Method for controlling morphology of phospholipid structure and phospholipid structure

Abstract

To provide a method for controlling morphology of a phospholipid structure and a phospholipid structure, which enable control of liposome association and fission by means of a compound that induces domain formation via self-assembly on a phospholipid membrane, and which are applicable to drug delivery systems or the like through encapsulation of biomolecules.SOLUTION: The present invention provides a method for controlling morphology of a phospholipid structure, comprising: adding a specific amphiphilic compound to a phospholipid aqueous solution comprising two or more phospholipid compounds with different phase transition temperatures so as to form a phospholipid structure; subsequently adding an anionic compound so as to be received by the amphiphilic compound; and thus controlling morphology of the phospholipid structure. The present invention also provides a phospholipid structure obtained by the method.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a method for controlling the shape of phospholipid structures that serve as models for structures related to in vivo substance transport, signal transduction, etc., and to phospholipid structures produced by this method. [Background technology]

[0002] Cells in the body perform biological phenomena such as substance transport and signal transduction by deforming their cell membranes. If such structural and morphological changes can be artificially realized, they could be used as tools for analyzing the functions of biomolecules and as carrier materials for drug delivery systems.

[0003] If we could form a phospholipid membrane, which has a cell membrane-like structure, and control the membrane deformation of phospholipid vesicles with artificial molecules, it would be possible to apply it to tools for analyzing the function of biomolecules, carrier materials with high-efficiency substance encapsulation, etc. For these medical and biological applications, it is necessary to control membrane deformation at a constant temperature, such as room temperature or the body temperature.

[0004] As a method for controlling phospholipid membranes, for example, Non-Patent Document 1 reports a membrane-deforming material that utilizes external stimuli such as osmotic pressure and light. Furthermore, Non-Patent Document 2 reports a technique for encapsulating a biopolymer in a liposome prepared by a static hydration method or the like. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] R. Lipowsky, Phys. A 1993, 194, 114-127. [Non-patent document 2] Y. Wang, et al. Mol. Pharmaceutics 2019, 16, 779-785. Summary of the Invention [Problem to be solved by the invention]

[0006] However, it has been thought that it is difficult to design a method to artificially control complex membrane behaviors similar to those occurring in living organisms. Membrane deformation phenomena are generally thermodynamically unstable processes, and can only be temporarily stabilized by external stimuli such as light, osmotic pressure, and electric fields (Non-Patent Document 1). This has made their widespread application to functional materials difficult. Furthermore, although Non-Patent Document 2 is capable of encapsulating biopolymers in liposomes, it does not rely on active transport and has the problem of low encapsulation efficiency.

[0007] The present invention has been made in consideration of the above-mentioned circumstances, and an object of the present invention is to provide a method for controlling the shape of phospholipid structures and phospholipid structures that can control the association and division of liposomes using a compound that induces domain formation by self-assembly on a phospholipid membrane, and that can be applied to drug delivery systems that encapsulate biomolecules, etc. [Means for solving the problem]

[0008] In order to solve the above problems, the present invention has the following aspects. [1] For a phospholipid aqueous solution containing two or more phospholipid compounds with different phase transition temperatures, An amphiphilic compound represented by the following general formula (1) is added to form a phospholipid structure, Next, an anionic compound is added to be accepted by the amphipathic compound, thereby controlling the shape of the phospholipid structure. [ka] (In the formula (1), R1 is hydrogen or a cationic peptide consisting of 1 to 10 amino acid residues, and R2 and R3 are hydrophobic alkyl groups.) [2] The method for controlling the morphology of a phospholipid structure according to [1], wherein the compound of general formula (1) includes any of the compounds represented by formulas (2) to (5). [ka] [ka] [ka] [ka] [3] The method for controlling the shape of a phospholipid structure according to [1] or [2], wherein the anionic compound is an acidic amino acid, a nucleic acid, or a polymer thereof. [4] The method for controlling the morphology of a phospholipid structure according to any one of [1] to [3], wherein the anionic compound is a compound having 2 to 4 aspartic acid groups bound thereto. [5] The method for controlling the shape of a phospholipid structure according to any one of [1] to [4], wherein the phospholipid structure contains the amphipathic compound at 0.8 to 5 mol %. [6] The method for controlling the shape of a phospholipid structure according to any one of [1] to [5], wherein the phospholipid structure further contains cholesterol. [7] The method for controlling the shape of a phospholipid structure according to any one of [1] to [6], wherein one of the phospholipid compounds has a phase transition temperature of 25°C or lower and the other has a phase transition temperature of above 25°C. [8] The method for controlling the shape of a phospholipid structure according to any one of [1] to [7], wherein the phospholipid compound contains dioleoylphosphatidylcholine (DOPC) and dipalmitoylphosphatidylcholine (DPPC). [9] The phospholipid structure forms a substantially uniform layer in an aqueous solution, The method for controlling the morphology of phospholipid structures according to any one of [1] to [8], wherein the layer of the phospholipid structure is made non-uniform and split by adding the anionic compound.

