Liposome-like complexes, and complexes of liposomes and proteins

Abstract

This invention provides a liposome-like complex capable of modifying the N-terminus of a protein. [Solution] A liposome-like complex comprising a compound of formula (I) [wherein A is an organic group] or a salt thereof, and a phospholipid. Also, formula (II) [wherein * is a liposome, # is a protein, R A R is the organic group or hydrogen atom of the amino acid side chain, and B A complex of liposomes and proteins containing the structure of an organic group or hydrogen atom in the amino acid side chain. TIFF2026064034000027.tif63106

Description

[Technical Field]

[0001] The present invention relates to a liposome-like complex capable of modifying the N-terminus of a protein, and to a method for producing the same. The present invention also relates to a liposome-protein complex using the liposome-like complex, and to a method for producing the same. The present invention also relates to compounds capable of forming the liposome-like complex. [Background technology]

[0002] Liposomes are known as a method for efficiently delivering drugs. For example, active ingredients can be incorporated into liposomes and targeted to the desired site. In liposome technology, attempts are also being made to impart the ability to target specific cells or tissues to liposome formulations by placing proteins or other substances on the surface of the liposome. Tissue targeting is expected to improve the therapeutic concentration range and the therapeutic time range, leading to higher drug efficacy and reduced burden on patients.

[0003] Surface-modified liposomes are commercially available (see, for example, Non-Patent Document 1). One such conventional technique involves immobilizing proteins on the surface of a bilayer membrane, such as a liposome, either directly or via a linker, using non-covalent bonds. However, because it relies on non-covalent bonds, stable immobilization is difficult to achieve. Aligning the orientation of the immobilized proteins is also challenging. Another conventional technique involves embedding a molecule containing a maleimide group in a bilayer membrane, such as a liposome, and linking it to the cysteine ​​side chain of a protein via a Michael addition reaction. However, while this method can form a more stable linkage than the non-covalent bond described above, it has the problem of not being able to selectively link proteins containing multiple cysteine ​​residues. Furthermore, it cannot link proteins or peptides that do not contain cysteine ​​(see, for example, Non-Patent Document 2). Another method involves linking via a click reaction between an azide group site and an alkyne site. By embedding a molecule containing either an azide group or an alkyne group in a bilayer and pre-binding the other end to a protein, it is possible to link a protein to a liposome (see, for example, Non-Patent Document 3). However, this method requires that the protein be pre-introduced with either an azide group or an alkyne group. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Drug Delivery System, 30-5, 2015, 486-488 [Non-Patent Document 2] Colloids and Surfaces B: Biointerfaces, 121, 2014, 141-149 [Non-Patent Document 3] Signal Transduction and Targeted Therapy, 7, 2022, 386 [Overview of the project] [Problems that the invention aims to solve]

[0005] The present invention has been made in view of the above circumstances and aims to provide a liposome-like complex capable of binding to proteins. The present invention can provide a liposome-like complex in which proteins and liposomes are covalently linked in a single step, regardless of the protein sequence and in a position-selective manner. [Means for solving the problem]

[0006] The present invention relates to the following embodiments. [1] Equation (I): [ka] [In the formula, A represents an organic group.] Compounds represented by or their salts, and Phospholipids, A liposome-like complex containing this complex. [2] The following structure in equation (I): [ka] However, the liposome-like complex described in [1] is arranged on the surface of the liposome-like complex. [3] A is selected from the group consisting of aliphatic hydrocarbon groups, phospholipids, and steroid skeleton-containing groups, as described in [1]. [4] A is C4-C 20 A liposome-like complex as described in [3], selected from the group consisting of alkyl groups, DOPE (1,2-dioleoyl-sn-glycero-phosphatidylethanolamine derivative group), DMPE (dimyristyl-sn-glycero-phosphatidylethanolamine derivative group), 1,2-dipalmitoyl-sn-glycero-3-phosphatidylethanolamine derivative group, 1,2-distearoyl-sn-glycero-3-phosphatidylethanolamine derivative group, and cholic acid-EG. [5] A method for producing a liposome-like complex according to any one of [1] to [4], comprising the step of mixing a compound of formula (I) or a salt thereof with a phospholipid. [6] A compound or a salt thereof represented by formula (I) according to [1], having the following formula: [7] The compound or a salt thereof selected from the group consisting of:

Chemical formula

Chemical formula

[10] A complex of a liposome and a protein according to [7], wherein the liposome encapsulates a pharmacologically active ingredient.

[11] A method for producing a complex of a liposome and a protein, comprising the structure represented by formula (II) according to [7], comprising: [1] A liposome-like complex according to any one of [1] to [4], represented by formula (III): [2] A liposome-like complex according to any one of [1] to [4], represented by formula (III):

Chemical formula

[12] [7]. [Effects of the Invention]

[0007] According to the present invention, it is possible to provide a liposome-like complex that can specifically bind to the N-terminus of a protein. Furthermore, according to the present invention, it is possible to provide a liposome-protein complex in which the protein is specifically bound to the liposome at its N-terminus. In the present invention, since the N-terminus of the protein can be specifically modified, it is possible to obtain a liposome-protein complex in which the protein is bound with its orientation aligned. [Brief explanation of the drawing]

[0008] [Figure 1] This is a schematic diagram of the structure of the liposome-like complex of the present invention and its enlarged view. The structural formula is shown as an example when the organic group A in formula (I) is DOPE (1,2-dioleoyl-sn-glycero-phosphatidylethanolamine derivative group). [Figure 2] This is a schematic diagram (synthesis scheme) showing how liposome-protein complexes are formed through the reaction of liposome-like complexes with proteins. [Figure 3] The 1H NMR spectra (400 MHz, DMSO-d6) of compound 1 (DOPE-TA4C) (top panel) and DOPE (bottom panel) are shown. [Figure 4] The ESI-TOF MS spectrum of compound 1 is shown. [Figure 5] The 1H NMR spectra (400 MHz, DMSO-d6) of compound 2 (DMPE-TA4C) (top panel) and DMPE (bottom panel) are shown. [Figure 6] The ESI-TOF MS spectrum of compound 2 is shown. [Figure 7] The 1H NMR spectra (400 MHz, DMSO-d6) of compound 3 (DPPE-TA4C) (top panel) and DPPE (bottom panel) are shown. [Figure 8] The 1H NMR spectra (400 MHz, CDCl3) of compound 4 (DSPE-TA4C) (top panel) and DSPE (bottom panel) are shown. [Figure 9] The ESI-TOF MS spectrum of compound 4 is shown. [Figure 10] The results of evaluating liposome particle size using dynamic light scattering are shown. [Figure 11] The top panel shows fluorescence microscopy results of liposomes modified with GFP via the N-terminal modification reaction between Liposome / 1 and GFP. The bottom panel also shows fluorescence microscopy results of a comparative example. [Figure 12] The following shows fluorescence microscopy results of liposome-GFP complexes prepared using liposomes with a (PC + DPPG + Cholesterol):DOPE-TA4C ratio of 10:1. [Figure 13] The following shows fluorescence microscopy results of liposome-GFP complexes prepared using liposomes with a (PC + DPPG + Cholesterol):DOPE-TA4C ratio of 10:0.0001. [Modes for carrying out the invention]

