Peptide cube

Abstract

To provide a nano-sized hollow cube material having shearing stress responsiveness.SOLUTION: Provided are a peptide cube in which a wall surface is constituted of a co-aggregate of a curved surface membrane made of a first amphiphatic polypeptide and a flat membrane made of a second amphiphatic polypeptide, a release-controlling composition which is made of the peptide cube, includes active ingredients in the peptide cube, and in which the included active ingredients are released by collapse of the cube shape caused by shearing stress loaded to the peptide cube, or the like.SELECTED DRAWING: Figure 2

Description

[Technical field]

[0001] The present invention relates to a peptide cube, the wall of which is formed by a co-assembly of a curved membrane made of a first amphipathic polypeptide and a planar membrane made of a second amphipathic polypeptide; and a composition for controlled release comprising the peptide cube, which contains an active ingredient and releases the encapsulated active ingredient when the cube shape collapses due to a shear stress applied to the peptide cube, and is useful in the fields of, for example, pharmaceuticals, cosmetics, detergents, foods, chemicals, functional materials, etc. [Background technology]

[0002] In recent years, research on nano-sized hollow objects has been actively conducted, and applications in various fields are expected. For example, as one method for treating vascular stenosis and imaging, a drug delivery system (DDS) using cubic materials that deform in response to shear stress and leak encapsulated substances has been proposed. There are two known reports on nano-sized hollow bodies with shear stress responsiveness (Non-Patent Documents 1 and 2). Non-Patent Document 1 describes shear stress responsive polymersomes. The polymersomes use unstable interactions between the constituent polymer molecules, and the interactions are broken by shear stress, destabilizing the polymersomes and leaking the contents. However, it is expected that it is very difficult to control the responsiveness. In addition, the reported size is as large as 400 nm, making it unsuitable for DDS, which is considered to be suitable for 10-200 nm, which is difficult to capture by the immune system. Non-Patent Document 2 describes shear stress responsive saddle-shaped liposomes. However, since it is a liposome, it is thought that the mechanical strength of the material is low, and the stability before administration to the subject and the long-term stability are questionable. The outline of Non-Patent Documents 1 and 2 is as above, but the shear stress responsive capsules described in these two documents do not have a cubic shape. There have been almost no examples of the development of nano-sized hollow cubic materials, and it was difficult to prepare them stably in the first place. Therefore, research and development of nano-sized hollow cubic materials that are responsive to shear stress has been eagerly awaited.

[0003] Patent Document 1 describes a nanostructure surrounded by walls composed of a plurality of amphiphilic molecules including a hydrophilic block and a hydrophobic block composed of peptides. Non-Patent Document 3 describes a hollow nanostructure composed of peptides that can encapsulate an anticancer drug. Non-Patent Document 4 describes a cubic vesicle consisting of two types of amphiphilic polypeptides. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] JP 2018-193308 A [Non-patent literature]

[0005] [Non-Patent Document 1] Angew. Chem. Int. Ed. 2021, 60, 904-909 [Non-Patent Document 2] Nat. Nanotech. 2012, 7, 536-543 [Non-Patent Document 3] ACS Nano 2019, 13, 305-312 [Non-Patent Document 4] Proceedings of the Society of Polymer Science, Vol. 71, No. 1, Presentation No. 2F04 Summary of the Invention [Problem to be solved by the invention]

[0006] In order to solve the above problems, the present invention aims to provide a peptide cube having a wall surface formed by a co-assembly of a curved membrane and a flat membrane; and a composition for controlled release comprising the peptide cube containing an active ingredient. [Means for solving the problem]

[0007] The inventors discovered that stable cubic hollow objects can be prepared by co-assembling an amphiphilic polypeptide that forms a curved sheet membrane with an amphiphilic polypeptide that forms a rigid, planar sheet membrane, and as a result of intensive research based on this novel finding, they have completed the present invention. Specific embodiments of the present invention are as follows, but the present invention is not limited to these.

[0008] [1] A peptide cube having a wall formed by co-assembly of a curved membrane made of a first amphipathic polypeptide and a planar membrane made of a second amphipathic polypeptide; The active ingredient is contained within the peptide cube. The peptide cube is collapsed by shear stress applied to the cube, and the encapsulated active ingredient is released. Controlled release compositions. [2] The shear stress applied to the peptide cube at which the shape begins to collapse is 5 to 20 Pa as evaluated by a rheometer. The composition described in [1] above. [3] The first amphipathic polypeptide has the following formula (I):

[0009] [ka]

[0010] (In the formula, R 1 is a hydrogen atom, C 1-6 represents an alkyl group or an aralkyl group, R 2 is a hydrogen atom or a C which may be substituted by a hydroxyl group. 1-4 C optionally substituted with an alkanoyl group or an aryl group 1-4 represents an alkoxycarbonyl group, m is an integer from 16 to 47, and (n is an integer between 5 and 10) A polypeptide represented by the formula: The second amphipathic polypeptide has the following formula (II):

[0011] [ka]

[0012] (In the formula, R 3 and R 4 each independently represents a hydrogen atom or a C which may be substituted by a hydroxyl group; 1-4 C optionally substituted with an alkanoyl group or an aryl group 1-4 represents an alkoxycarbonyl group, Q is NH or NH(CH 2 ) r NH (wherein r is an integer of 1 to 3), o and q each independently represent an integer of 6 to 15, and p represents an integer of 5 to 10. A polypeptide represented by the formula: The composition described in [1] or [2] above. [4] The composition according to any one of the above [1] to [3], wherein the active ingredient is an active ingredient used in any one selected from pharmaceuticals, cosmetics, detergents, foods, chemicals and functional materials. [5] The composition described in [4] above, wherein the active ingredient is an active ingredient used in pharmaceuticals. [6] The composition described in [5] above, wherein the pharmaceutical agent is a drug for improving vascular stenosis or vascular occlusion, or an anticancer drug.