[10] The phospholipid structure forms a liposome containing a phospholipid bilayer in an aqueous solution, The method for controlling the shape of a phospholipid structure according to any one of [1] to [9], wherein the phospholipid structure is made into a liposome having a particle size smaller than that of the liposome by adding the anionic compound.

[11] The method for controlling the shape of phospholipid structures according to

[10] , wherein the anionic compound is added to the liposomes having a small particle size to incorporate a component containing the anionic compound.

[12] A phospholipid structure whose shape is controlled by the method for controlling the shape of a phospholipid structure according to any one of [1] to

[11] .

[13] The phospholipid structure according to

[12] , wherein the component containing the anionic compound is incorporated into a liposome containing a phospholipid bilayer membrane.

[14] The phospholipid structure according to

[12] to

[13] , in which an amino acid, a protein, a virus, or a nucleic acid is incorporated into a liposome comprising a phospholipid bilayer membrane. [Effects of the Invention]

[0009] According to the present invention, it is possible to provide a method for controlling the shape of phospholipid structures and phospholipid structures that can control the association and division of liposomes using a compound that induces domain formation by self-assembly on a phospholipid membrane, and that can be applied to drug delivery systems that encapsulate biomolecules. [Brief explanation of the drawings]

[0010] [Figure 1] 1 shows a schematic diagram and a microscopic image of the effect of atomic force when Asp4 is added to a phospholipid structure containing cationic pep. [Figure 2] FIG. 1 is a graph showing the results of potential measurement when Asp4 was added to a phospholipid structure containing cationic pep formed by static hydration. [Figure 3] 1A and 1B are schematic diagrams and microscopic images showing liposome fission by unilamellar vesicles. [Figure 4] FIG. 1 is a schematic diagram showing liposome fission and DNA encapsulation by unilamellar vesicles and a graph of the FACS results. DETAILED DESCRIPTION OF THE INVENTION

[0011] Hereinafter, the morphology control method of phospholipid structures and the phospholipid structures according to the present invention will be described with reference to embodiments, although the present invention is not limited to the following embodiments.

[0012] (Method for controlling the morphology of phospholipid structures) (Phospholipid structures and compounds used to control morphology) The method for controlling the morphology of phospholipid structures of this embodiment involves adding an amphipathic compound represented by the following general formula (1) to an aqueous phospholipid solution containing two or more types of phospholipid compounds having different phase transition temperatures to form phospholipid structures, and then adding an anionic compound to be accepted by the amphipathic compound to control the morphology of the phospholipid structures.

[0013] [ka] (In the formula (1), R1 is hydrogen or a cationic peptide consisting of 1 to 10 amino acid residues, and R2 and R3 are hydrophobic alkyl groups.)

[0014] Two or more types of phospholipid compounds having different phase transition temperatures can be selected as appropriate, but compounds that have been used to form lipid bilayer membranes or liposomes using conventional techniques can be preferably selected. The combination of two or more phospholipid compounds having different phase transition temperatures is preferably such that when the phospholipid compounds are mixed alone, phase separation occurs. That is, with this configuration, although a combination of phospholipid compounds would normally cause phase separation between them, the inclusion of an amphipathic compound described below can suppress phase separation, and the addition of an anionic compound can further promote phase separation, thereby controlling the association and separation of the phospholipid structures.

[0015] The two or more phospholipid compounds preferably include a combination of two compounds, one of which has a phase transition temperature of 25°C or lower and the other of which has a phase transition temperature of more than 25°C.

[0016] The phospholipid compound preferably contains two or more of dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), and 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC). More preferably, the phospholipid compounds include DOPC and DPPC.