[0009] The following describes embodiments for carrying out the present invention. However, the present invention is not limited to the following embodiments.

[0010] Liposome-like complex The liposome-like complex according to the present invention is of formula (I): [ka] [In the formula, A represents an organic group.] Compounds represented by or their salts, and Phospholipids, This invention relates to a liposome-like complex that includes (hereinafter also referred to as "the liposome-like complex of the present invention").

[0011] The compound of formula (I) is a derivative of 1H-1,2,3-triazole-4-carboaldehyde (TA4C). In this compound, substituent A is substituted on the nitrogen atom at position 1 of the triazole in 1H-1,2,3-triazole-4-carboaldehyde. Hereinafter, the compound of formula (I) will also be referred to as the "TA4C compound".

[0012] Figure 1 shows a schematic diagram of the structure of the liposome-like complex of the present invention. In the liposome-like complex, phospholipids form liposomes, and the compound of formula (I) is incorporated into these liposomes and complexed to form the liposome-like complex. Figure 1 shows how liposomes are formed by phospholipids, and how the A group of the TA4C compound is embedded within these liposomes. The triazole ring (TA4C structure) of the TA4C compound is located on the surface of the liposome. Thus, the following structure in formula (I): [ka] It is preferable that the TA4C structure is located on the surface of the liposome-like complex. This positions the TA4C structure on the outer surface of the liposome-like complex, making it more reactive and facilitating the formation of a complex with the protein. Generally, liposomes have a lipid bilayer structure, with the hydrophobic groups of phospholipids located on the inside of the lipid bilayer and the hydrophilic groups of phospholipids located on the outside. The A group may be a group that has an affinity for the hydrophobic group, and as a result, the A group is incorporated into the hydrophobic portion within the liposome. On the other hand, the TA4C structure is a hydrophilic portion and can be located on the outside of the liposome. This allows the triazole ring (TA4C structure) of the TA4C compound to be located on the surface of the liposome. Note that Figure 1 shows a schematic structure, and in reality, multiple TA4C structures may be located on the surface of the liposome. Furthermore, while Figure 1 depicts the TA4C structure being complexed with liposomes via a methylene group (CH2) (i.e., the TA4C end of group A is a methylene group), the group adjacent to the TA4C structure may be a group or structure other than a methylene group (CH2).

[0013] In the compound of formula (I), group A is an organic group. The organic group is not particularly limited as long as it is a group derived from an organic molecule, for example, a group obtained by removing one or more atoms from an organic molecule. The organic molecule is not particularly limited and may be natural, synthetic, or artificial. As described above, it is preferable that group A is a group that has the property of being embedded inside liposomes. Specifically, it is preferable that group A is hydrophobic. It is also preferable that group A has affinity for the hydrophobic group of phospholipids.

[0014] The above A group is preferably selected from the group consisting of aliphatic hydrocarbon groups, phospholipids, and steroid skeleton-containing groups. These are hydrophobic groups or hydrophobic groups, and have affinity with the hydrophobic portion of the liposome-like complex, making it easier for the TA4C compound to form a complex with liposomes. Furthermore, this can lead to the formation of a stable liposome-like complex.

[0015] Furthermore, the above A group is C4-C 20 It is preferable to select from the group consisting of alkyl groups, DOPE (1,2-dioleoyl-sn-glycero-phosphatidylethanolamine derivative group), DMPE (dimyristyl-sn-glycero-phosphatidylethanolamine derivative group), 1,2-dipalmitoyl-sn-glycero-3-phosphatidylethanolamine derivative group, 1,2-distearoyl-sn-glycero-3-phosphatidylethanolamine derivative group, 1,2-dioleoyl-sn-glycero-phosphatidylethanolserine derivative group, and cholic acid-EG. This makes it possible to form liposome-like complexes more easily. Furthermore, this may lead to the formation of more stable liposome-like complexes. As will be described later, EG in cholic acid-EG means ethylene glycol. Preferred embodiments of group A will be described below.

[0016] An aliphatic hydrocarbon group is a non-aromatic, chain-like hydrocarbon group composed of carbon atoms and hydrogen atoms. The aliphatic hydrocarbon group may be linear or may have a branched chain. Further, the aliphatic hydrocarbon group may be a saturated hydrocarbon group or a hydrocarbon group having an unsaturated bond (specifically, a carbon-carbon double bond or triple bond). Specifically, the aliphatic hydrocarbon group may be an alkyl group, an alkenyl group, or an alkynyl group. Also, when having an unsaturated bond, the unsaturation may be one or two or more. The aliphatic hydrocarbon group is more preferably an alkyl group, and 20 even more preferably a C4-C 20 alkyl group. Thereby, a stable liposome-like complex can be easily formed. Note that the numbers of C4 and C 20 mean the number of carbon atoms, and the C4-C 20 alkyl group means an alkyl group having 4 to 20 carbon atoms. Examples of the C4-C 20 alkyl group include, but are not limited to, a butyl group, a 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 hexadecyl group, an octadecyl group, and the like.