[0013] [7] A peptide cube comprising a co-assembly of a curved membrane made of a first amphipathic polypeptide and a planar membrane made of a second amphipathic polypeptide, The first amphipathic polypeptide has the following formula (I):

[0014] [ka]

[0015] (In the formula, R 1 is a hydrogen atom, C 1-6 represents an alkyl group or an aralkyl group, R 2 is a hydrogen atom or a C which may be substituted by a hydroxyl group. 1-4 C optionally substituted with an alkanoyl group or an aryl group 1-4 represents an alkoxycarbonyl group, m is an integer from 16 to 47, and (n is an integer between 5 and 10) A polypeptide represented by the formula: The second amphipathic polypeptide has the following formula (II):

[0016] [ka]

[0017] (In the formula, R 3 and R 4 each independently represents a hydrogen atom or a C which may be substituted by a hydroxyl group; 1-4 C optionally substituted with an alkanoyl group or an aryl group 1-4 represents an alkoxycarbonyl group, Q is NH or NH(CH 2 ) r NH (wherein r is an integer of 1 to 3), o and q each independently represent an integer of 6 to 15, and p represents an integer of 5 to 10. A peptide cube, which is a polypeptide represented by the formula: [8] m is an integer between 20 and 30; n is an integer from 6 to 8; o and q are each an integer of 8 to 13; p is an integer from 5 to 7; The peptide cube according to [7] above. [9] m is 25 or 26; n is an integer from 6 to 8; o and q are each 10; p is 6 or 7; The peptide cube according to [7] above.

[10] m is 25 and n is 6, or m is 26 and n is 7; o and q are each 10; p is 6; The peptide cube according to [7] above.

[11] The peptide cube according to any one of [7] to

[10] above, wherein r is 2.

[12] The mixing ratio (molar ratio) of the first amphipathic polypeptide to the second amphipathic polypeptide is within the range of 1:1 to 1:8; The peptide cube according to any one of [7] to

[11] above.

[13] The length of one side is 10 to 300 nm. The peptide cube according to any one of the above [7] to

[12] .

[0018] (*) In the peptide cube constituting the composition for controlled release described in any one of the above [1] to [6], reference can be made to any one of the above [7] to

[13] as a preferred embodiment. Effect of the Invention

[0019] The present invention provides, as an embodiment thereof, a peptide cube capable of encapsulating a variety of ingredients and expected to be utilized in a wide range of fields, the wall of which is formed by a co-assembly of a curved membrane made of a first amphipathic polypeptide and a planar membrane made of a second amphipathic polypeptide; and a composition for controlled release comprising the peptide cube, which encapsulates an active ingredient within the peptide cube, and which is characterized in that the encapsulated active ingredient is released when the cube shape is collapsed due to shear stress applied to the peptide cube. [Brief description of the drawings]

[0020] [Figure 1]1 shows the results of TEM observation of the peptide cube (mixed molar ratio = 1:1) produced in Example 1 (2) described below. The left image shows a TEM image, and the right image shows a Cryo-TEM image. [Diagram 2] FIG. 2 shows a TEM image of the peptide cube (mixed molar ratio=1:2) produced in Example 1(2) described below. [Diagram 3] FIG. 3 shows a TEM image of the aggregates derived from S26L14 produced in Example 1(3) described below. [Figure 4] FIG. 4 shows a TEM image of the aggregates derived from S10L12S10 produced in Example 1(3) described below. [Diagram 5] FIG. 5 shows a TEM image of the co-assembly resulting from the combination of S16L14 and S10L12S10 produced in Example 1(4) below. [Figure 6] FIG. 6 shows a TEM image of the co-assembly resulting from the combination of S37L14 and S10L12S10 produced in Example 1(4) below. [Figure 7] FIG. 7 shows a TEM image of the co-assembly resulting from the combination of S47L14 and S10L12S10 produced in Example 1(4) below. [Figure 8] FIG. 8 shows a TEM image of the co-assembly resulting from the combination of S26L14 and S13L12S13 produced in Example 1(4) below. [Figure 9] 9 shows the results of evaluating the stability of the peptide cubes (mixed molar ratio = 1:1) produced in Example 1 (2) described below. The left image shows a TEM image on "0 days of storage", and the right image shows a TEM image on "60 days of storage". [Figure 10] FIG. 10 shows a TEM image visualizing the localization of S26L14 in a peptide cube, obtained in Example 3 described below. [Figure 11] 11 shows the observation results of the shape change of a peptide cube after loading a shear stress of 1 Pa in Example 5 described below. The left image shows the shape after loading for 20 minutes, and the right image shows the shape after loading for 2 hours. [Figure 12]12 shows the observation results of the shape change of a peptide cube after loading a shear stress of 10 Pa in Example 5 described below. The left image shows the shape after loading for 20 minutes, and the right image shows the shape after loading for 2 hours. [Figure 13] FIG. 13 shows the results of the evaluation of the release of the active ingredient by the shear stress response in Example 6 described later. FIG. 13 shows an outline of the test. FIG. 13 shows TEM images of the peptide cube before and after 20 minutes of pipetting, respectively. FIG. 13 shows the time course of calcein release from the peptide cube with and without pipetting. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0021] The present invention is described in detail below. Unless otherwise specified in the text, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described below. All publications and patents mentioned herein are incorporated by reference for the purpose of describing and disclosing, for example, the constructs and methodologies described in the publications that can be used in connection with the described invention. In the following, we will first explain the peptide cube provided by the present invention, and then explain the controlled release composition in which the peptide cube encapsulates an active ingredient.