[0017] The amphiphilic compound of this embodiment is a compound represented by general formula (1). In the formula (1), R1 is hydrogen or a cationic peptide consisting of 1 to 10 amino acid residues, and R2 and R3 are hydrophobic alkyl groups. In summary, the hydrophobic alkyl groups of R2 and R3 interact (associate) with the hydrophobic lipid portion of the phospholipid compound to form a lipid bilayer membrane, while the cationic peptide of R1 is configured to interact (bind, accept) with the anionic compound described below. When the cationic peptide R1 interacts with an anionic compound, the charge of the amphipathic compound changes, causing a structural change in the phospholipid structure containing the amphipathic compound in the lipid bilayer. For example, the phospholipid structure undergoes phase separation and splits into smaller units. Hereinafter, the amphipathic compound is also referred to as "cationic pep."

[0018] In other words, R1 consists of 0 to 10 amino acid residues, and R1 is hydrogen when the number of amino acid residues is 0. The number of amino acid residues in R1 is preferably 2 to 5.

[0019] The hydrophobic alkyl groups of R2 and R3 may be appropriately selected so as to be easily associated with the phospholipid compound, depending on the structure of the phospholipid compound. The alkyl group is preferably linear or linear with a partial branch, and although there is no limitation on its length, it is preferably an alkyl group having 10 to 20 carbon atoms.

[0020] More specifically, the amphiphilic compound preferably contains any of the compounds represented by the following formulas (2) to (5).

[0021] [ka] [ka] [ka] [ka]

[0022] The compounds represented by formulas (3) and (4) have NBD (nitrobenzoxadiazole) at the end of R3 in formula (1), which functions as a fluorescent label, but the amphiphilic compound may or may not have NBD. The phospholipid structure preferably contains 0.8 to 5 mol% of the amphipathic compound relative to the total number of moles of the phospholipid structure. If the amount of the amphipathic compound is too small, it becomes difficult to control the morphology by adding an anionic compound, which will be described later. If the amount of the amphipathic compound is too small, it becomes difficult to maintain the morphology of the phospholipid structure and also to control the morphology by adding an anionic compound.

[0023] The phospholipid structure may contain other organic compounds, particularly lipid compounds. For example, the phospholipid structure preferably further contains cholesterol.

[0024] The water used for the phospholipid aqueous solution containing the phospholipid compound may be pure water or may contain ions as appropriate. When the phospholipid compound is not dissolved in the water but the lipid is dispersed in the water, the "phospholipid aqueous solution" can also be called a "phospholipid dispersion." The pH of the water used for the aqueous phospholipid solution can be selected from the range of about 4 to 10. The pH is preferably near neutral (pH 7), 6.5 to 7.5, and more preferably 6.8 to 7.2. Generally, when an amphipathic compound is added to the phospholipid compound contained in the aqueous phospholipid solution, the amphipathic compound and the phospholipid compound associate with each other to form a phospholipid structure.

[0025] The anionic compound to be added to the aqueous solution containing the phospholipid structure can be appropriately selected as long as it is a compound having anionic properties (negative ionic properties), but it is preferable that it is an anionic compound that can be used in vivo. More preferably, it is an acidic amino acid, a nucleic acid, or a polymer thereof.

[0026] The anionic compound is also preferably an oligomer in which several amino acid or nucleic acid residues are polymerized, for example, a compound in which 1 to 20 amino acids or nucleic acids are bound. The anionic compound is more preferably a compound having 2 to 4 aspartic acid groups bonded thereto.

[0027] The anionic compound may be added separately from the substance to be incorporated into the phospholipid structures, or the anionic compound may be the substance itself to be incorporated into the phospholipid structures. For example, when the substance to be incorporated into the phospholipid structures is a nucleic acid or an acidic oligopeptide or polypeptide as a whole, the incorporated substance can function as an anionic compound.

[0028] (Morphology control process) The method for controlling the morphology of phospholipid structures of the present embodiment may include a step of causing the phospholipid structures to form substantially uniform layers in an aqueous solution, and may also include a step of making the layers of the phospholipid structures non-uniform and splitting them by adding the anionic compound.

[0029] In the step of controlling the phospholipid structures, the phospholipid structures are preferably dispersed in an aqueous solution, which preferably contains 1% by mass or less of the phospholipid structures, more preferably 0.1% by mass or less. The temperature can be selected appropriately as long as the aqueous solution is at or below 100°C. It is preferably about 37°C, which corresponds to the in vivo environment. It is also preferably about 25°C, which corresponds to room temperature.

