which involves bonding bismaleimide-PEG2 to the thiol group of a cysteine ​​residue in the two amino acid residues starting from the C-terminus of the TMR-β-Annulus-Cys peptide. First, DMSO (68 μL) was added to 0.2 mg of bismaleimide-PEG2 to dissolve it. Next, 612 μL of deionized water was added to 0.6 mg of TMR-β-Annulus-Cys peptide to dissolve it, and then this solution was added to the bismaleimide-PEG2 / DMSO solution. The mixture was allowed to react at 25°C with shaking for 12 hours (final concentration: 333 μM TMR-β-Annulus-Cys peptide, 1 mM bismaleimide-PEG2). The reaction solution was purified by reversed-phase HPLC as described in (1) above, and the molecular weight of the target analyte was identified by MALDI-TOF-MS (m / z = 3071, matrix: α-CHCA), thus yielding purified TMR-β-Annulus-PEG2-maleimide (theoretical yield: 166.5 nmol, yield: 14.24 nmol, percentage: 8.6%). The reversed-phase HPLC plot is shown in [image / image / data]. Figure 4 (A) shows the results of MALDI-TOF-MS. Figure 4 (B)

[0167] (5) CAD C8F17 F-peptide synthesis

[0168] CAD C8F17 F peptide (H-Cys-Ala-Asp(C8F)) 17 )-Phe-NH2) is synthesized as follows.

[0169] First, the synthesis involves using -Ph-C8F to remove one hydrogen atom from the amino group of the side chain of aspartic acid. 17 The amino acid Asp (C8F) is a group in which one hydrogen atom of a phenyl group is replaced by a perfluoroalkyl group with eight carbon atoms. 17 (D) C8F17 ).

[0170]

[0171] 4-(perfluorooctyl)aniline (compound (1)) was synthesized according to the method described in Org. Lett., 2019, 21, 6481.

[0172] In addition, in a dry 25 mL double-necked flask, 150 mg (0.3 mmol) of compound (1) and 130 mg (0.33 mmol, 1.1 equivalent) of Fmoc-Asp-OAll were dissolved in 6 mL of dichloromethane. 141 mg (0.33 mmol, 1.1 equivalent) of COMU, 47 mg (0.33 mmol, 1.1 equivalent) of oxyma, and 85.3 mg (0.66 mmol, 2.2 equivalent) of DIPEA were added, and the mixture was stirred at room temperature for 16 hours. The reaction mixture was then quenched with HCl (1N) and extracted three times with dichloromethane. The combined organic phases were concentrated under reduced pressure, diluted with ethyl acetate, and washed with HCl (1N), saturated sodium bicarbonate solution, and saturated brine. The washed organic phases were dried with sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product of compound (2a). The crude product was dissolved in acetone and then purified by reprecipitation with hexane to give pure compound (2a) (Fmoc-Asp(C8F17)-OAll) as a white solid (173 mg, 0.19 mmol). (Yield 64.9%)

[0173] Compound (2a):

[0174] 1 H NMR (400MHz, DMSO-D6) δ=7.87-7.77 (m, 5H), 7.65-7.57 (m, 4H), 7.37-7.22 (m, 4H), 5.82 (m, 1H), 5 .26(d,J=17.1Hz,1H),5.12(d,J=17.1Hz,1H),4.54(m,3H),4.29-4.18(m,3H),2.94-2.54(m,2H).

[0175] 19 F NMR (376MHz, DMSO-D6) δ=-80.0 (s, 3F), -108.8 (s, 2F), -121.0 (s, 2F), -121.6 (s, 6F), -122.3 (s, 2F), -125.7 (s, 2F).

[0176]

[0177] Tris(dibenzylacetone)dipalladium (0) (10 mol%) and phosphine (20 mol%) were added to a THF (2 mL) solution of compound (2a) (173 mg, 0.2 mmol) at 0 °C and stirred. Phenylsilane (2 equivalents) was then added, and the mixture was heated to room temperature and stirred for 2 hours. The reaction mixture was then quenched with HCl (1N) (10 mL) and extracted twice with dichloromethane. The organic phase was concentrated under reduced pressure, and the crude product was purified by silica gel column chromatography (chloroform:methanol = 8:1 (volume ratio)) to give a yellow solid (139 mg, 0.16 mmol, yield 82%) of Fmoc-Asp(C8F17)-OH (compound (2b)).