[0022] [Regarding the peptide cube of the present invention] According to one embodiment of the present invention, "A peptide cube whose wall is constituted by a co-assembly of a curved membrane made of a first amphipathic polypeptide and a planar membrane made of a second amphipathic polypeptide." (First embodiment) is provided. The peptide cube is described in detail below.

[0023] (Cube shape) The nano-sized hollow body (nanocapsule) made of the polypeptide of the present invention is characterized by its cube shape. The "cube shape" is not particularly limited as long as it is a shape that a person skilled in the art can recognize as a hexahedron having six walls, and examples of the shape include a cube shape and a rectangular parallelepiped shape. More specifically, the peptide cube of the present invention is a cube-shaped hollow body surrounded by six walls formed by the co-assembly of a curved membrane made of a first amphipathic polypeptide and a flat membrane made of a second amphipathic polypeptide, and various ingredients such as pharmaceuticals can be encapsulated inside the hollow body depending on the purpose. The "cube shape" that is a characteristic of the nanocapsules of the present invention is formed by bonding together two types of membranes with different curvatures, a curved membrane formed by co-assembling a first amphipathic polypeptide and a flat membrane formed by co-assembling a second amphipathic polypeptide, to form a membrane structure in which the curved membranes are primarily assembled to form the edges and vertices, while the flat membranes are primarily assembled to form the wall surfaces, resulting in a sealed hollow body.

[0024] (cube size) The size of the peptide cube of the present invention is not particularly limited and can be appropriately designed according to the purpose of use. In one embodiment, from the viewpoint of application to DDS, the length of one side can be, for example, 10 to 300 nm, preferably 10 to 250 nm, more preferably 10 to 200 nm. In the peptide cube of the present invention, the size of the cube can be appropriately controlled by appropriately changing the degree of polymerization of the sarcosine portion of the second amphiphilic polypeptide that forms the planar membrane, thereby adjusting the size of the planar membrane.

[0025] (Amphipathic Polypeptides) As used herein, the term "polypeptide" refers to a compound in which two or more amino acids are linked together via peptide bonds. As used herein, the term "amino acid" includes natural amino acids, unnatural amino acids, and derivatives thereof obtained by modification and / or chemical alteration, and also includes α-amino acids, β-amino acids, γ-amino acids, etc. Here, α-amino acids are preferred. As used herein, "amphiphilic" refers to the presence of both a "hydrophilic portion" that is compatible with water (aqueous phase) and a "lipophilic portion" (hydrophobic portion) that is compatible with oil (organic phase) within a single molecule.

[0026] (First amphipathic polypeptide) In the peptide cube of the present invention, the curved membrane is formed by a first amphipathic polypeptide. The "first amphipathic polypeptide" includes a polypeptide including a hydrophilic polypeptide block formed by polymerization of hydrophilic peptide units and a hydrophobic polypeptide block formed by polymerization of hydrophobic peptide units. Preferably, * (hydrophilic polypeptide block)-(hydrophobic polypeptide block) ** (wherein, * indicates the N-terminus, ** indicates the C-terminus). The first amphipathic polypeptide may contain a component other than the polypeptide as a part thereof, and such a component may be, for example, a modification of the N-terminus or C-terminus. The hydrophilic polypeptide block and the hydrophobic polypeptide block may be bonded directly or via a linker, which may be composed of a peptide or a non-peptide.

[0027] The amino acids constituting the hydrophilic peptide unit are not particularly limited, and examples thereof include N-methylglycine (sarcosine), lysine, and histidine, and preferably N-methylglycine (sarcosine). The hydrophilic peptide unit may be composed of one amino acid, or may be composed of two or more kinds of amino acids. In addition, it may contain a component other than an amino acid. The amino acids constituting such hydrophilic peptide units can be appropriately selected by a person skilled in the art so that the hydrophilic polypeptide block after polymerization has hydrophilicity as a whole. The degree of hydrophilicity is not particularly limited, but it is sufficient that the hydrophilic polypeptide block is a region that is relatively more hydrophilic than other regions of the amphiphilic polypeptide, and has hydrophilicity to such an extent that the amphiphilic polypeptide as a whole can realize amphiphilicity by forming the amphiphilic polypeptide together with the other regions.

[0028] The amino acids constituting the hydrophobic peptide unit are not particularly limited, and examples thereof include glycine, alanine, valine, leucine, isoleucine, proline, methionine, tyrosine, tryptophan, aminoisobutyric acid, norleucine, α-aminobutyric acid, and cyclohexylalanine, and preferably include alanine, valine, leucine, isoleucine, aminoisobutyric acid, norleucine, α-aminobutyric acid, and more preferably include leucine and aminoisobutyric acid. The hydrophobic peptide unit may be composed of one amino acid, or may be composed of two or more kinds of amino acids. It may also contain a component other than an amino acid. The amino acids constituting such hydrophobic peptide units can be appropriately selected by a person skilled in the art so that the hydrophobic polypeptide block after polymerization has hydrophobicity as a whole. The degree of hydrophobicity is not particularly limited, but it is sufficient that the hydrophobic polypeptide block is a region that is relatively more hydrophobic than other regions of the amphiphilic polypeptide and has a degree of hydrophobicity that makes it possible for the amphiphilic polypeptide as a whole to realize amphiphilicity by forming the amphiphilic polypeptide together with the other regions. In carrying out the present invention, it is preferable that the hydrophobic polypeptide block is designed so as to form an α-helical structure.

[0029] The "first amphipathic polypeptide" has been described in detail above. One preferred embodiment of the first amphipathic polypeptide is represented by the following formula (I):

[0030] [ka]

[0031] (In the formula, R 1 is a hydrogen atom, C 1-6 represents an alkyl group or an aralkyl group, R 2 is a hydrogen atom or a C which may be substituted by a hydroxyl group. 1-4 C optionally substituted with an alkanoyl group or an aryl group 1-4 represents an alkoxycarbonyl group, m represents an integer of 16 to 47, and n represents an integer of 5 to 10. The polypeptides preferably form a curved structure when assembled by themselves.