[0030] The method for controlling the morphology of phospholipid structures may include a step of causing the phospholipid structures to form a substantially uniform layer in an aqueous solution. A substantially uniformly formed layer refers to a lipid bilayer membrane with a substantially uniform composition and membrane shape with little bias in composition. For example, it refers to a state in which the two or more phospholipid compounds and amphipathic compounds are distributed substantially uniformly to form a substantially flat membrane. It also refers to a state in which this bilayer membrane has a large phospholipid structure, such as a lipid bilayer membrane vesicle or liposome with a diameter of 0.05 to 50 μm. For example, when an amphipathic compound represented by the general formula (1) is added to an aqueous phospholipid solution containing two or more types of phospholipid compounds having different phase transition temperatures as described above to form a phospholipid structure, the phospholipid structure forms a substantially uniform layer in the aqueous solution.

[0031] The method for controlling the morphology of phospholipid structures may include a step of making the layer of the uniform phospholipid structures non-uniform or disrupting it by adding the anionic compound. Making the layer of uniform phospholipid structures non-uniform includes a process of making the lipid bilayer membrane have a bias in composition depending on the region, such as a patchwork structure in which a certain region contains more of a certain type of phospholipid compound or amphipathic compound than another region. The term "dividing the layer" refers to a process in which the lipid bilayer membrane no longer forms a large membrane or structure and is physically separated, for example, when the layer becomes non-uniform and physically separates due to differences in the properties of the components, and the phospholipid structures are divided and move into an assembly of smaller structures.

[0032] The method for controlling the shape of phospholipid structures of the present embodiment may include a step of forming liposomes containing a phospholipid bilayer membrane from the phospholipid structures in an aqueous solution, and may also include a step of converting the phospholipid structures into liposomes having a smaller particle size than the liposomes by adding the anionic compound.

[0033] The method for controlling the shape of phospholipid structures of the present embodiment may include a step of adding the anionic compound to allow the liposomes having a small particle size to incorporate a component containing the anionic compound. When the anionic compound is added to form liposomes with small particle sizes, the anionic compound is incorporated into the liposomes, but other molecules present in the vicinity are also incorporated into the liposomes. This effect can be used to incorporate the anionic compound and other molecules, particles, etc. into the liposomes.

[0034] (phospholipid structures) The phospholipid structure of this embodiment has its shape controlled by the above-described method for controlling the shape of a phospholipid structure.

[0035] The phospholipid structure has a structure in which a component (component to be incorporated) containing the anionic compound is incorporated into a liposome containing a phospholipid bilayer membrane. The component to be incorporated containing the anionic compound may contain only the anionic compound as described above, or may contain other components. The anionic compound may contain acidic amino acids, proteins, viruses, nucleic acids (DNA, RNA), etc. The other components to be incorporated together with the anionic compound may similarly contain amino acids, proteins, viruses, or nucleic acids, and these may not be acidic compounds as a whole. The component to be incorporated may also be a complex of these.

[0036] Liposomes are small structures made up of lipid bilayer membranes. The phospholipid structure may be, for example, a sac-like structure (vesicle), such as a unilamellar vesicle made of one lipid bilayer membrane or a vesicle made of multiple lipid bilayer membranes.

[0037] The phospholipid structure may have a structure in which the component to be incorporated, that is, an amino acid, a protein, a virus, or a nucleic acid, is incorporated into a liposome comprising a phospholipid bilayer membrane. As described above, if the protein or virus is anionic as a whole, it may be incorporated as an anionic compound. Alternatively, the protein or virus may be incorporated separately from the anionic compound.

[0038] (Effects of this embodiment) According to the present embodiment, it is possible to provide a method for controlling the shape of a phospholipid structure and a phospholipid structure that can be applied to, for example, a drug delivery system that encapsulates biomolecules, by controlling the association and division of liposomes using a compound that induces the formation of domains by self-assembly on a phospholipid membrane. Hydrophilic compounds can be encapsulated inside the phospholipid structures, and hydrophobic compounds can be encapsulated in the membrane of the hydrophobic part of the phospholipid structures.