[0178] Compound (2b):

[0179] 1 H NMR (500MHz, DMSO-D6) δ=10.37 (s, 1H), 7.85-7.21 (m, 12H), 4.44 (brs, 1H), 4.52-4.22 (m, 3H), 2.90-2.68 (m, 2H).

[0180] 19 F NMR (376MHz, DMSO-D6) δ=-80.06 (s, 3F), -108.83 (s, 2F), -122.36~-120.96 (m, 10F), -125.41 (s, 2F).

[0181]

[0182] As condensed amino acids, Fmoc-Phe-OH and Fmoc-Asp (C8F) were used. 17 CAD was obtained from Fmoc solid-state synthesis methods using Fmoc-Ala-OH, Fmoc-Cys(Trt)-OH, and Fmoc-Cys(Trt)-OH. C8F17 F peptide (16 mg, 17 μmol, yield 22%).

[0183] H-Cys-Ala-Asp (C8F) 17 )-Phe-NH2:

[0184] LRMS (LC-MS) [M+H]+: m / z calcd for C 33 H 32 F 17 N6O5S + 947.69, found 947.54

[0185] (6) β-Annulus-CAD C8F17 F-peptide synthesis

[0186]

[0187] In using CAD C8F17 F peptide (7.1 mg) was dissolved in a MeCN / 10 mM NaHCO3 aqueous solution (MeCN / 10 mM NaHCO3 = 1 / 1 (volume ratio)) (750 μL). β-Annulus-PEG2-maleimide powder (2.0 mg) was then added to the solution, and the mixture was stirred at 25°C for 24 hours to allow the reaction to proceed (final concentration: 1 mM β-Annulus-PEG2-maleimide, 10 mM CAD). C8F17 F-peptide). The reaction solution was dialyzed against deion-exchanged water for 24 hours (Spectra / por7, cutoff Mw: 1000, SPECTRUM). Purification was then performed using reversed-phase HPLC (MeCN / 0.1% TFA aqueous solution linear gradient (100 min, from 5 / 95 to 100 / 0) on a C4 column (Inertsil WP300 C4 column; GL Science). The molecular weight of the target analyte was identified by MALDI-TOF-MS (m / z = 3604, matrix: α-CHCA), thus yielding purified β-Annulus-CAD. C8F17 F-peptide (theoretical yield: 2.7 mg, actual yield: 1.0 mg, yield: 37%). Reversed-phase HPLC illustration is shown in [image / image / data]. Figure 5 (A) shows the results of MALDI-TOF-MS. Figure 5 (B)

[0188] (7) TMR-β-Annulus-CAD C8F17 F-peptide synthesis

[0189] Make CAD C8F17 F-peptide (0.12 mg) was mixed with TMR-β-Annulus-PEG2-maleimide / DMSO solution (100 μL) and reacted at 25 °C for 24 hours with stirring (final concentration: 132 μM TMR-β-Annulus-PEG2-maleimide, 1320 μM CAD). C8F17F-peptide). The reaction solution was purified by reversed-phase HPLC (MeCN / 0.1% TFA aqueous solution) using a C4 column (Inertsil WP300 C4 column; GL Science) with a linear gradient (100 min, from 5 / 95 to 100 / 0) over a C4 column (MeCN / 0.1% TFA aqueous solution). The molecular weight of the target analyte was identified by MALDI-TOF-MS (m / z = 4017, matrix: α-CHCA), thus yielding purified TMR-β-Annulus-CAD. C8F17 F peptide (theoretical yield: 13.2 nmol, yield: 2.1 nmol, recovery rate: 15.6%). Reversed-phase HPLC plotted on... Figure 6 (A) shows the results of MALDI-TOF-MS. Figure 6 (B)

[0190] (8) TMR-CAD C8F17 F-peptide synthesis

[0191] In CAD C8F17 F peptide (0.0946 mg) was added to 67.6 μL of 1.48 mM TMR-maleimide / DMSO solution and 932.4 μL of 200 mM phosphate buffer (pH 7.4), and the reaction was carried out at 25 °C with stirring for 24 hours (final concentration: 100 μM CAD). C8F17 F peptide, 100 μM TMR-maleimide). The reaction solution was purified by reversed-phase HPLC as described in (7) above, and the molecular weight of the target analyte was identified by MALDI-TOF-MS (m / z = 1427, matrix: α-CHCA), thus obtaining purified TMR-CAD. C8F17 F peptide (theoretical yield: 100 nmol, yield: 8.49 nmol, recovery rate: 8.5%). Reversed-phase HPLC chromatogram is shown below. Figure 7 (A) shows the results of MALDI-TOF-MS. Figure 7 (B)