[0017] Furthermore, in group A, phospholipid refers to a lipid compound having a phosphate ester moiety in its structure. Phospholipids are amphiphilic and can form a lipid bilayer. A portion of the phospholipid, preferably the terminal end of one group in the phosphate ester, can bind to the TA4C structure. In this embodiment, the TA4C compound itself can become a phospholipid. When the TA4C compound has a phospholipid, its affinity for the phospholipids constituting the liposome structure of the liposome-like complex (phospholipids without TA4C) increases, thus enabling the formation of a better liposome-like complex. Moreover, when group A is a phospholipid, the TA4C compound can function as one of the phospholipids forming the liposome structure. The phospholipid may be a glycerophospholipid or a sphingophospholipid. A glycerophospholipid is a phospholipid with a glycerol backbone. A sphingophospholipid is a phospholipid with a sphingosine backbone. Glycerophospholipids are more preferred as the phospholipid in group A. This allows for the formation of a more stable liposome structure. While not limited to these, preferred phospholipids include, for example, DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine derivative group), DMPE (1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine derivative group), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine derivative group, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine derivative group, and 1,2-dioleoyl-sn-glycero-phosphatidylethanolserine derivative group.

[0018] Furthermore, in group A, a steroid skeleton-containing group is a group that contains a steroid skeleton as its main skeleton. The steroid skeleton generally has a tetracyclic ring structure consisting of three 6-membered rings and one 5-membered ring as its basic skeleton. Examples of steroid skeleton-containing groups include groups derived from compounds having a steroid skeleton, and while not limited to these, examples of compounds having a steroid skeleton include cholesterol, cholic acid, deoxycholic acid, testosterone, androgens, estradiol, aldosterol, progesterone, aldosterone, cortisol, corticosterone, cortisone, hydrocortisone, and stigmasterol. The steroid skeleton-containing group may be linked to the TA4C structure using EG (ethylene glycol) as a linker. Cholesterol and cholic acid-EG are particularly preferred as steroid skeleton-containing groups. This allows for the formation of a stable liposome-like complex. Here, EG refers to ethylene glycol and ethylene glycol polymers (e.g., 2-10 mers, i.e., ethylene glycol chains). Cholic acid-EG refers to a group consisting of cholic acid and an ethylene glycol chain. Preferably, the EG (ethylene glycol) portion of cholic acid-EG can be bonded to the TA4C structure.

[0019] The compound of formula (I) may also be in the form of a salt. The salt of the compound of formula (I) is not particularly limited and may be either an acidic salt or a basic salt. Examples of acidic salts include inorganic acid salts such as hydrochloride, hydrobromide, sulfate, nitrate, perchlorate, and phosphate; and organic acid salts such as acetate, propionate, tartrate, fumarate, maleate, malate, citrate, methanesulfonate, and p-toluenesulfonate. Examples of basic salts include alkali metal salts such as sodium salt and potassium salt; alkaline earth metal salts such as calcium salt and magnesium salt; salts with ammonia; and salts with organic amines such as morpholine, piperidine, pyrrolidine, monoalkylamine, dialkylamine, trialkylamine, mono(hydroxyalkyl)amine, di(hydroxyalkyl)amine, and tri(hydroxyalkyl)amine.

[0020] In one embodiment of the present invention, preferred specific compounds of the compound represented by formula (I) are provided, and in this embodiment, the present invention is a compound represented by formula (I) or a salt thereof, [ka] This relates to compounds, or salts thereof, selected from the group consisting of the following.

[0021] The compound of formula (I) can be synthesized by referring to previously reported methods (A. Onoda, N. Inoue, E. Sumiyoshi, T. Hayashi, ChemBioChem, 2020, 21, 1274-1278, and WO 2020 / 175680 A1). An example of the synthesis scheme is shown below. In this reaction, a TA4C compound in which the lipid structure and the TA4C structure are bonded can be obtained by reacting an amine compound having the desired lipid structure with a compound having a TA4C structure and a leaving group. This reaction is an application of Dimroth rearrangement (JT Fletcher, MDHanson, JA Christensen, EM Villa, Beilstein J. Org. Chem. 2018, 14, 2098-2105). This reaction has the advantage of introducing the TA4C structure in one step. Examples of leaving groups, though not limited to these, include the p-nitrophenyl group and the 4-cyano-2,3,5,6-tetrafluorophenyl group.

[0022] General synthesis scheme [ka]

[0023] As a specific example of the above synthesis scheme, the synthesis of alkylTA4C from a precursor TA4C compound is shown below. In the example below, alkylTA4C (hexyl-TA4C, nonyl-TA4C, and dodecyl-TA4C) is obtained by the reaction of an alkylamine with p-nitrophenyl-TA4C.

[0024] Synthesis scheme of alkylTA4C [ka]

[0025] The phospholipids that constitute the liposome-like complex are not particularly limited, as long as they are capable of forming a liposome structure. The phospholipids are distinct from the phospholipids in the TA4C compound described above and do not necessarily have a TA4C structure. The phospholipids may be natural phospholipids, synthetic phospholipids, or artificial phospholipids derived from nature. A phospholipid is defined as a lipid compound having a phosphate ester moiety in its structure. Phospholipids are amphiphilic and capable of forming a lipid bilayer (or multilayer). Phospholipids include, but are not limited to, phosphatidic acid (PA), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidic acid (PA), phosphatidylglycerol (PG), diphosphatidylglycerol (cardiolipin) (CL), sphingomyelin (SM), phospholipids of these with different chain lengths, and their salts, but are not limited to these. For example, phosphatidylglycerol (PG) and its salts (e.g., sodium salt) are examples. Specifically, examples include 1,2-dipalmitoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DPPG) (also known as L-α-dipalmitoylphosphatidylglycerol), 1,2-dimiristoyl-sn-glycero-3-phosphorylglycerol sodium salt (DMPG), distearoylphosphatidylglycerol sodium salt (DSPG), and mixtures of two or more of these. Furthermore, fluorescent or staining phospholipids may be added for analysis or research purposes. An example of such a phospholipid is rhodamine 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (Rhodamine-DHPE).

[0026] The liposome-like complex may or may not contain any other appropriate components. For example, other components may include substances that stabilize the liposome structure (organic compounds, salts, etc.).

[0027] The liposome-like complex may contain a pharmacologically active component within its liposome structure. This allows for the creation of a liposome-protein complex in which the pharmacologically active component is encapsulated within the liposome, as described later. This liposome-protein complex enables the delivery of the pharmacologically active component protected by the liposome, resulting in a liposome-protein complex with excellent targeting capabilities.