[0039] If it were possible to control the membrane deformation of phospholipid structures such as phospholipid vesicles using artificial molecules, it could be applied to tools for functional analysis of biomolecules, carrier materials with highly efficient substance encapsulation, and so on. For these medical and biological applications, it is necessary to control membrane deformation at a constant temperature, such as room temperature or the body's internal temperature. This embodiment is characterized by the ability to induce membrane deformation at room temperature through the self-assembly of peptide receptors in response to targets.

[0040] In previously reported cases, membrane deformation was often induced by changing the fluidity of phospholipid membranes through temperature changes, making it difficult to use at room temperature or body temperature, and limiting its practical use. In this embodiment, it is possible to control the phospholipid structure at room temperature, body temperature, etc. Furthermore, it is possible to control the complex membrane behavior, namely membrane deformation, by a simple method in which the phospholipid structure is simply incorporated into a phospholipid membrane and self-assembled. Furthermore, since control can be achieved by having the amphipathic compound accept an anionic compound and changing the charge, control can be achieved even by adding a small amount of anionic compound, and the phospholipid structure can be controlled without significantly changing the pH from the environment inside the organism. This method allows us to design membrane-deforming materials that respond to various targets by designing self-assembling receptors. The molecules of the present invention are the first to be able to control membrane deformation in response to stimuli, achieving effects that could not be achieved with conventional techniques.

[0041] Furthermore, by changing the molecular structure of the hydrophilic portion of the amphiphilic compound (e.g., peptide receptor), the morphology control method and phospholipid structure of this embodiment can design interactions with various targets, making it possible to design membrane-deforming materials that respond to various stimuli. Based on this concept, we have actually succeeded in incorporating anionic biopolymers into vesicles, and by utilizing this to incorporate other biopolymers such as DNA and viruses, the method has industrial applicability as a medical material.

[0042] Although the embodiment of the present invention has been described above, the present invention is not limited to the above embodiment and various modifications can be made. [Example]

[0043] The effects of the present invention will be made clearer by the following examples and comparative examples. Note that the present invention is not limited to the following examples, and can be practiced by making appropriate changes within the scope of the present invention.

[0044] (Compounds used) The peptide-containing amphiphiles used were synthesized by Fmoc solid-phase peptide synthesis. A condensation reagent cocktail of HBTU (2.28 g, 6.01 mmol) and HOBt-HO (0.94 g, 6.12 mmol) was prepared immediately prior to synthesis. A cleavage cocktail of a mixture of DMF (12 mL), DIEA (2.24 mL) and NMP (12 mL), TIS (125 μL), TFA (4.75 mL) and water (125 μL) was prepared immediately prior to synthesis. Fmoc-NH-SAL resin (0.08 mmol) in a polypropylene tube was soaked in DMF (2 mL) for 3 hours at 25 °C. After removing the DMF, 2 mL of 20% piperidine in DMF was added and mixed on a vortex device for 1 minute. After removing the reaction mixture, 2 mL of 20% piperidine in DMF was added, and the reaction tube was shaken at 25 °C for 10 minutes. After removing the reaction mixture, the resin was washed with DMF (2 mL, 5 times), CHCl (2 mL, 3 times), and DMF (2 mL, 3 times). To the resin, 0.24 mmol of Fmoc-protected amino acid dissolved in 560 μL of condensation-reagent cocktail and 560 μL of a mixture of DIEA and NMP were added. After shaking for 20 min at 25° C., the reaction solution was emptied and the resin was washed with DMF (2 mL, 5 times), CH 2 Cl 2 (2 mL, 3 times), and DMF (2 mL, 3 times).

[0045] The piperidine-based Fmoc deprotection and Fmoc-protected amino acid coupling reactions were repeated in the designated order. After the final Fmoc deprotection and washing, acetic anhydride in CHCl (25%, 2 mL) was added to the resin, and the reaction tube was shaken at 25 °C for 10 min. After removing the reaction solution, the resin was washed with CHCl (2 mL, 3 times), DMF (2 mL, 5 times), and CHCl (2 mL, 5 times). Cleavage cocktail (2.5 mL) was added to the resin, and the reaction tube was left at 25 °C for 90 min, with gentle shaking every 30 min. The solution was filtered and collected in a polypropylene centrifuge tube. The reaction tube was washed with TFA (500 μL, 3 times), which was also collected by filtration. EtO (40 mL) was added to the centrifuge tube, mixed on a vortex for 1 min, and centrifuged at 3500 × g for 5 min at 4 °C. The supernatant was then removed. This process was repeated three times, and the peptide was then dried under vacuum at 25°C for 2 hours, dispersed in water, and lyophilized.