[0192] (9) Formation of fluorinated peptide-modified capsid

[0193] Make β-Annulus-CAD C8F17 F peptides undergo self-assembly to form a fluorinated peptide-modified capsid. Specifically, this is achieved by using 1 mM β-Annulus-CAD... C8F17 F peptide (5 μL) was mixed with DMSO solution (5 μL) and PBS (pH 7.4, 90 μL) and stirred for 30 seconds to form a capsid (final concentration: 50 μM β-Annulus-CAD). C8F17F peptide in 10% DMSO / PBS (pH 7.6) solution. Additionally, by using 1 mM β-Annulus-CAD... C8F17 F peptide (1 μL) was mixed with DMSO solution (9 μL) and PBS (pH 7.4, 90 μL) and stirred for 30 seconds to form a capsid (final concentration: 10 μM β-Annulus-CAD). C8F17 F peptide in 10% DMSO / PBS (pH 7.6) solution.

[0194] The obtained capsid was analyzed using DLS to determine the particle size distribution (number function distribution). (β-Annulus-CAD) C8F17 The number-average particle size of the fluorinated peptide-modified capsid formed in a 50 μM solution of F peptide was 97 ± 26 nm. Figure 8 (A)), in β-Annulus-CAD C8F17 The number-average particle size of the fluorinated peptide-modified capsid formed in a 10 μM solution of F peptide was 81 ± 76 nm. Figure 8 (B)). Additionally, in β-Annulus-CAD C8F17 TEM images of fluorinated peptide-modified capsids formed in a 50 μM solution of F peptide are shown below. Figure 9 .exist Figure 9 In the right figure, arrows indicate isolated, dispersed fluorinated peptide-modified capsids.

[0195] (10) Formation of TMR-labeled fluorinated peptide-modified capsid

[0196] Make TMR-β-Annulus-CAD C8F17 F peptides undergo self-assembly to form a TMR-labeled fluorinated peptide-modified capsid. Specifically, this is achieved by using 1 mM β-Annulus-CAD... C8F17 F peptide (5 μL) and 1 mM TMR-β-Annulus-CAD C8F17 F peptide (0.5 μL) was mixed with DMSO solution (5 μL) and PBS (pH 7.4, 90 μL) and stirred for 30 seconds to form a capsid (final concentration: 50 μM β-Annulus-CAD). C8F17 F peptide and 5μM TMR-β-Annulus-CAD C8F17 F peptide in 10% DMSO / PBS (pH 7.6) solution.

[0197] The obtained capsid was analyzed using DLS to determine its particle size distribution (number function distribution). The number-average particle size of the formed TMR-labeled fluorinated peptide-modified capsid was 209 ± 61 nm. Figure 10 ).

[0198] (11) Intracellular delivery of fluorinated peptide-modified capsids (part 1)

[0199] In a single-well glass-bottomed petri dish, at 2.0 × 10⁻⁶... 4 Add 100 μL of HepG2 cells per well and incubate at 37°C for 24 hours in a 5% CO2 incubator. Next, add a TMR-labeled fluorinated peptide-modified capsid (50 μM β-Annulus-CAD). C8F17 F peptide and 5μM TMR-β-Annulus-CAD C8F17 F peptide), TMR-labeled fluorinated peptides (50 μM β-Annulus and 5 μM TMR-CAD) C8F17 F peptide), or TMR-labeled capsid (50 μM CAD) C8F17 F peptide and 5μM TMR-CAD C8F17 100 μL of DMEM (containing 10% FBS) solution of F peptide was added and incubated at 37°C in a 5% CO2 incubator for 3 hours. After incubation, the culture medium was removed, cells were washed with PBS, and 80 μL of Hoechst 33342 (10 μg / mL) nuclear staining agent was added. The cells were then incubated at 25°C for 10 minutes. After incubation, the nuclear staining agent solution was removed, cells were washed with PBS, and 100 μL of DMEM (containing 10% FBS) was added. The cells were then observed using a confocal scanning microscope (CLSM, FLUOVIEW FV10i, Olympus). TMR detection was performed using the filters used for rhod-2 detection (Ex: 553 nm, Em: 577 nm, sensitivity: 50%, laser intensity: 15%), and Hoechst 33342 detection was performed using a blue fluorescence detection filter (Ex: 352 nm, Em: 455 nm, sensitivity: 50%, laser intensity: 15%). The fluorescence intensity of TMR from capsids or peptides introduced into HepG2 cells was obtained by subtracting the fluorescence intensity outside the cell from the fluorescence intensity inside the cell using ImageJ image analysis software (N=60).