[0028] The molar ratio of the compound of formula (I) to the phospholipid is preferably 1:1 to 1:1,000,000. This allows the TA4C structure to be positioned on the liposome surface in a more appropriate amount, enabling the formation of the liposome-protein complex described later. Furthermore, the molar ratio of the compound of formula (I) to the phospholipid is preferably 1:1 to 1:1,000,000, more preferably 1:1 to 1:100,000, and even more preferably 1:10 to 1:100,000.

[0029] In liposome-like complexes, one or more TA4C structures may be arranged in a single liposome-like complex. The number of TA4C structures arranged in a single liposome-like complex may be, for example, one or more, two or more, three or more, five or more, ten or more, or twenty or more. Alternatively, the number of TA4C structures arranged in a single liposome-like complex may be, for example, 1,000 or less, 500 or less, 100 or less, or 50 or less. Furthermore, the number of TA4C structures arranged in a single liposome-like complex can be, for example, 1 to 100 billion, 2 to 1 billion, or 3 to 100,000. The number of TA4C structures can be adjusted by the ratio of the compound of formula (I) to the phospholipid.

[0030] The liposome-like complex of the present invention can be produced by mixing a compound of formula (I) or a salt thereof with the phospholipid. That is, in one embodiment of the present invention, a method for producing the liposome-like complex is provided, comprising the step of mixing a compound of formula (I) or a salt thereof with a phospholipid.

[0031] The mixing of the compound of formula (I) and the phospholipid can be carried out in a solvent. The solvent is preferably capable of dissolving or dispersing the compound of formula (I) and the phospholipid. Furthermore, for the formation of liposome structures, the solvent is preferably a volatile solvent. Volatile solvents are easy to remove. Suitable solvents include, but are not limited to, alcohols such as methanol, ethanol, propanol, and isopropanol, alkyl halides such as chloroform, dichloromethane, and carbon tetrachloride, and mixtures of two or more of these. As a solvent, for example, a mixed solvent of chloroform and methanol (1:10 to 10:1) can be used, and specifically, a chloroform / methanol (2:1) solvent can be used.

[0032] In the production of liposome-like complexes, a mixed solution of the compound of formula (I) and phospholipids can be heated to a temperature of, for example, room temperature (e.g., 20°C) to about 80°C and stirred for 1 to 60 minutes, and then the solvent can be removed to form a lipid film. A buffer solution (e.g., potassium phosphate buffer) is added to this, and after stirring, the mixture is processed with a liposome preparation device (e.g., Avanti Mini-Extruder) to obtain liposome-like complexes in the form of a solution or dispersion. In the liposome preparation device, the liposome-containing solution can be applied to a membrane of an appropriate pore size (e.g., pore size 0.01 to 100 μm, specifically 0.1 μm or 10 μm).

[0033] The liposome-like complex may exist in the solvent in the form of particles. The liposome-like complex may be dispersed in the solvent. Examples of solvents include water, and specifically, buffer solutions (e.g., potassium phosphate buffer) can be used. The average particle size of the liposome-like complex may be, for example, 10 to 100,000 nm, preferably 50 to 10,000 nm, and more preferably 100 to 10,000 nm. The liposome-like complex may have a negative zeta potential. The zeta potential may be, for example, -1 to -100 mV, and more preferably -10 to -50 mV.

[0034] To produce a liposome-like complex containing a pharmacologically active component, the pharmacologically active component can be further added to and mixed with a solution or dispersion of the compound of formula (I) and a phospholipid.

[0035] The liposome-like complexes prepared in this way can serve as material for forming liposome-protein conjugates.

[0036] Liposomes and protein complexes The liposome-protein complex according to the present invention is given by formula (II): [ka] [In the formula, * represents liposomes, # represents proteins, and R represents proteins.] A represents the organic group or hydrogen atom of the amino acid side chain, and R B [This represents an organic group or hydrogen atom in the amino acid side chain.] It is a complex of liposomes and proteins containing the structure represented by [the formula shown].

[0037] The liposome-protein complex described above can be produced by using the liposome-like complex described above. That is, the present invention is a method for producing a liposome-protein complex comprising the structure represented by formula (II) described above, The above liposome-like complex is given by formula (III): [ka] [In the formula, R A represents the organic group or hydrogen atom of the amino acid side chain, and R B [This represents an organic group or hydrogen atom in the amino acid side chain.] The present invention relates to a method for producing a liposome-protein complex, which includes a step of reacting a protein containing a peptide linker represented by [a specific formula] with the liposome.

[0038] Furthermore, the present invention relates to the use of the above-mentioned liposome-like complex for producing the above-mentioned liposome-protein complex.

[0039] Figure 2 shows a schematic diagram (synthesis scheme) illustrating how a liposome-protein complex is formed by the reaction of a liposome-like complex with a protein. As shown in Figure 2, a liposome-like complex having a TA4C structure (triazole-carbaldehyde structure) reacts with a protein to form a liposome-protein complex. Here, the protein that reacts with the liposome-like complex contains the peptide linker of formula (III) above at its N-terminus, and this peptide linker reacts with the carbaldehyde of the TA4C structure to form a triazole-carbaldehyde-linker structure (TA4C-linker structure) that links the liposome and the protein. Specifically, as can be seen from the structures of formulas (II) and (III), the nitrogen atoms of the first and second amino acid residues at the N-terminus react with the carboaldehyde in the TA4C structure to form a five-membered ring (oxoimidazolidine ring), which constitutes the linker structure.

[0040] In equation (II), * represents a liposome, and this liposome is derived from the liposome structure of the liposome-like complex described above. Therefore, the description of the liposome is applied mutatis mutandis to the description of the liposome-like structure described above.

[0041] In equations (II) and (III), R A represents the organic group or hydrogen atom of the amino acid side chain, and R B R represents an organic group or hydrogen atom in the amino acid side chain. A and R B They may be the same or they may be different. A R may be a hydrogen atom. A If R is a hydrogen atom, A The amino acid residue containing R becomes glycine. B R may be a hydrogen atom. B If R is a hydrogen atom, B The amino acid residue having becomes glycine. In a preferred embodiment, R A R can be an organic group in the side chain of an amino acid. B This can be an organic group of an amino acid side chain. The organic group of an amino acid side chain may be an organic group of a naturally occurring amino acid (L-type amino acid and / or D-type amino acid), or it may be an organic group of an artificial amino acid side chain.