[0046] The synthesized molecules were identified by MALDI-TOF MS or ESI-TOF MS. MALDI-TOF MS (2,5-dihydroxybenzoic acid, reflector positive): m / z calculated for Cationic pep (C 66 H 117 N 15 O 10 + ) (Compound represented by the above formula (5)) 1190.05; found 1191.18, Cationic pep (with NBD) (C 66 H 121 N 15 O 10 + ) (compound represented by formula (2) above) 1280.911; found 1280.979, for CaRL Ole (C 66 H 117 N 15 O 10 + ) (Compound represented by the above formula (4)) 1275.677; found 1275.940, CaRL Br (C 74 H 135 N 17 O 12 + ) (Compound represented by the above formula (3)) 1454.08; found 1455.93

[0047] (Test Example 1: Formation of membrane domains by self-assembly of cationic pep) In this example, a compound of formula (5) was used as the cationic peps, i.e., an amphiphilic compound that is positively charged in the near-neutral pH range. A lipid membrane was formed in an aqueous solution containing DOPC, DPPC, and cholesterol. To this lipid membrane, an anionic compound, aspartic acid tetrapolymer (Asp4), was added to attempt to control the properties of the lipid membrane.

[0048] Figure 1 shows a schematic diagram and a microscopic image of the effect of atomic force when Asp4 is added to a phospholipid structure containing cationic pep. A lipid thin film was prepared by mixing DOPC, DPPC, cholesterol, and cationic pep in a ratio of 4:4:2:0.1, and 0.5 mM Asp4 was added. The solvent was an aqueous dispersion, and the test was performed at room temperature (25°C). (a) is a schematic diagram of the positively charged cationic pep and the negatively charged (anionic compound) Asp4. (b) is a schematic diagram and microscopic image of a lipid thin film made by mixing DOPC, DPPC, cholesterol, and cationic pep in a ratio of 4:4:2:0.1. (c) is a schematic diagram and microscopic image of the lipid thin film in (b) with Asp4 added.

[0049] As shown in (b), no domains are observed in the thin film to which cationic pep has been added, indicating that lipid phase separation is suppressed by cationic pep. As shown in (c), when Asp4 is further added, domain shapes due to phase separation are observed, indicating that the electrostatic aggregation of cationic pep has eliminated its function of suppressing phase separation.

[0050] (Test Example 2: Change in liposome surface charge due to self-assembly of cationic pep) Aqueous dispersions of liposomes containing DOPC, DPPC, and cholesterol were prepared by the static hydration method using Cationic Pep, and the potential was measured by adding Asp.

[0051] The static hydration method for preparing vesicle solutions was as follows: 2 mM DOPC / CHCl3 solution (8 μL), 2 mM DPPC / CHCl3 solution (8 μL), 2 mM cholesterol / CHCl3 solution (4 μL), and 0.8 mM cationic pep / MeOH solution (0.5 μL) were added to a 1 mL microtube and dried overnight for at least 5 hours while rotating in a centrifugal evaporator. MilliQ (10 μL) was added and the mixture was shaken in a shaking thermostatic bath at 45°C or higher for 5 minutes. MilliQ (200 μL) was then added, the microtube was capped, and the mixture was shaken at 45°C or higher for at least 5 hours to prepare a vesicle solution.

[0052] FIG. 2 is a graph showing the results of potential measurement when Asp4 was added to a phospholipid structure containing cationic pep formed by static hydration. Zeta potential measurements were performed on an aqueous dispersion of liposomes composed of DOPC (0.08 mM), DPPC (0.08 mM), cholesterol (0.04 mM), and cationic pep (0.0015 mM) with the addition of Asp4 (0.5 mM). The solvent was an aqueous dispersion, and the test was performed at room temperature (25°C). As shown in the figure, it can be seen that the surface charge decreases with the addition of Asp4.