[0200] The TMR fluorescence images of each cell, the combined image of the TMR fluorescence image and the Hoechst 33342 fluorescence image, and the transmitted light image are shown below, all captured by confocal scanning microscopy. Figure 11 . Figure 11 The top section shows cells with TMR-labeled fluorinated peptide-modified capsids, the middle section shows cells with TMR-labeled fluorinated peptide-modified capsids, and the bottom section shows cells with TMR-labeled capsids. Additionally, the results of measuring the TMR fluorescence intensity of each cell type are shown below. Figure 12 . Figure 12In the middle column, the left column shows cells with capsids modified with TMR-labeled fluorinated peptides, the middle column shows cells with capsids modified with TMR-labeled fluorinated peptides, and the right column shows cells with capsids modified with TMR-labeled peptides.

[0201] like Figure 11 As shown, red fluorescence of TMR was observed intracellularly in cells with capsids modified with TMR-labeled fluorinated peptides and in cells with capsids modified with TMR-labeled fluorinated peptides, confirming their intracellular uptake. Strong TMR fluorescence was observed particularly in cells that took up the TMR-labeled fluorinated peptides, thus indicating that the fluorinated peptides (CAD) were indeed taken up by the cells. C8F17 F-peptides themselves possess cell membrane permeability. On the other hand, no TMR fluorescence was observed within cells with TMR-labeled capsids, indicating that the capsid without TMR-labeled peptide modification was almost entirely taken up into the cell. Modifying the surface with TMR-labeled peptides significantly improved cell membrane permeability. In the TMR fluorescence intensity measurements obtained through image analysis, although the uptake was lower in cells with TMR-labeled fluorinated capsids compared to cells without TMR-labeled fluorinated capsids, a significant increase in uptake was confirmed compared to cells with TMR-labeled capsids.

[0202] (12) Intracellular delivery of fluorinated peptide-modified capsids (part 2)

[0203] HepG2 cells were divided into 3.0 × 10⁻⁶ cells. 5 500 μL of the culture was seeded into each well of a 24-well plate using a 1-to-2 method and incubated in a CO2 incubator for 48 hours. Next, a TMR-labeled fluorinated peptide-modified capsid (50 μM β-Annulus-CAD) was added. C8F17 F peptide and 5μM TMR-β-Annulus-CAD C8F17 F-peptide) solution, TMR-labeled fluorinated peptides (50 μM β-Annulus and 5 μM TMR-CAD) C8F17 F-peptide) solution, or TMR-labeled capsid (50 μM CAD) C8F17 F peptide and 5μM TMR-CAD C8F17500 μL of FACS (F-peptide) solution was incubated at 37°C in a 5% CO2 incubator for 3 hours. After incubation, the culture medium was removed, and cells were detached from the culture plate by trypsin treatment and collected into 1.5 mL plastic tubes to obtain a cell suspension. The obtained cell suspension was centrifuged (800 g, 10 min, 4°C) to remove the supernatant. Subsequently, 500 μL of ice-cold FACS buffer (PBS (-) containing 2% FBS) was added to resuspend the cells, and centrifugation was performed under the same conditions to remove the supernatant. This operation was repeated twice. After removing the supernatant, 500 μL of FACS buffer was added to resuspend the cells. The resulting cell suspension was subjected to flow cytometry three times (detection wavelength: 577 nm, Gallios, Beckman Coulter). As a control, untreated cells were also subjected to flow cytometry.

[0204] For TMR-labeled fluorinated peptide-modified capsid solutions, 2.5 μL of β-Annulus-CAD was added. C8F17 F-peptide / DMSO stock solution (10 mM), 0.25 μL of TMR-β-Annulus-CAD C8F17 F-peptide / DMSO stock solution (10 mM) and 122.25 μL of PBS (pH 7.4) were mixed in a 1.5 mL plastic tube. The resulting solution was dialyzed for 12 hours (Dialysis tube, cutoff Mw: 1000 (1 kDa)) to remove DMSO. Subsequently, 375 μL of DMEM (containing 10% FBS) was added to prepare a TMR-labeled fluorinated peptide-modified capsid solution. The TMR-labeled fluorinated peptide solution and the TMR-labeled capsid solution were prepared by dialyzing in the same manner.