[0032] Each symbol and definition above will be explained below. "Ca-b" (e.g., C 1-6 ) or "Ca-Cb" (e.g., C 1 -C 6 ) indicates that the group comprises a to b carbon atoms (for example, 1 to 6). "C 1-6 Examples of the "alkyl group" include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, 1-ethylpropyl, hexyl, isohexyl, 1,1-dimethylbutyl, 2,2-dimethylbutyl, 3,3-dimethylbutyl, and 2-ethylbutyl. As the "aralkyl group", "C 7-16aralkyl groups, such as benzyl, phenethyl, naphthylmethyl, and phenylpropyl. "C 1-4 Examples of the "alkanoyl group" include acetyl, propanoyl, and butanoyl. 1-4 The "alkanoyl group" may be substituted with 1 to 3 hydroxy groups at substitutable positions. Here, "C optionally substituted with a hydroxy group" 1-4 A preferred embodiment of the alkanoyl group is -COCH 2 An example of such a group is the OH group. "C 1-4 Examples of the "alkoxycarbonyl group" include methoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, isopropoxycarbonyl, butoxycarbonyl, isobutoxycarbonyl, sec-butoxycarbonyl, and tert-butoxycarbonyl. 1-4 The "alkoxycarbonyl group" may be substituted with 1 to 3 aryl groups at substitutable positions. Here, the "aryl group" is any of C 6-14 Examples of the aryl group include aryl groups, and more specifically, examples of the aryl groups include phenyl, naphthyl, and fluorenyl. m is as defined above, but is preferably an integer of 20-30, and more preferably an integer of 25-26. Although n is as defined above, it is preferably an integer of 6 to 8, and more preferably 6 or 7. Specifically, it is particularly preferable that m is 25 and n is 6, that m is 26 and n is 7, and the like.

[0033] (Second Amphipathic Polypeptide) In the peptide cubes of the invention, the planar membrane is formed by a second amphipathic polypeptide. The "second amphipathic polypeptide" includes a polypeptide including a hydrophilic polypeptide block formed by polymerization of hydrophilic peptide units and a hydrophobic polypeptide block formed by polymerization of hydrophobic peptide units. Preferably, * (hydrophilic polypeptide block)-(hydrophobic polypeptide block)-(hydrophilic polypeptide block) ** Examples of the block copolymer include those containing a block copolymer structure represented by the following formula: (In the above, * and ** indicates the N-terminus, respectively. That is, two (hydrophilic polypeptide blocks) are directly or indirectly bound to the (hydrophobic polypeptide block) from their respective C-terminus. The second amphipathic polypeptide may contain a component other than the polypeptide as a part thereof, and such a component may be, for example, a modification of the N-terminus or C-terminus. The hydrophilic polypeptide block and the hydrophobic polypeptide block may be directly bonded to each other or may be bonded to each other via a linker. The linker may be composed of a peptide or a non-peptide. The linker can be used to reverse the bonding direction of the peptide blocks.

[0034] For details on the "amino acids constituting the hydrophilic peptide unit," "hydrophilic polypeptide block," "amino acids constituting the hydrophobic peptide unit," and "hydrophobic polypeptide block," please refer to the above for the "first amphipathic polypeptide."

[0035] The "second amphipathic polypeptide" has been described in detail above. One preferred embodiment of the second amphipathic polypeptide is represented by the following formula (II):

[0036] [ka]

[0037] (In the formula, R 3 and R 4 each independently represents a hydrogen atom or a C which may be substituted by a hydroxyl group; 1-4C optionally substituted with an alkanoyl group or an aryl group 1-4 represents an alkoxycarbonyl group, Q is NH or NH(CH 2 ) r NH (wherein r is an integer of 1 to 3), o and q each independently represent an integer of 6 to 15, and p represents an integer of 5 to 10. The polypeptides preferably form a planar structure when assembled by themselves. R 3 and R 4 "C optionally substituted with a hydroxy group" 1-4 "Alkanoyl group", and "C optionally substituted with an aryl group" 1-4 For "alkoxycarbonyl group", R 2 The explanation of each group in can be referred to. R 3 and R 4 is as described above, but is preferably a hydrogen atom or a C substituted with hydroxy 1-4 An alkanoyl group, more preferably a hydrogen atom or -COCH 2 OH, and more preferably -COCH 2 It is OH.

[0038] In the formula, o and q are as defined above, but preferably each independently represents an integer of 8 to 13, and more preferably 10. r is as defined above, but is preferably 1 or 2, and more preferably 2. Furthermore, p is as described above, but is preferably an integer of 5 to 7, more preferably 6 or 7, and even more preferably 6.

[0039] (shear stress response) As described in detail in the examples below, the peptide cube of the present invention has a responsiveness to shear stress, and has the characteristic that under a certain shear stress, the cube shape starts to collapse and the encapsulated matter is gradually released to the outside (shear stress responsiveness). The degree of responsiveness to shear stress is not particularly limited and can be appropriately determined according to the purpose of use of the peptide cube, and can be controlled, for example, by adjusting the type and blending ratio of the first amphipathic polypeptide and the second amphipathic polypeptide. The degree of responsiveness of such peptide cubes to shear stress is, for example, a shear stress at which collapse of the shape starts of 5 Pa to 20 Pa, preferably 8 Pa to 12 Pa. In one preferred embodiment, the shear stress at which collapse of the shape starts of 10 Pa.