[0053] (Test Example 3: Liposome fission due to self-assembly of cationic pep on unilamellar vesicles) An anionic compound, Asp4, was added to an aqueous dispersion of liposomes made of unilamellar vesicles, which are phospholipid structures containing cationic pep, and a test was conducted to disrupt the liposomes (a test to control the structure of phospholipid structures). FIG. 3 is a schematic diagram and a microscopic image showing liposome fission by unilamellar vesicles. (a) is a schematic diagram of this test. In this example, the phospholipid structures form unilamellar vesicles, i.e., sac-shaped (e.g., hollow, roughly spherical) liposomes formed by one layer of lipid bilayer membrane. When the anionic compound Asp4 is added to these liposomes, the cationic pep accepts Asp4, changing the charge of the cationic pep contained in the liposomes and changing the structure of the phospholipid structures, causing the liposomes formed by unilamellar vesicles to fission into smaller liposomes.

[0054] First, DOPC (0.64 mM), DPPC (0.64 mM), cholesterol (0.64 mM), and cationic peptide (7.2 × 10 -3 An aqueous dispersion of liposomes consisting of 100 mM each was prepared. Vesicle solution preparation by reverse-phase centrifugation was performed as follows. 1 g / L DOPC / Et2O solution (400 μL), 7.47 g / L DPPC / CHCl3 solution (50 μL), and 3.94 g / L cholesterol / CHCl3 solution (50 μL) were added to an Eppendorf tube and mixed. 0.3 M sucrose solution (490 μL) was gently added, and 1 g / L cationic pep / sucrose solution (10 μL) was added to the aqueous phase. Droplets were formed by repeating 5 minutes of shaking and a 50°C water bath twice. The mixture was centrifuged at 21,500 × g and 20°C for 3 minutes, and the organic supernatant was removed. 0.3 M glucose solution (400 μL) was added and pipetted, followed by centrifugation at 100 × g and 20°C for 10 minutes to prepare a vesicle solution. To this vesicle solution, Asp4 (0.5 mM) was added. The solvent was an aqueous dispersion, and the temperature was kept at room temperature (25°C).

[0055] Figure 3 (b) shows a microscopic image before the addition of Asp4, and (c) shows that after the addition of Asp4. Before the addition of Asp4, large liposomes were observed, whereas after the addition of Asp4, small liposomes were observed. These results demonstrated that the addition of the anionic compound Asp4 to phospholipid structures containing cationic pep can disrupt the phospholipid structures and control their structure.

[0056] (Test Example 4: Encapsulation of DNA using liposome fission caused by self-assembly of cationic pep on unilamellar vesicles) Using the same structural control of liposomes made of unilamellar vesicles as in Test Example 3, a test was carried out to incorporate (encapsulate) DNA into liposomes. Figure 4 shows a schematic diagram of liposome fission and DNA encapsulation by unilamellar vesicles, along with a graph of the FACS results. (a) is a schematic diagram of this test. In this example, phospholipid structures form unilamellar vesicle liposomes. When DNA, an anionic compound and the target for encapsulation, is added to these liposomes, the cationic pep accepts the DNA, the charge of the cationic pep contained in the liposomes changes, the structure of the phospholipid structures changes, and the unilamellar vesicle liposomes are fissioned into smaller liposomes. During this process, the DNA is incorporated into the liposomes.

[0057] First, an aqueous dispersion of liposomes consisting of DOPC (0.3 mM), DPPC (0.3 mM), cholesterol (0.16 mM), and cationic pep (0.029 mM) was prepared by reverse-phase centrifugation. Vesicle solutions were prepared by reverse-phase centrifugation as follows: 15 μL of 8 g / L DOPC / CHCl3 solution, 15 μL of 8 g / L DPPC / CHCl3 solution, and 15 μL of 2 g / L cholesterol / CHCl3 solution were added to an Eppendorf tube and heated in a 45°C water bath for 2 minutes. After returning to room temperature, 205 μL of Et2O and 250 μL of 0.3 M sucrose solution were gently added, and 25 μL of 1 g / L cationic pep / sucrose solution was added to the aqueous phase. After pipetting, the mixture was shaken for 15 minutes to form droplets. The mixture was centrifuged at 21,500 × g and 20°C for 2 minutes to remove the organic supernatant. The Eppendorf tube was then left at room temperature with the lid open for 30 minutes to remove the organic phase. A 0.3 M glucose aqueous solution (250 μL) was added and pipetted, followed by centrifugation at 100×g and 20° C. for 10 minutes to prepare a vesicle solution. DNA (Sheared Salmon Sperm DNA, manufactured by Funakoshi, catalog number F012, 0.46% by mass) was added to this vesicle solution. The solvent was an aqueous dispersion, and the temperature was kept at room temperature (25°C).