[0205] The scatter plot (left) and histogram (right) obtained by flow cytometry are shown below. Figure 13 . Figure 13 (A) represents the result for untreated cells. Figure 13 (B) shows the results of introducing TMR-labeled fluorinated peptides into the capsid of cells. Figure 13 (C) represents the results of introducing TMR-labeled fluorinated peptides into cells. Figure 13 (D) represents the results for cells inoculated with TMR-labeled capsids. The histogram is calculated from the portion enclosed by the elliptical boxes within the scatter plot. Additionally, the percentage (%) of cells with TMR uptake in each sample is shown in the figure. Figure 14 (A) shows the median value of the TMR fluorescence intensity of each cell. Figure 14 (B)

[0206] like Figure 13 As shown, firstly, in the scatter plot of all samples, normal cell populations were identified ( Figure 13The portion enclosed by ellipses in the scatter plot on the left). A histogram of fluorescence intensity was constructed for this normal cell population. The results showed that cells with TMR-labeled fluorinated peptide-modified capsids or cells with TMR-labeled fluorinated peptides showed higher intracellular fluorescence compared to untreated cells or cells with TMR-labeled capsids. Figure 13 (Right). For example Figure 14 As shown, in cells with capsids modified with TMR-labeled fluorinated peptides, approximately 90% of the cells took up the TMR-labeled fluorinated peptides. This confirmed that although the intracellular fluorescence intensity was lower than in cells with TMR-labeled fluorinated peptides, it was significantly higher than in cells with TMR-labeled capsids. These results also indicate that modifying the capsid surface with fluorinated peptides increases cell membrane permeability and improves intracellular delivery efficiency.

[0207] [Example 2]

[0208] The drug was encapsulated within a fluorinated peptide-modified capsid, and its intracellular delivery efficiency was investigated. β-Annulus-CAD, synthesized in Example 1, was used as the subunit of the fluorinated peptide-modified capsid. C8F17 F-peptide. Doxorubicin (Dox) was used as the pharmaceutical agent. In addition, as a comparative example, the β-Annulus-Cys peptide synthesized in Example 1 was used as the subunit for forming the capsid that was not modified with the fluorinated peptide.

[0209] (1) Formation of a fluorinated peptide-modified capsid containing Dox

[0210] Mix 10 mM Dox / DMSO solution (1 μL) and 10 mM β-Annulus-CAD C8F17 After mixing F-peptide / DMSO solution (1 μL), add PBS (pH 7.4, 98 μL) and stir for 30 seconds (final concentration: 100 μM Dox, 100 μM β-Annulus-CAD). C8F17 F-peptide). The resulting solution was dialyzed for 12 hours (Dialysis tube, cutoff Mw: 1000 (1 kDa)) to remove free Dox and DMSO, yielding a fluorinated peptide-modified capsid containing Dox. The Dox concentration after encapsulation (Dox binding rate (%)) was calculated by UV-vis spectroscopy of the dialyzed solution using a Nanodrop (Thermo Scientific) micro-spectrophotometer (εmolar extinction coefficient of Dox). 480 =11500M -1 cm -1 (Non-Patent Literature 5). Using the β-Annulus-Cys peptide, a fluorinated peptide-unmodified capsid containing Dox was also obtained without modification by the fluorinated peptide.

[0211] The fluorinated peptide-modified capsid containing Dox was analyzed using DLS to determine the particle size distribution (number function distribution). The number-average particle size was 78 ± 26 nm. Figure 15 Additionally, TEM images of the fluorinated peptide-modified capsid containing Dox are shown below. Figure 16 .like Figure 15 and 16 As shown, this indicates that even with an inner Dox, β-Annulus-CAD C8F17 F peptides also stably self-assemble to form a capsid.

[0212] Furthermore, the results obtained by measuring the UV-vis spectra and Dox binding rate (%) of the fluorinated peptide-modified capsid containing Dox and the unmodified capsid containing Dox are presented in the figure. Figure 17 The UV-vis measurements before and after dialysis were compared, and the Dox binding rate of each sample was calculated. The results showed that the Dox binding rate of the fluorinated peptide-modified capsid containing Dox was 41%, while the Dox binding rate of the unmodified fluorinated peptide capsid containing Dox was 14%. These results confirm that the fluorinated peptide-modified capsid is useful as a DDS carrier for encapsulating Dox and other drugs.

[0213] (2) Cytotoxicity evaluation of the fluorinated peptide-modified capsid containing Dox

[0214] The cytotoxicity of the fluorinated peptide-modified capsid containing Dox prepared in (1) above and the fluorinated peptide-unmodified capsid containing Dox was investigated using the WST-assay. The investigation also investigated whether Dox taken up into cells by modifying the capsid with fluorinated peptide exerted its pharmacological effect.