[0040] [About the method for producing the peptide cube of the present invention] The peptide cube of the present invention can be easily prepared by mixing a first amphipathic polypeptide with a second amphipathic polypeptide, dispersing the mixture in an aqueous solvent, and then heating the mixture. In one embodiment, when mixing the two polypeptides, it is preferable to first dissolve them in an appropriate solvent (eg, an alcohol such as ethanol) to prepare a solution, and then mix them. The temperature during mixing and dispersing is not particularly limited, but mixing may be performed at room temperature (for example, 5° C. to 30° C.). The heating temperature is not particularly limited, but may be, for example, within a temperature range of 60 to 100° C. (preferably 85 to 95° C., more preferably 90° C.) for one to several hours (preferably 1 to 3 hours). Heating may be accompanied by stirring. In preparing the peptide cube of the present invention, the mixing ratio of the first amphipathic polypeptide to the second amphipathic polypeptide can be in the range of 1:1 to 1:8 in terms of molar ratio. Here, the range of 1:1 to 1:4 is preferred, the range of 1:1 to 1:2 is more preferred, and the range of 1:2 is particularly preferred. By hydrophobically interacting a first amphipathic polypeptide and a second amphipathic polypeptide, each of which has an α-helix structure in its hydrophobic portion, the first amphipathic polypeptide and the second amphipathic polypeptide each form a co-assembly starting from the α-helix structure to form a curved membrane and a planar membrane, and the two types of membranes, the curved membrane and the planar membrane, are further co-assembled and bonded together to form membranes with different curvatures that become the wall, thereby forming a cube-shaped nano-sized hollow object made of the peptide of the present invention. In addition, a person skilled in the art can appropriately prepare and use the first amphipathic polypeptide and the second amphipathic polypeptide, which are raw materials, starting from known raw materials using methods known in the art (see, for example, Chem. Commun. 47, 3204-3206 (2011)).

[0041] [Regarding the controlled release composition comprising the peptide cube of the present invention] According to another embodiment of the present invention, "The wall surface is composed of a peptide cube formed by co-assembly of a curved membrane made of a first amphipathic polypeptide and a planar membrane made of a second amphipathic polypeptide, The active ingredient is contained within the peptide cube. The peptide cube is collapsed by shear stress applied to the cube, and the encapsulated active ingredient is released. A composition for controlled release." (Second embodiment) is provided. The controlled release composition of this embodiment (hereinafter also abbreviated as "the composition") will be described in detail below.

[0042] (Peptide Cube) This embodiment is based on the finding that by forming a composition in which an active ingredient is encapsulated in a "peptide cube, the wall of which is formed by a co-assembly of a curved membrane made of a first amphipathic polypeptide and a planar membrane made of a second amphipathic polypeptide" as described in the first embodiment, the cube shape collapses in response to shear stress applied to the peptide cube, and the active ingredient is released from the composition. Therefore, with regard to the "peptide cube whose wall is constituted by a co-assembly of a curved membrane made of a first amphipathic polypeptide and a flat membrane made of a second amphipathic polypeptide" itself, reference can be made to the details described in the first embodiment with regard to each component of the cube such as its shape, size, amphipathic polypeptide, and preferred aspects thereof, as well as its manufacturing method.

[0043] (Active ingredient) The "peptide cube" constituting this composition can encapsulate various active ingredients inside the hollow body. Examples of such active ingredients include a wide variety of active ingredients used in pharmaceuticals (e.g., drugs for improving vascular stenosis or vascular blockage, or anticancer drugs), cosmetics, detergents, foods, chemicals, functional materials, etc. Therefore, this composition can be used for various purposes depending on the properties of the active ingredients to be encapsulated. When the inside of the peptide cube is filled with water, highly hydrophilic ingredients such as hydrophilic drugs can be encapsulated as they are.

[0044] (Manufacturing method) The incorporation of the active ingredient to be encapsulated into the peptide cube, which is necessary for producing the present composition, can be carried out, for example, by mixing the first amphipathic polypeptide and the second amphipathic polypeptide together with the ingredient to be encapsulated, as described in the Examples below. However, the present composition is not limited to this method, and a person skilled in the art can produce the present composition by appropriately encapsulating the active ingredient into the peptide cube. In addition, the amount of active ingredient contained in the peptide cube cannot be determined uniformly because it varies depending on the active ingredient used, the purpose of use, and the form of use. However, a person skilled in the art can make an appropriate determination and manufacture the present composition. Here, the phrase "an active ingredient is encapsulated" means that the active ingredient is present inside the hollow body without being bound to the inner wall of the peptide cube; more specifically, for example, the active ingredient is encapsulated in the hollow body formed by the peptide cube in the form of molecules or in the form of a solution or suspension.

[0045] (shear stress response) As described in the Examples below, the peptide cubes constituting this composition have excellent shear stress responsiveness due to their cube shape, and the walls break in response to shear stress, causing the cube shape to collapse, allowing the encapsulated contents to be gradually released. The degree of responsiveness to shear stress is not particularly limited and can be appropriately determined depending on the purpose of use of the peptide cube, and can be controlled, for example, by adjusting the type and blending ratio of the first amphipathic polypeptide and the second amphipathic polypeptide. The degree of responsiveness to shear stress of such peptide cube is such that the shear stress at which the shape starts to collapse is 5 Pa to 20 Pa, preferably 8 Pa to 12 Pa. In one preferred embodiment, the shear stress at which the shape starts to collapse is 10 Pa. The present composition is useful as a release control composition because it can control the release of the active ingredient by appropriately adjusting the shear stress required for the collapse of the cube shape by utilizing the above characteristics. For example, when a peptide cube containing an active ingredient of a pharmaceutical is administered into the blood vessel of a patient suffering from vascular stenosis or vascular occlusion, the cube moves along the blood flow, and at the site of vascular stenosis or vascular occlusion, the blood pressure rising at that site acts as a shear stress, causing the cube shape to collapse, allowing the active ingredient to act effectively at that site. The peptide cube of the present invention is expected to be used in the pharmaceutical field as such a DDS technology (drug carrier). However, the above is merely one example, and by incorporating useful ingredients other than pharmaceuticals, the composition can be used as a composition for controlled release of active ingredients not only in the pharmaceutical field but also in a wide range of other fields. For example, by encapsulating chemical substances such as pigments or luminescent substances, they can be used as pressure / shear stress responsive materials or pressure / shear stress sensors; by encapsulating active ingredients of detergents, they can be used as detergents; and by encapsulating active ingredients of chemical seasonings, they can be used as food.