[0058] Figure 4(b) shows the FACS distribution diagram after the addition of DNA. When the area corresponding to the liposomes with high side scattering (right panel, enlarged view of the left panel) was selected and observed from the entire FACS distribution diagram (left panel), FITC-A fluorescence was confirmed, indicating that DNA was encapsulated in the liposomes. These results showed that adding DNA, an anionic compound, to phospholipid structures containing cationic pep can disrupt the phospholipid structures and allow DNA to be incorporated into the phospholipid structures.

[0059] Although the embodiments of the present invention have been described above, these embodiments are presented as examples and are not intended to limit the scope of the invention. These embodiments can be embodied in various other forms, and various omissions, substitutions, and modifications can be made without departing from the spirit of the invention. These embodiments and their modifications are included within the scope and spirit of the invention, as well as within the scope of the invention and its equivalents as defined in the claims. [Industrial Applicability]

[0060] According to the present invention, it is possible to provide a method for controlling the shape of phospholipid structures and phospholipid structures that can control the association and division of liposomes using a compound that induces domain formation by self-assembly on a phospholipid membrane, and that can be applied to drug delivery systems that encapsulate biomolecules.

Claims

1. For a phospholipid aqueous solution containing two or more phospholipid compounds having different phase transition temperatures, An amphiphilic compound represented by the following general formula (1) is added to form a phospholipid structure, Next, an anionic compound is added to be accepted by the amphipathic compound, thereby controlling the shape of the phospholipid structure. 【Chemical 1】 (In the formula (1), R1 is hydrogen or a cationic peptide consisting of 1 to 10 amino acid residues, and R2 and R3 are hydrophobic alkyl groups.)

2. The method for controlling the morphology of phospholipid structures according to claim 1, wherein the compound of general formula (1) includes any of compounds represented by formulas (2) to (5). 【Chemistry 2】 【Chemistry 3】 【Chemistry 4】 【Chemistry 5】

3. The method for controlling the shape of phospholipid structures according to claim 1 or 2, wherein the anionic compound is an acidic amino acid, a nucleic acid, or a polymer thereof.

4. The method for controlling the morphology of phospholipid structures according to claim 1 or 2, wherein the anionic compound is a compound having 2 to 4 aspartic acid groups bound thereto.

5. The method for controlling the shape of a phospholipid structure according to claim 1 or 2, wherein the phospholipid structure contains 0.8 to 5 mol % of the amphipathic compound.

6. The method for controlling the shape of a phospholipid structure according to claim 1 or 2, wherein the phospholipid structure further contains cholesterol.

7. The method for controlling the shape of a phospholipid structure according to claim 1 or 2, wherein one of the phospholipid compounds has a phase transition temperature of 25°C or lower and the other has a phase transition temperature of more than 25°C.

8. 3. The method for controlling the shape of phospholipid structures according to claim 1 or 2, wherein the phospholipid compound comprises dioleoylphosphatidylcholine (DOPC) and dipalmitoylphosphatidylcholine (DPPC).

9. the phospholipid structures form a substantially uniform layer in an aqueous solution, The method for controlling the morphology of phospholipid structures according to claim 1 or 2, wherein the layers of the phospholipid structures are made non-uniform and split by adding the anionic compound.

10. the phospholipid structure forms a liposome containing a phospholipid bilayer in an aqueous solution, 3. The method for controlling the shape of phospholipid structures according to claim 1 or 2, wherein the phospholipid structures are converted into liposomes having a particle size smaller than that of the liposomes by adding the anionic compound.

11. The method for controlling the shape of phospholipid structures according to claim 10, wherein the anionic compound is added to cause the small-particle liposomes to incorporate a component containing the anionic compound.

12. A phospholipid structure whose morphology has been controlled by the method for controlling the morphology of a phospholipid structure according to claim 1 or 2.

13. The phospholipid structure according to claim 12, wherein the component containing the anionic compound is incorporated into a liposome containing a phospholipid bilayer membrane.

14. The phospholipid structure according to claim 12, wherein an amino acid, a protein, a virus, or a nucleic acid is incorporated into a liposome comprising a phospholipid bilayer membrane.

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