[0215] First, the dialysis-prepared Dox-containing fluorinated peptide-modified capsid solution and the Dox-containing unmodified capsid solution prepared in (1) were diluted 4 times with DMEM (+) to prepare capsid dilution solutions.

[0216] HepG2 cells were divided into 2.0 × 10⁻⁶ cells. 4100 μL of the above-mentioned capsid dilution solution was seeded into each well of a 96-well culture plate at a rate of 100 cells / well, and cultured at 37°C for 24 hours in a CO2 incubator. Next, after removing the culture medium, 100 μL of the capsid dilution solution was added to each well, and the plates were cultured at 37°C for 24 hours in a CO2 incubator. After culture, the culture supernatant was removed, and a viability assay solution (a mixture of 10 μL of Cell Counting Kit diluted with 8 μL of culture medium) was added, and the plates were cultured for 4 hours. Subsequently, the supernatant was recovered and diluted 10-fold, and UV-Vis spectroscopy was performed using a Nanodrop instrument. Cell viability (N=3) was calculated from the absorbance at 460 nm. As a comparison, solutions containing only Dox dissolved in DMEM(+) or Dox and the CAD synthesized in Example 1 were used. C8F17 Cell viability was also calculated for cells cultured in DMEM(+) solution with F peptide dissolved in it. The Dox concentration for all samples was standardized to 0.1 μM for comparison. The cell viability (%) results are shown below. Figure 18 .

[0217] Based on cytotoxicity evaluation, cells that were only introduced with Dox monomer (labeled "Dox" in the figure) served as a control, while cells introduced with both Dox and CAD were also included. C8F17 Cells containing F peptide (labeled as "2.4 μM CAD" in the figure) C8F17 Cells with capsids modified with fluorinated peptides containing Dox (labeled "7.1 μM β-Annulus+Dox") showed survival rates exceeding 85%, with almost no toxicity observed. Conversely, cells with capsids modified with fluorinated peptides containing Dox (labeled "2.4 μM β-Annulus-CAD") exhibited high survival rates. C8F17 The cell survival rate of "F+Dox") was 65%, which was low, indicating cytotoxicity. Figure 18 Based on these results, it was confirmed that even at low Dox concentrations of 0.1 μM, encapsulation in a fluorinated peptide-modified capsid still exhibits the cytotoxicity induced by Dox. These results indicate that the fluorinated peptide-modified capsid is useful as a delivery carrier for anticancer agents.

[0218] [Example 3]

[0219] To investigate the intracellular uptake pathway of fluorinated peptide-modified capsids, intracellular uptake of fluorinated peptide-modified capsids was performed in the presence of endocytosis inhibitors. As the fluorinated peptide-modified capsid, the TMR-labeled fluorinated peptide-modified capsid (50 μM β-Annulus-CAD) prepared in Example 1 was used. C8F17 F peptide and 5μM TMR-β-Annulus-CAD C8F17 F peptide).

[0220] First, in a single-well glass-bottomed petri dish, the culture medium was prepared to a density of 2.0 × 10⁻⁶. 4 Add 100 μL of HepG2 cells per well and incubate at 37°C in a 5% CO2 incubator for 24 hours. Next, add 60 μL of DMEM (containing 10% FBS) to each well, containing 80 μM EIPA (5-(N-ethyl-N-isopropyl)-amiloride, a macropinocytosis inhibitor), 20 μM Pitstop2 (N-[5-(4-bromobenzyl)-4-oxo-4,5-dihydro-1,3-thiazo-2-yl]naphthalene-1-sulfonamide, a clathrin-dependent endocytosis inhibitor), and 160 μM genistein (5,7-dihydroxy-3-(4-hydroxyphenyl)-4H-1-benzopyran-4-one, a cavitation-dependent endocytosis inhibitor). Incubate at 37°C in a 5% CO2 incubator for 30 minutes. Afterward, remove the culture medium and wash the cells with PBS(-). Next, each well was added with an inhibitor containing 80 μM EIPA, 20 μM Pitstop2, or 160 μM genistein, along with a TMR-labeled fluorinated peptide-modified capsid (50 μM β-Annulus-CAD). C8F17 F peptide and 5μM TMR-β-Annulus-CAD C8F17 60 μL each of DMEM containing DMSO (containing 10% FBS and 5% DMSO) was incubated at 37°C in a 5% CO2 incubator for 3 hours. As a control, DMEM containing only TMR-labeled fluorinated peptide-modified capsids and containing DMSO (containing 10% FBS and 5% DMSO) was used as the culture medium, and the cells were similarly incubated at 37°C in a 5% CO2 incubator for 3 hours. After incubation, the culture medium was removed, the cells were washed with PBS, and 80 μL of Hoechst 33342 (10 μg / mL) was added, and the cells were incubated at 25°C for 10 minutes. After incubation, the cells were washed with PBS as in Example 1, stained with Hoechst 33342, and observed using a confocal scanning microscope to determine the fluorescence intensity of the TMR from the capsid introduced into HepG2 cells.