[0042] While not limited to these, L-type amino acids are examples of naturally occurring organic groups in amino acid side chains. Specifically, these include groups selected from those circled with dashed lines below. These also encompass the corresponding D-type amino acids. Below, the amino acids from which the side chains originate are shown, with the amino acid side chain groups circled with dashed lines and the amino acid names listed together. [ka]

[0043] The amino acid side chain groups described above may be further modified, such as by being protected by a protecting group.

[0044] Other organic groups for amino acid side chains include, for example, alkyl groups such as ethyl, propyl, and butyl groups, hydroxyalkyl groups, phenylalkyl groups, hydroxyphenylalkyl groups, and 4-azidophenylmethyl groups. In addition to the α-amino acids listed above, other examples such as β-amino acids and γ-amino acids can also be listed, but are not limited to these. [ka]

[0045] In equation (II), # represents a protein. This protein corresponds to the protein portion of the liposome-protein complex in Figure 2 (which is depicted as a three-dimensional chain structure in Figure 2). The peptide linker shown in equation (III) is bound to the protein represented by #.

[0046] The peptide linker represented by formula (III) can be the N-terminal region of a protein. The TA4C structure described above can selectively and specifically react with the N-terminal region of the protein represented by formula (III).

[0047] The method of the present invention allows the TA4C structure to be specifically bound to the N-terminus, making it possible to form a complex with any protein whose N-terminus is unmodified. However, since this involves a cyclization reaction between the first and second amino acids of the N-terminus, proteins whose second amino acid residue from the N-terminus is proline are excluded. Even for proteins with an N-terminal portion that cannot be directly used in the above method, the N-terminal portion may be modified or altered to prepare a protein that is N-terminally unmodified and whose second amino acid residue is not proline, so that it can bind liposomes by the above method and form a complex between the liposome and the protein. Proteins are generally composed of linked amino acid residues and are also called peptides or polypeptides. Therefore, the present invention makes it possible to form a complex between liposomes and peptides, or between liposomes and polypeptides.

[0048] The proteins used may be those derived from living organisms. Artificial proteins may also be used. Examples of proteins, though not limited to those listed, include enzymes, antibodies, plasma components, fluorescent proteins, peptides, and hormones. Specifically, examples of enzymes include RNases (e.g., RNase A), aldolases, lipases, esterases, and alkaline phosphatases. Examples of antibodies include trastuzumab (anti-HER2 humanized monoclonal) and anti-transferrin receptor antibodies. Examples of plasma components include bovine serum albumin and human serum albumin (HSA). Examples of fluorescent proteins include green fluorescent protein (GFP), red fluorescent protein (mCherry), yellow fluorescent protein (YFP), insulin, and growth hormone.

[0049] In formula (II), # represents a protein, and this protein includes the enzymes, antibodies, plasma components, fluorescent proteins, peptides, hormones, etc. For example, peptides or polypeptides composed of linked amino acid residues are included. Here, the amino acid residue may be an organic group of a side chain of a naturally occurring amino acid (L-type amino acid and / or D-type amino acid) or an organic group of an artificial amino acid side chain.

[0050] In one preferred embodiment, the protein is green fluorescent protein (GFP). In this case, targeting can be confirmed by green fluorescence. This allows for the acquisition of a liposome-protein complex for analysis.

[0051] In one preferred embodiment, the protein is an antibody. In this case, a complex with targeting properties capable of binding the antibody to an antigen can be obtained. Furthermore, if the liposome encapsulates a drug, it becomes possible to deliver the liposome and the drug within the liposome to the target site. This makes it possible to obtain a liposome-protein complex with excellent targeting properties.

[0052] In one preferred embodiment, the protein is an enzyme. In this case, it is possible to target the enzyme. Furthermore, if the liposomes encapsulate a drug, it becomes possible to deliver the liposomes and the drug within the liposomes to the target site. This makes it possible to obtain a liposome-protein complex with excellent targeting capabilities.

[0053] In the liposome-protein complex described above, the substance delivered by targeting may be the protein itself, the liposome itself, or the active substance within the liposome. In one embodiment of the present invention, the protein has targeting properties, causing the liposome-protein complex to reach a target site, and the liposome or the active substance within the liposome can exert a pharmacological effect. In another embodiment of the present invention, the liposome has targeting properties, causing the liposome-protein complex to reach a target site, and the protein can exert a pharmacological effect. Alternatively, in yet another embodiment of the present invention, both the protein and the liposome have targeting properties, and both the protein and the liposome or the active substance within the liposome can exert a pharmacological effect. Thus, by varying the protein, liposome, and active substance, it is possible to obtain liposome-protein complexes with various functionalities.

[0054] In a liposome-protein complex, it is preferable that 1 to 100 proteins are bound to one liposome, more preferably 1 to 50 proteins, even more preferably 1 to 10 proteins, and even more preferably 1 to 5 proteins. Alternatively, 1 to 3 proteins (e.g., 1, 2, or 3) may be bound to one liposome. The number of proteins per liposome may vary depending on the size of the liposome. As described above, the liposome-like complex can have multiple TA4C structures that act as linkers, and since it can bind to proteins with these multiple TA4C structures, it is possible to bind to multiple proteins.

[0055] In a liposome-protein complex, it is preferable that the liposome encapsulates a pharmacologically active component. This allows the active component to be released from the liposome to impart a pharmacological effect to the living organism (e.g., treatment, prevention, procedure, inhibition, activation, etc.). The pharmacologically active component may be a drug. Any drug that can be encapsulated in the liposome can be used in the above-described liposome-protein complex. The pharmacologically active component may be a hydrophobic compound or a hydrophilic compound. Furthermore, the pharmacologically active component may be a low-molecular-weight compound (e.g., molecular weight less than 1000) or a high-molecular-weight compound (e.g., molecular weight 1000 or more). Furthermore, the pharmacologically active component may be a biologically derived substance or an artificial bio-like substance (e.g., protein, peptide, polypeptide, nucleic acid, etc.). Examples of pharmacologically active components, though not limited to these, include doxorubicin, paclitaxel, irinotecan, and verteporfin.