[0046] (Use form of the composition) This composition can be used for various purposes in the form of a dispersion in which peptide cubes containing an active ingredient are dispersed. Those skilled in the art can use this composition by appropriately designing the form of the composition according to the purpose of use. When the composition is administered to a subject such as a human, for example, using it as a drug carrier, the peptide cube containing the active ingredient of a pharmaceutical can be used either alone or in the form of a pharmaceutical composition containing the peptide cube together with a medicamentously acceptable carrier. Such pharmaceutical compositions include, for example, liquid preparations such as injections (eg, subcutaneous injections, intravenous injections (eg, bolus), intramuscular injections, intraperitoneal injections), drip infusions, and the like. As the "pharmaceutical acceptable carrier", various carriers commonly used in the field of pharmaceutical formulation technology can be used. Specific examples of "pharmaceutical acceptable carriers" that can be used in liquid preparations include solvents (e.g., water for injection, isotonic saline, alcohol, propylene glycol, macrogol, sesame oil, etc.), solubilizing agents (e.g., polyethylene glycol, propylene glycol, D-mannitol, benzyl benzoate, ethanol, triethanolamine, sodium carbonate, sodium citrate, etc.), suspending agents (e.g., surfactants such as stearyl triethanolamine, sodium lauryl sulfate, lauryl aminopropionic acid, lecithin, benzalkonium chloride, glycerin monostearate, etc.; hydrophilic polymers such as polyvinyl alcohol, polyvinylpyrrolidone, sodium carboxymethylcellulose, methylcellulose, hydroxymethylcellulose, hydroxypropylcellulose, etc.), isotonic agents (e.g., glucose, D-sorbitol, sodium chloride, glycerin, D-mannitol, etc.), buffering agents (e.g., buffer solutions such as phosphates and citrates), and soothing agents (e.g., benzyl alcohol, etc.). The subjects of administration vary depending on the components encapsulated, but for example, the composition can be safely administered to mammals (particularly humans). EXAMPLES

[0047] The present invention will be further illustrated by the following examples, which are not intended to limit the present invention and may be modified without departing from the scope of the present invention.

[0048] Example 1: Preparation of peptide cubes (1) Preparation of amphiphilic polypeptides In this example, the first amphipathic polypeptide is (Sar) 26 -(L-Leu-Aib) 14 (S 26 L 14 As a second amphipathic polypeptide, (Sar) 10 -(L-Leu-Aib) 12 -(Sar) 10(S 10 L 12 S 10 (also abbreviated as S) was used. 26 L 14 can be prepared by, for example, the method described in J. Am. Chem. Soc., 2018, 140(51), 17956-17961. or a method equivalent thereto. 10 L 12 S 10 was produced by, for example, the method described in Int. J. Mol. Sci. 2021, 22(21), 10275. or a method similar thereto. In the first and second amphipathic polypeptides described above, the N-terminus is protected with glycolic acid and the C-terminus is protected with a methoxy group by a method known in the art or a method similar thereto. Similarly, as the first amphipathic polypeptide, (Sar) 16 -(L-Leu-Aib) 14 (S 16 L 14 (also abbreviated as (Sar) 37 -(L-Leu-Aib) 14 (S 37 L 14 (also abbreviated as (Sar) 47 -(L-Leu-Aib) 14 (S 47 L 14 (also abbreviated as was manufactured. Similarly, the second amphipathic polypeptide may be (Sar) 13 -(L-Leu-Aib) 12 -(Sar) 13 (S 13 L 12 S 13 (also abbreviated as was manufactured. In the above, the N-terminus is also protected with glycolic acid, and the C-terminus is protected with a methoxy group.

[0049] (2) Preparation of peptide cubes S26 L 14 Ethanol solution of and S 10 L 12 S 10 The ethanol solutions of the above were mixed to a specified molar ratio (1:1 or 1:2) and injected in one go into stirred water, saline, or other aqueous solvents using a micropipette (ethanol injection method). After stirring at room temperature for several minutes (approximately 1 to 30 minutes), the resulting suspension was heated at 90°C for 1 hour to obtain peptide cubes, which are co-assembled bodies. TEM images of the resulting peptide cubes are shown in FIG. 1 (left) (mixing molar ratio=1:1) and FIG. 2 (mixing molar ratio=1:2). TEM images were acquired using a JEOL JEM-1230 at an accelerating voltage of 80 kV. A drop (2 μL) of the dispersion was placed on a carbon-coated Cu grid, negatively stained with 2% samarium acetate, and excess liquid was soaked up with filter paper. Frozen-Hydrated / Cryogenic-TEM (Cryo-TEM) observations were performed. The dispersion in the buffer solution was rapidly frozen in liquid ethane cooled with liquid nitrogen. The samples were evaluated at liquid nitrogen temperature at an accelerating voltage of 100 kV. Figure 1 (right) shows a Cryo-TEM image of a peptide cube (mixed molar ratio = 1:1).