[0221] The TMR fluorescence images of each cell, captured using a confocal scanning microscope, are shown below. Figure 19 In addition, the results of intracellular TMR fluorescence intensity measurements in each cell will be shown in [the table / document / etc.]. Figure 20 The p-value of TMR fluorescence intensity was calculated using the Kruskal-Wallis test (Dunn test). Figure 19 and 20As shown, a significant reduction in intracellular red fluorescence was confirmed in cells cultured in a medium containing the membrane-dependent endocytosis inhibitor genistein and a TMR-labeled fluorinated peptide-modified capsid. These results suggest that the fluorinated peptide-modified capsid is primarily taken up into the cell via membrane-dependent endocytosis.

[0222] [Example 4]

[0223] Make β-Annulus-CAD C6F13 F peptides self-assemble to form CAD. C6F13 F peptide was used to modify the capsid of an artificial virus to investigate cell membrane permeability.

[0224] (1) β-Annulus-CAD C6F13 F-peptide synthesis

[0225]

[0226] Add 2.5 μL of 10 mM β-Annulus-PEG2-maleimide DMF solution and 10 mM CAD C6F13 A 2.5 μL solution of the F peptide in DMF and 35 μL of DMF were mixed and reacted at 25 °C for 24 hours with stirring. After distilling off the DMF using a Smart Evaporator (BioChromato), the solution was dissolved in 50 μL of trifluoroethanol and purified using reversed-phase HPLC (MeCN / 0.1% TFA aqueous solution) on a C4 column (Inertsil WP300 C4 column (GL Science) with a linear gradient (100 min, from 5 / 95 to 100 / 0) over a 0.1% TFA aqueous solution). The molecular weight of the target analyte was determined by ESI-MS (m / z = 877.1357, Calcd. [M+4H]). 4+ = 877.1362), thus obtaining purified β-Annulus-CAD. C6F13 F-peptide (theoretical yield: 1.75 mg, actual yield: 0.2 mg, recovery rate: 27%). Reversed-phase HPLC plotted on... Figure 21 (A) shows the ESI-MS results. Figure 21 (B)

[0227] (2) Preparation and intracellular introduction of artificial viral capsids containing mRNA

[0228]

[0229] dT 20The β-Annulus peptide conjugate was prepared using the method described in the literature (Applied Sciences, 2020, 10, 8004). 500 μM β-Annulus-CAD was added to a microcentrifuge tube. C6F13 1 μL of F peptide aqueous solution, 500 μM; 1.28 μL of unmodified β-Annulus peptide aqueous solution, 10 μM dT 20 11 μL of SS-β-Annulus aqueous solution was freeze-dried. 20 μL of 1.1 μM mCherry mRNA (OZBIOSCIENCES) dissolved in nuclease-free water was added to the peptide powder and slowly pipetted. Subsequently, the mixture was incubated at 37°C for 30 minutes to prepare CAD. C6F13 F peptide-modified artificial viral capsids containing mRNA.

[0230] In a culture dish containing HepG2 cells (cell density: 4.0 × 10⁶ cells / year) 4 50 μL of the prepared sample was seeded into each of the following solutions (cells / 100 μL) and incubated at 37°C and 5% CO2 for 3 hours. After removing the solvent and washing with PBS, 80 μL of 1 μg / mL Hoechst 33342 in DMEM medium (containing 10% FBS) was added, and the mixture was incubated at 37°C and 5% CO2 for 10 minutes. After removing the solvent and washing with PBS, 100 μL of DMEM medium (containing 10% FBS) was added, and the mixture was observed using a confocal scanning microscope (CLSM, FLUOVIEW FV10i, Olympus). The detection of mCherry expression in HepG2 cells was performed using Ex: 584 nm, Em: 610 nm, sensitivity: 50%, laser intensity: 15%. The detection of Hoechst 33342 was performed using Ex: 352 nm, Em: 455 nm, sensitivity: 50%, laser intensity: 15%. The fluorescence intensity of mCherry expressed in HepG2 cells was obtained by subtracting the fluorescence intensity outside the cell from the fluorescence intensity inside the cell using ImageJ image analysis software (N=60).