[0056] A liposome-protein complex containing a pharmacologically active component can be obtained by reacting the aforementioned liposome-like complex containing the pharmacologically active component with a protein. Alternatively, a liposome-protein complex containing a pharmacologically active component can also be obtained by forming a liposome-protein complex containing empty liposomes (empty liposomes), and then encapsulating the pharmacologically active component within the liposomes of this liposome-protein complex. The former method (using a liposome-like complex containing the pharmacologically active component) is easier and preferable for encapsulating the component into liposomes.

[0057] In the production of liposome-protein complexes, a liposome-like complex and a protein are mixed and reacted to bind together, thereby obtaining a liposome-protein complex. For the synthesis method, refer to WO 2020 / 175680 A1, which discloses compounds (TA4C compounds) that can modify the N-terminus of proteins and specifically bind to the N-terminus of the protein. The reaction can be carried out in the presence of a solvent. The solvent is not particularly limited, but examples include water and water-soluble organic solvents (alcohols, ketones, etc.). The solvent may be used alone or in combination of multiple solvents. It is also preferable to add a buffer such as a phosphate buffer to the solvent. When water is used, the pH of this reaction is preferably near neutral from the viewpoint of N-terminus selectivity, specifically 6 to 8.5, more preferably 6.5 to 8, and even more preferably 7 to 7.5. In addition to the above components, additives may be used as appropriate in the reaction, as long as they do not significantly impair the progress of the reaction.

[0058] The reaction can be carried out under heating, room temperature, or cooling conditions, and is generally preferred at a temperature that does not significantly denature the protein or peptide, for example, 0 to 45°C (particularly 0 to 40°C). The reaction time is not particularly limited and can be 8 to 36 hours, particularly 12 to 24 hours. The progress of the reaction can be monitored by conventional methods such as chromatography. After the reaction is complete, the product can be isolated and purified as needed by conventional methods such as chromatography or dialysis. For example, size exclusion chromatography can be used as the chromatography method.

[0059] According to the present invention, by preparing liposomes in which lipid molecules containing the 1H-1,2,3-triazole-4-carbaldehyde (TA4C) moiety, a protein N-terminal modifier, are pre-embedded in a lipid bilayer, a desired protein can be linked to the bilayer surface with its N-terminus aligned. Compared to conventional techniques, this technique allows for more efficient targeting of specific cells or tissues because the protein orientation can be aligned when linked to the bilayer. Another advantage is the simplicity of complex preparation, as no other reagents such as condensing agents are required for linking to the bilayer.

[0060] In this context, the present inventors have previously discovered a method for forming liposome-protein complexes in which a hydrophobic molecule is ligated to the N-terminus of a protein, and then mixed with a bilayer such as a liposome to align the protein's orientation and immobilize it on the liposome. However, this method has problems such as the need to purify the protein to which the hydrophobic molecule is ligated to the N-terminus, and the difficulty in ligating particularly highly hydrophobic molecules. Therefore, there were limitations on the proteins that could be used. Furthermore, there were limitations such as the limited selection of hydrophobic molecules for binding liposomes and proteins. In contrast, the method of the present invention first forms a liposome-like complex (i.e., a liposome) having a TA4C structure capable of binding to proteins, and then binds the liposome and protein. This makes it possible to use a wide variety of proteins, and to obtain liposome-protein complexes with a wide range of applications. [Examples]

[0061] The present invention will be described below with reference to examples, but the present invention is not limited to these examples.

[0062] Experimental Example 1 Synthesis of triazole compounds (TA4C compounds) Equipment used Nuclear magnetic resonance (NMR) spectra were measured using Bruker ULTRASHIELD 300 and JEOL RESONANCE ECZ500R nuclear magnetic resonance spectrometers, and chemical shifts were calculated using the residual signal of the measurement solvent as an internal reference. For electrospray ionization-based time-of-flight mass spectrometry (ESI-TOF MS), a Bruker micrOTOF focus III mass spectrometer was used, with methanol or acetonitrile (both HPLC grade) as the mobile phase. Liposomes were prepared using an Avanti Mini-Extruder and a polycarbonate membrane with a pore size of 0.1 μm. Liposome particle size and zeta potential were measured using a Malvern Zetasizer Nano Series. The prepared liposomes were also observed using a Leica TCS SP5 confocal microscope.

[0063] Reagents and solvents, etc. The reagents and solvents used in the synthesis were commercially available products. The precursor compounds for the triazole compounds (precursor triazoles) were synthesized based on previously reported methods (A. Onoda, N. Inoue, E. Sumiyoshi, T. Hayashi, ChemBioChem, 2020, 21, 1274-1278, and WO 2020 / 175680 A1).

[0064] Example 1-1 Synthesis of 1,2-dioleoyl-sn-glycero-3-phospho-2-(4-formyl-1H-1,2,3-triazol-1-yl)ethanol (DOPE-TA4C, Compound 1) Synthesis scheme of compound 1 [ka]

[0065] Compound 1 (1,2-dioleoyl-sn-glycero-3-phospho-2-(4-formyl-1H-1,2,3-triazole-1-yl)ethanol) (DOPE-TA4C) was synthesized based on previously reported information (A. Onoda, N. Inoue, E. Sumiyoshi, T. Hayashi, ChemBioChem, 2020, 21, 1274-1278, and WO 2020 / 175680 A1). The specific synthesis procedure and the results of compound identification are shown below.

[0066] Compound 1 (DOPE-TA4C) was obtained by adding a THF solution (200 μL) of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) (15 mg, 20 μmol), a THF solution (600 μL) of 4-cyano-2,3,5,6-tetrafluorophenyl-1,2,3-triazole-4-carbaldehyde (16 mg, 60 μmol) (600 μL) and an aqueous solution of 3-morpholinopropanesulfonic acid (1 M, 6 μL) to a reactor and stirring at 60°C for 12 hours. As a byproduct, 4-cyano-2,3,5,6-fluoroaniline may be formed, but this may or may not be removed.

[0067] Figure 3 shows DOPE's 1 1H NMR spectrum and DOPE-TA4C (compound 1) 1 The 1H NMR spectrum is shown. Figure 4 shows the MS spectrum of DOPE-TA4C (compound 1).