[0050] (3)S 26 L 14 Single / S 10 L 12 S 10 Single-origin assemblage As an amphipathic polypeptide, S 26 L 14 and S 10 L 12 S 10 The aggregates were obtained by the same procedure as in (2) above, except that each of the above was used alone. TEM images of each assembly are shown in Fig. 3(S 26 L 14 4 (S 10 L 12 S 10 The following is a collection of origins: (4) In the same manner as in (2) above, a peptide cube was obtained as a co-assembly using a combination of the first amphipathic polypeptide and the second amphipathic polypeptide shown in Table 1 below (mixing molar ratio = 1:2).

[0051] [Table 1]

[0052] The TEM images for each of the above peptide cubes are shown in Figure 5(S 16 L 14 and S. 10 L 12 S 10 ), Fig. 6 (S 37 L 14 and S. 10 L 12 S 10 ), Fig. 7 (S 47 L 14 and S. 10 L 12 S 10 combination), Fig. 8(S 26 L 14 and S. 13 L 12 S 13 The combination of

[0053] As described above, it was confirmed that the co-assembly of the first amphipathic polypeptide and the second amphipathic polypeptide forms a "peptide cube" having the desired cube shape (Figs. 1-2, 5-8). On the other hand, it was confirmed that the assembly of only the first amphipathic polypeptide formed a curved membrane (Fig. 3), while the assembly of only the second amphipathic polypeptide formed a flat membrane (Fig. 4).

[0054] Example 2: Evaluation of the stability of peptide cubes The peptide cubes (mixed molar ratio = 1:2) prepared in Example 1(2) were stored in a refrigerator at 4°C for 2 months, and then observed again by TEM to check whether there was any change in their shape.

[0055] The results are shown in Figure 9. In Figure 9, the left image is a TEM image on "0th day of storage", and the right image is a TEM image on "60th day of storage". From the above, it was revealed that the peptide cube of the present invention maintained its cube structure for over two months, and thus the cube structure has excellent stability.

[0056] Example 3: S 26 L 14 Localization of S in peptide cubes 26 L 14 To visualize the localization of S 10 L 12 S 10 , S 26 L 14 , L.A.-S. 26 L 14 A mixture of these (mixing ratio = 2:0.9:0.1) was prepared by the ethanol injection method, and then heated at 90°C for 1 hour to prepare a LA-labeled peptide cube dispersion. 26 L 14 is S 26 L 14 was prepared by capping the N-terminus with lipoic acid in a conventional manner. A 5 nm gold colloid dispersion was mixed with a LA-labeled peptide cube dispersion at room temperature for 10 min. A drop (5 μL) of the resulting dispersion was placed on a carbon-coated Cu grid, washed three times with Milli-Q water on the grid, and after negative staining with 2% samarium acetate, the excess liquid was soaked up with filter paper. The resulting sample was observed by TEM.

[0057] The results are shown as TEM images in FIG. The black dots in the image are LA-S 26 L 14 1 shows gold nanoparticles bound to the lipoic acid portion of the first amphiphilic polypeptide S 26 L 14 It was revealed that the peptide was localized at the corners of the peptide cube.

[0058] Example 4: Evaluation of drug encapsulation ability S 26 L 14 and S. 10 L 12 S 10 A solution of 0.2 mg of each of these was dissolved in 4 μl of ethanol, and added to a Dox (doxorubicin) solution (0.75 mg / ml) (amount of solution was 990 ml) and stirred. After stirring at 90°C for 1 hour, this solution was purified using Amicon Ultra 10 kDa (Merck) to obtain a peptide cube encapsulating Dox.

[0059] The efficiency of Dox encapsulation in the peptide cubes was about 3%. The efficiency of Dox encapsulation was evaluated by measuring the absorbance of light at the absorption wavelength of Dox. Furthermore, no leakage of Dox from the peptide cubes was observed over 48 hours after preparation. From the above, it was found that the peptide cube of the present invention has a closed hollow shape and has a function of encapsulating components such as pharmaceuticals inside. Therefore, it was revealed that the peptide cube of the present invention is useful as a composition containing a pharmaceutical component by encapsulating the component.

[0060] Example 5: Evaluation of shear stress response To investigate the mechanosensitivity of peptide cubes, the change in the intrinsic shape of the peptide cubes as a function of shear stress was examined using an AR-G2 rheometer (TA Instruments-Waters LLC., New Castle, UK). A certain amount of peptide cube dispersion was loaded into the rheometer, equilibrated for 2 minutes, and then shear stress was applied. Two shear stresses, 1 Pa and 10 Pa, were applied, which correspond to the shear stress in normal and pathologically contracted blood vessels, respectively. Under each stress, the peptide cubes were subjected to shear stress for 20 minutes or 2 hours. Finally, the shape of the peptide cubes was examined by visualization with a TEM, and the size of the aggregates was determined by the following DLS measurement. In this test, the peptide cubes produced in Example 1(2) with a mixed molar ratio of 1:2 were used. (DLS measurement) The hydrodynamic diameter of the aggregates in saline was measured with an ELSZ-2PL instrument (Photal Otsuka Electronics Co. Ltd., Osaka, Japan) using a He-Ne laser (633 nm). Sample measurements were performed in triplicate at 25°C.

[0061] The change in shape of the peptide cube in this evaluation is shown in FIG. 11 (1 Pa shear stress loading; the left figure shows the shape after 20 minutes loading, and the right figure shows the shape after 2 hours loading, respectively) and FIG. 12 (10 Pa shear stress loading; the left figure shows the shape after 20 minutes loading, and the right figure shows the shape after 2 hours loading, respectively). As is clear from FIG. 11, when the shear stress loaded was 1 Pa (corresponding to the shear stress in normal blood vessels), there was no change in the shape of the peptide cube, and no collapse of the cube shape was observed. On the other hand, when the shear stress loaded was 10 Pa (corresponding to the shear force in pathologically contracted blood vessels), the collapse of the shape of the peptide cube progressed over time, and almost complete collapse was observed at the 2-hour point. From the above, it was revealed that the peptide cube of the present invention containing pharmaceutical ingredients can release the encapsulated ingredients by collapsing its cube shape under shear stress simulating that in a pathologically constricted blood vessel, and is therefore useful as a composition for controlled release.