[0231] Images of Hoechst 33342 fluorescence, mCherry fluorescence, a combined image of mCherry and Hoechst 33342 fluorescence, and transmitted light images of each cell, taken using confocal scanning microscopy, are shown below. Figure 22 In addition, the results of measuring the mCherry fluorescence intensity of each cell are shown in... Figure 23 . Figure 23In the middle column, the left column shows cells transfected with mCherry mRNA using a transfection reagent (Lipofectamine MessengerMAX), and the middle column shows cells transfected using CAD. C6F13 Cells with F-peptide-modified artificial viral capsids used to introduce mCherry mRNA are shown in the right column, while cells with unmodified artificial viral capsids used to introduce mCherry mRNA are shown in the left column.

[0232] like Figure 22 As shown, for the use of transfection reagents and CAD C6F13 F-peptide-modified artificial viral capsids were introduced into cells containing mCherry mRNA. Red fluorescence of mCherry was observed inside the cells, confirming that the virus was taken up into the cells and that mCherry was expressed.

[0233] In addition, such as Figure 23 As shown, although using CAD C6F13 The fluorescence intensity of the artificial viral capsid modified with F peptide introduced into mCherry mRNA was slightly lower than that using the transfection reagent, but still showed almost the same level of fluorescence intensity. Furthermore, compared to the fluorescence intensity when introducing mCherry mRNA using the unmodified artificial viral capsid, it showed approximately 5 times the fluorescence intensity. Based on these results, it is confirmed that modifying the artificial viral capsid with CAD... C6F13 F can fully deliver mRNA into the cell and express it.

Claims

1. An artificial viral capsid, formed by the self-assembly of multiple subunits. The subunit contains a β-Annulus peptide of tomato dwarf virus, a group derived from a fluorine-containing compound, and a divalent linker connecting the β-Annulus peptide and the group derived from the fluorine-containing compound. The divalent linker is attached to the C-terminus of the β-cyclic peptide.

2. The artificial virus capsid according to claim 1, wherein, The fluorinated compound is a fluorinated peptide.

3. The artificial virus capsid according to claim 2, wherein, At least one side chain of the amino acid residue constituting the fluorinated peptide is a group represented by the following general formula (1). In equation (1), Z 1 It is a divalent, trivalent, or tetravalent linking group other than an alkylene group; Rf is a C group substituted with at least two fluorine atoms. 1-30 Alkyl, -SF5, or -SF4-CR 101 R 102 -CR 103 R 104 Cl, where the C 1-30 When an alkyl group has two or more carbon atoms, it can have 1 to 5 oxygen atoms with ether-like bonds between the carbon atoms, R 101 R 102 R 103 and R 104 Each can be independently a hydrogen atom, a fluorine atom, or a chlorine atom, but R 101 R 102 R 103 and R 104 Two or more of them are fluorine atoms; n3 is 1, 2 or 3; n4 is 0 or 1; black dots represent bonding sites.

4. The artificial virus capsid according to claim 3, wherein, The Rf is a group represented by the following general formula (f-1) or (f-2). In equation (f-1) or (f-2), Rf P This indicates a fully halogenated C containing at least two fluorine atoms. 1-10 Alkyl, wherein the fully halogenated C 1-10 When an alkyl group has 2 or more carbon atoms, it can have oxygen atoms with ether-like bonds between carbon atoms; n1 is an integer from 0 to 10; n2 is an integer from 0 to 9; black dots represent bonding sites.

5. The artificial virus capsid according to claim 3, wherein, The group represented by the general formula (1) is an amino acid residue with a side chain that has 1 to 3 amino acid residues of the Rf directly or indirectly attached to the side chain of a natural amino acid.

6. The artificial virus capsid according to claim 1, wherein, The divalent linker is a bismaleimide group containing maleimide groups at both ends.

7. The artificial virus capsid according to claim 1, wherein, The β-cyclic peptide has a cysteine ​​residue at or near its C-terminus, and the divalent linker is linked to a thiol group from that cysteine ​​residue.

8. A pharmaceutical composition comprising an artificial viral capsid as described in any one of claims 1 to 7.

9. A drug delivery carrier composition comprising an artificial viral capsid according to any one of claims 1 to 7.

Images