[0068] Examples 1-2 Synthesis of 1,2-Dimyristoyl-sn-glycero-3-phospho-2-(4-formyl-1H-1,2,3-triazol-1-yl)ethanol (DMPE-TA4C, Compound 2) [ka]

[0069] Compound 2 (1,2-dimiristoyl-sn-glycero-3-phospho-2-(4-formyl-1H-1,2,3-triazole-1-yl)ethanol) (DMPE-TA4C) was synthesized using 1,2-dimiristoyl-sn-glycero-3-phosphoethanolamine as a precursor, by the same method as DOPE-TA4C (Compound 1) described above. Compound 2 (DMPE-TA4C) was obtained by adding a chloroform-methanol mixed solution (200 μL) of 1,2-dimiristoylglycero-3-phosphoethanolamine (DMPE) (13 mg, 20 μmol), a chloroform-methanol mixed solution (200 μL) of 4-cyano-2,3,5,6-tetrafluorophenyl-1,2,3-triazole-4-carbaldehyde (5 mg, 20 μmol), and an aqueous solution of 3-morpholinopropanesulfonic acid (1 M, 2 μL), and stirring at 60°C for 6 hours. The by-product 4-cyano-2,3,5,6-fluoroaniline was not removed. Figure 5 shows Compound 2 and DMPE. 1 The 1H NMR spectrum is shown. Figure 6 shows the ESI-TOF MS spectrum of compound 2.

[0070] Examples 1-3 Synthesis of 1,2-Dipalmitoyl-sn-glycero-3-phospho-2-(4-formyl-1H-1,2,3-triazol-1-yl)ethanol (Compound 3) [ka]

[0071] Compound 3 (1,2-dipalmitoyl-sn-glycero-3-phospho-2-(4-formyl-1H-1,2,3-triazole-1-yl)ethanol) was synthesized using 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine as a precursor, by the same method as for DPPE-TA4C (compound 3) described above. Figure 7 shows the results for compound 3 and DPPE. 1 The 1H NMR spectrum is shown.

[0072] Examples 1-4 Synthesis of 1,2-Distearoyl-sn-glycero-3-phospho-2-(4-formyl-1H-1,2,3-triazol-1-yl)ethanol (Compound 4) [ka]

[0073] Compound 4 (1,2-distearoyl-sn-glycero-3-phospho-2-(4-formyl-1H-1,2,3-triazole-1-yl)ethanol) was synthesized using 1,2-distearoyl-sn-glycero-3-phosphoethanolamine as a precursor, by the same method as for DPPE-TA4C (compound 4) described above. Figure 8 shows the results for compound 4 and DPPE. 1 The 1H NMR spectrum is shown. Figure 9 shows the ESI-TOF MS spectrum of compound 4.

[0074] Examples 1-5 Synthesis of cholic acid-EG3-TA4C (compound 5) [ka]

[0075] Cholic acid-EG3-TA4C (compound 5) was synthesized based on previously reported information (A. Onoda, N. Inoue, E. Sumiyoshi, T. Hayashi, ChemBioChem, 2020, 21, 1274-1278, and WO 2020 / 175680 A1). The specific synthesis procedure and compound identification results are shown below.

[0076] DMSO (111 μL), cholic acid (6.7 mg, 1.75 eq.), DMT-MM (4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium Chloride) (9.1 mg, 3.5 eq.), triethylamine (2.28 mL, 1.75 eq.), and Boc-TOTA (N-Boc-4,7,10-trioxa-1,13-tridecanediamine) (3 mg, 1 eq.) were added in that order, and the mixture was reacted at 25°C for 1 hour. After that, water was added to the reaction solution, and the target product was extracted with ethyl acetate. After removing the solvent from the organic phase under reduced pressure, the resulting crude product was purified by silica gel chromatography (methanol:dichloromethane = 1:9) to obtain cholic acid-EG3 amine. Next, 50 mM cholic acid-EG3 amine (1.11 mL, 55.5 μmol) and 4-cyano-2,3,5,6-tetrafluorophenyl-1,2,3-triazole-4-carbaldehyde (15 mg, 55.5 μmol) were added to 1 M MOPS aqueous solution (5.55 μL), and the mixture was reacted at 37 °C for 12 hours. Water was added to the reaction solution, and extraction was performed with ethyl acetate. The solvent from the organic phase was then removed under reduced pressure to obtain compound 5 as an oil.

[0077] Examples 1-6 Synthesis of alkylTA4C Alkyl TA4C was synthesized using the previously reported methods (A. Onoda, N. Inoue, E. Sumiyoshi, T. Hayashi, ChemBioChem, 2020, 21, 1274-1278, and WO 2020 / 175680 A1). Three types of alkyl TA4C (with 6, 9, or 12 carbon atoms) were obtained. [ka]

[0078] Example 2 Preparation of liposome-like complexes Equipment used Liposome-like complexes (also simply called liposomes) were prepared using an Avanti Mini-Extruder liposome preparation device and a polycarbonate membrane with a pore size of 0.1 μm. The particle size and zeta potential of the liposomes were measured using a Malvern Zetasizer Nano Series particle size and theta potential analyzer. The prepared liposomes were also observed using a Leica TCS SP5 confocal microscope.

[0079] Reagents and solvents, etc. Ultrapure water purified using Milli-Q IQ 7005 was used. Other reagents and solvents were commercially available and used as is.

[0080] Preparation of DOPE-TA4C-containing liposome-like complexes All lipids used below were dissolved in chloroform and methanol (2:1). A solution of DOPE-TA4C (compound 1) (40 μL, 2 mM, 80 nmol), a cholesterol solution (200 μL, 2 mM, 400 nmol), a phosphatidylcholine (PC) solution (2 mM, 160 μL, 320 nmol), and an L-α-dipalmitoylphosphatidylglycerol (DPPG) solution (2 mM, 80 μL, 80 nmol) were mixed. To this, a rhodamine 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (Rhodamine-DHPE) solution (10 μL, 0.1 mM, 1 nmol) was added and the mixture was thoroughly stirred. The solvent was removed using an evaporator, and a lipid film was formed on the flask wall. Potassium phosphate buffer (100 mM, pH 7.5, 4 mL) was added to this mixture, and it was hydrated by standing at 4°C overnight. Then, using an Avanti Mini-Extruder, the mixture was passed through a membrane with pore sizes of 0.1 μm to 10 μm to prepare liposome-like complexes (i.e., liposomes). The particle size and zeta potential of the prepared liposomes were measured and determined using a Malvern Zetasizer Nano Series. The average particle size of the liposomes was 100 nm, and the zeta potential was -30 mV. The results of the particle size measurement by DLS are shown in Figure 10.