[0062] Example 6: Evaluation of release of active ingredients by shear stress response The possibility of peptide cubes as shear stress responsive DDS carriers was examined. Specifically, in order to simulate the pathological flow state in a stenosed blood vessel, pipetting was repeated using a syringe and a 27G needle, and the release behavior of calcein dye as a hydrophilic drug model from the peptide cube was evaluated. As the peptide cube, the peptide cube with a mixed molar ratio of 1:2 produced in Example 1 (2) was used. The overview of this test is shown in Figure 13A. Calcein was encapsulated in the peptide cubes at a level lower than the self-quenching concentration without affecting the inherent shape of the peptide cubes. Each time the dye was released from the peptide cubes into the surrounding medium, the dye was diluted, resulting in a decrease in fluorescence intensity. To evaluate the release rate of calcein from the peptide cubes, the fluorescence intensity of calcein-encapsulated peptide cubes digested with Triton X-100 (TX-100) was measured as the intensity when 100% of the encapsulated calcein was released.

[0063] Even after 20 min at 37°C without pipetting, more than 95% of calcein remained inside the peptide cube (Fig. 13D). On the other hand, when the peptide cube containing calcein was continuously aspirated and discharged with a syringe and a 27G needle, the fluorescence intensity significantly decreased. As a result, 36%, 64%, and 84% of calcein were released after 1, 5, and 20 min, respectively (Fig. 13D). TEM images after 20 min also confirmed that the shear stress-responsive deformability of the peptide cube was maintained even after calcein encapsulation. As a result, it was revealed that the peptide cube was completely stable and retained its inclusions under static conditions, but showed a unique release behavior in which the inclusions leaked out when exposed to shear flow. For reference, TEM images of the peptide cube before and after 20 min of pipetting are shown in Fig. 13B (before pipetting) and Fig. 13C (after pipetting), respectively. From the above, it was revealed that the peptide cube of the present invention containing pharmaceutical ingredients can release the encapsulated ingredients by collapsing its cube shape under shear stress simulating the flow conditions in a pathologically constricted blood vessel, and is therefore useful as a controlled release composition. [Industrial Applicability]

[0064] The present invention relates to a peptide cube, the wall of which is constituted by a co-assembly of a curved membrane made of a first amphipathic polypeptide and a planar membrane made of a second amphipathic polypeptide; and a composition for controlled release comprising the peptide cube, which contains an active ingredient and releases the encapsulated active ingredient when the cube shape collapses due to shear stress applied to the peptide cube, and is useful, for example, in the field of medicine.

Claims

1. The peptide cube has a wall formed by co-assembly of a curved membrane made of a first amphipathic polypeptide and a planar membrane made of a second amphipathic polypeptide; The active ingredient is contained within the peptide cube. The peptide cube is collapsed by shear stress applied to the cube, and the encapsulated active ingredient is released. Controlled release compositions.

2. The shear stress applied to the peptide cube at which the shape begins to collapse is 5 to 20 Pa as evaluated by a rheometer. The composition of claim 1.

3. The first amphipathic polypeptide has the following formula (I): 【Chemistry 1】 (In the formula, R １ is a hydrogen atom, C １－６ represents an alkyl group or an aralkyl group, R ２ is a hydrogen atom or a C optionally substituted by a hydroxy group １－４ C optionally substituted with an alkanoyl group or an aryl group １－４ represents an alkoxycarbonyl group, m represents an integer from 16 to 47, and n represents an integer of 5 to 10. A polypeptide represented by the formula: The second amphipathic polypeptide has the following formula (II): 【Chemistry 2】 (In the formula, R ３ and R ４ each independently represents a hydrogen atom or a C optionally substituted by a hydroxyl group; １－４ C optionally substituted with an alkanoyl group or an aryl group １－４ represents an alkoxycarbonyl group, Q is NH or NH(CH ２ ) ｒ NH (wherein r is an integer of 1 to 3), o and q each independently represent an integer of 6 to 15, and p represents an integer of 5 to 10. A polypeptide represented by the formula: The composition according to claim 1 or 2.

4. A peptide cube comprising a co-assembly of a curved membrane made of a first amphipathic polypeptide and a planar membrane made of a second amphipathic polypeptide, The first amphipathic polypeptide has the following formula (I): 【Chemistry 3】 (In the formula, R １ is a hydrogen atom, C １－６ represents an alkyl group or an aralkyl group, R ２ is a hydrogen atom or a C optionally substituted by a hydroxy group １－４ C optionally substituted with an alkanoyl group or an aryl group １－４ represents an alkoxycarbonyl group, m represents an integer from 16 to 47, and n represents an integer of 5 to 10. A polypeptide represented by the formula: The second amphipathic polypeptide has the following formula (II): 【Chemistry 4】 (In the formula, R ３ and R ４ each independently represents a hydrogen atom or a C optionally substituted by a hydroxyl group; １－４ C optionally substituted with an alkanoyl group or an aryl group １－４ represents an alkoxycarbonyl group, Q is NH or NH(CH ２ ) ｒ NH (wherein r is an integer of 1 to 3), o and q each independently represent an integer of 6 to 15, and p represents an integer of 5 to 10. A peptide cube, which is a polypeptide represented by the formula:

5. The mixing ratio (molar ratio) of the first amphipathic polypeptide to the second amphipathic polypeptide is within the range of 1:1 to 1:

8. A peptide cube according to claim 4.

6. The length of one side is 10 to 300 nm. A peptide cube according to claim 4.

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