[0081] Example 3 Production of liposome-protein complexes

[0082] Example 3-1 Liposomes and green fluorescent protein (GFP) complex As a concrete example of a liposome-protein complex, we fabricated a conjugate generated by binding the above-mentioned liposome-like complex with green fluorescent protein (GFP). The green fluorescent protein (GFP) had an unmodified N-terminal amino group.

[0083] The amino acid sequence of green fluorescent protein (GFP) is shown. MSKGEELFTG VVPILVELDG DVNGHKFSVR GEGEGDATNG KLTLKFICTT GKLPVPWPTL VTTLTYGVQC FSRYPDHMKR HDFFKSAMPE GYVQERTISF KDDGTYKTRA EVKFEGDTLV NRIELKGIDF KEDGNILGHK LEYNFNSHNV YITADKQKNG IKANFKIRHN VEDGSVQLAD HYQQNTPIGD GPVLLPDNHY LSTQSVLSKD PNEKRDHMVL LEFVTAAGIT HGMDELYK (Sequence ID 1).

[0084] Protein N-terminal modifications were performed based on previously reported methods (A. Onoda, N. Inoue, E. Sumiyoshi, T. Hayashi, ChemBioChem, 2020, 21, 1274-1278 and WO 2020 / 175680 A1). The specific experimental procedure is shown below.

[0085] The DOPE-TA4C-containing liposome-like complex prepared in Example 2 above was diluted with potassium phosphate buffer (100 mM, pH 7.5, 2 mL) (100 μM, 200 μL), and a GFP-containing aqueous solution (0.1 μM, 17 μL, 0.0017 nmol, final concentration 8.5 × 10⁻¹⁴) was added to this solution. -15 A μM solution was added and the mixture was shaken at 37°C for 16 hours. This resulted in GFP modification and the production of a liposome-GFP complex (a complex with a TA4C linker).

[0086] Here, as a comparative example to Example 3-1, liposomes (liposomes without a TA4C structure) were prepared using the same materials and methods as above, except that DOPE-TA4C was not used. These liposomes were then mixed with green fluorescent protein (GFP) to produce a comparative example liposome-GFP complex (a complex without a TA4C linker) (referred to as Comparative Example 3-1).

[0087] Figure 11 shows the results of fluorescence microscopy observation of liposome-GFP complexes. The upper panel shows the complex from Example 3-1 (containing TA4C linker), and the lower panel shows the complex from Comparative Example 3-1 (not containing TA4C). From left to right, rhodamine (red), GFP fluorescence (green), and their combined (merged) are shown. From the fluorescence microscopy observation results, it was confirmed that GFP accumulated (bound) to the liposome surface in the complex from Example 3-1. In contrast, no accumulation (binding) of GFP was observed in Comparative Example 3-1.

[0088] Example 3-2 Liposomes were prepared by varying the ratio of DOPE-TA4C. (PC + DPPG + Cholesterol):DOPE-TA4C ratios were 10:1 and 10:0.0001. The DOPE-TA4C-containing liposome-like complex was diluted with potassium phosphate buffer (100 mM, pH 7.5, 2 mL) (100 μM, 200 μL), and GFP aqueous solution (0.1 μM, 10 μL, 0.0017 nmol, final concentration 8.5 × 10⁻¹⁴) was added. -15 A μM solution was added and the mixture was shaken at 37°C for 16 hours. The results of fluorescence microscopy observation of the liposome-GFP complex are shown in Figure 12 for 10:1 and in Figure 13 for 10:0.0001. The fluorescence microscopy results clearly showed that GFP accumulated on the liposome surface in both 10:1 and 10:0.0001 conditions. [Industrial applicability]

[0089] The present invention provides liposome-like complexes and liposome-protein complexes using the same.

Claims

1. Equation (I): 【Chemistry 1】 [In the formula, A represents an organic group.] Compounds represented by or their salts, and Phospholipids, A liposome-like complex containing this complex.

2. The following structure in equation (I): 【Chemistry 2】 The liposome-like complex according to claim 1, wherein the liposome-like complex is arranged on the surface of the liposome-like complex.

3. The liposome-like complex according to claim 1, wherein A is selected from the group consisting of aliphatic hydrocarbon groups, phospholipids, and steroid skeleton-containing groups.

4. A is C ４ -C ２０ A liposome-like complex according to claim 3, selected from the group consisting of alkyl groups, DOPE, DMPE, a 1,2-dipalmitoyl-sn-glycero-3-phosphatidylethanolamine derivative group, a 1,2-distearoyl-sn-glycero-3-phosphatidylethanolamine derivative group, and cholic acid-EG.

5. A method for producing a liposome-like complex according to any one of claims 1 to 4, A method for producing a liposome-like complex, comprising the step of mixing a compound of formula (I) or a salt thereof with a phospholipid.

6. A compound represented by formula (I) according to claim 1, or a salt thereof, wherein the following formula: 【Transformation 3】 A compound, or a salt thereof, selected from the group consisting of the following.

7. Formula (II): 【Chemistry 4】 [In the formula, * represents liposomes, # represents proteins, and R represents proteins.] Ａ represents an organic group or hydrogen atom of the amino acid side chain, and R Ｂ [This represents an organic group or hydrogen atom in the amino acid side chain.] A complex of liposomes and proteins containing the structure represented by [the formula shown].

8. The liposome-protein complex according to claim 7, wherein the protein is green fluorescent protein (GFP).

9. The liposome-protein complex according to claim 7, wherein the protein is an antibody.

10. The liposome-protein complex according to claim 7, wherein the liposome encapsulates a pharmacologically active component.

11. A method for producing a liposome-protein complex comprising a structure represented by formula (II) as described in claim 7, A liposome-like complex according to any one of claims 1 to 4, formula (III): 【Transformation 5】 [In the formula, R Ａ represents an organic group or hydrogen atom of the amino acid side chain, and R Ｂ [This represents an organic group or hydrogen atom in the amino acid side chain.] A method for producing a liposome-protein complex, comprising the step of reacting a protein containing a peptide linker represented by .

12. Use of the liposome-like complex according to any one of claims 1 to 4 for producing the liposome-protein complex according to claim 7.

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