WO2012117587A1 - Procédé de fabrication d'une structure lipidique - Google Patents

Procédé de fabrication d'une structure lipidique Download PDF

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Publication number
WO2012117587A1
WO2012117587A1 PCT/JP2011/069337 JP2011069337W WO2012117587A1 WO 2012117587 A1 WO2012117587 A1 WO 2012117587A1 JP 2011069337 W JP2011069337 W JP 2011069337W WO 2012117587 A1 WO2012117587 A1 WO 2012117587A1
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lipid
liposome
nanoparticle
particles
nanoparticles
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PCT/JP2011/069337
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English (en)
Japanese (ja)
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善浩 佐々木
一成 秋吉
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国立大学法人東京医科歯科大学
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Publication of WO2012117587A1 publication Critical patent/WO2012117587A1/fr

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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J13/00—Colloid chemistry, e.g. the production of colloidal materials or their solutions, not otherwise provided for; Making microcapsules or microballoons
    • B01J13/02—Making microcapsules or microballoons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • A61K9/1274—Non-vesicle bilayer structures, e.g. liquid crystals, tubules, cubic phases, cochleates; Sponge phases

Definitions

  • the present invention relates to a method for producing a lipid structure.
  • Liposomes self-assemble to form stable molecular aggregates and are used as basic materials in basic biology, medicine, pharmacy and engineering. Liposomes are known as lipid molecule aggregates formed from lipids including naturally derived phospholipids. Liposomes are prepared by an ultrasonic irradiation method, a stationary hydration method, or the like. However, since liposomes are spherical, their fields of use are limited.
  • lipid nanotubes which are aggregates of lipid molecules in the form of nanotubes
  • they can be applied to 3D artificial cell arrays, biochips, microreactors, drug carriers, templates for producing inorganic nanostructures, nerve cells, immune cells, etc.
  • studies have been actively conducted because of expectations for elucidating the mechanism of intercellular communication such as the above.
  • lipid nanotubes nanotubes
  • a method of producing a lipid nanotube by self-assembling a synthetic lipid having a specific structure is known (see, for example, Chemistry of Materials 2008, 20, 625 and Nature Communications 2010, 1, 20).
  • a method is known in which a part of a lipid membrane of a liposome is sucked with a micropipette using a micropipette suction technique, and the part is physically stretched to form a lipid nanotube part one by one ( For example, see Japanese translations of PCT publication No. 2004-509778 and Langmuir 2001, 17, 6754).
  • a specific biomolecule is added to the liposome, and the movement of the biomolecule is used to grow part of the lipid membrane into a lipid nanotube structure (see, for example, The EMBO Journal 2005, 24, 1537).
  • a technique for adding a specific substance such as ganglioside to grow a part of a lipid membrane into a lipid nanotube structure is known.
  • an object of the present invention is to produce a lipid structure having a lipid tube part such as a lipid nanotube part by a simple method with high efficiency and controllability, and having a wide range of choice of lipid to be used. Is to provide a method.
  • ⁇ 1> preparing a particle-encapsulating liposome comprising a liposome and particles encapsulated in the liposome; By moving particles in the particle-encapsulating liposomes by an external field, by extending a part of the lipid membrane of the liposomes by the particles, and forming a lipid tube part; Have When the lipid tube part is formed by extending a part of the lipid membrane of the liposome, the liposome is restricted from moving in the external field, so that the particle pushes out the lipid membrane of the liposome.
  • a method for producing a lipid structure that acts on the skin is
  • ⁇ 2> The method for producing a lipid structure according to ⁇ 1>, wherein the external field is an electric field, a magnetic field, or an inertial force.
  • the lipid tube part is formed by extending a part of the lipid membrane of the liposome, the liposome is held on a substrate or in a gel according to ⁇ 1> or ⁇ 2>.
  • ⁇ 4> The method for producing a lipid structure according to any one of ⁇ 1> to ⁇ 3>, wherein the particles have a volume average particle diameter of 10 nm to 500 nm.
  • ⁇ 5> The method for producing a lipid structure according to any one of ⁇ 1> to ⁇ 4>, wherein the particle-encapsulating liposome has a volume average particle diameter of 2 ⁇ m to 100 ⁇ m.
  • ⁇ 6> The method for producing a lipid structure according to any one of ⁇ 1> to ⁇ 5>, wherein the external field is an electric field having a strength of 2.0 kV / m to 10.0 kV / m.
  • ⁇ 8> preparing liposomes; Forming a lipid tube part by extending a part of the lipid membrane of the liposome by deforming the liposome by an external fluid force; and Have When the lipid tube part is formed by extending a part of the lipid membrane of the liposome, the liposome is restricted from moving in the fluid, and a shear flow acts on the lipid membrane of the liposome.
  • a method for producing a lipid structure that is adapted to: ⁇ 9> The method for producing a lipid structure according to ⁇ 8>, wherein when the lipid tube part is formed by extending a part of the lipid membrane of the liposome, the liposome is held on a base material.
  • the lipid structure which has a lipid tube part can be produced with sufficient efficiency and controllability by a simple method, and the manufacturing method of a lipid structure with a wide selection range of the lipid to be used can be provided. .
  • the method for producing a lipid structure of the present invention comprises preparing a particle-encapsulated liposome containing a liposome and particles encapsulated in the liposome (hereinafter also referred to as “preparation step”), and particles in the particle-encapsulated liposome.
  • Preparation step a particle-encapsulated liposome containing a liposome and particles encapsulated in the liposome
  • particles in the particle-encapsulated liposome is formed by extending a part of the lipid membrane of the liposome by the particles by moving by an external field.
  • Another method for producing the lipid structure of the present invention is to prepare liposomes (hereinafter also referred to as “preparing a liposome”), and deforming the liposomes by external fluid force, Extending a part of the lipid membrane of the liposome to form a lipid tube part (hereinafter also referred to as a “lipid tube part forming step”).
  • the liposome comprises the Movement in the fluid is restricted, and a shear flow acts on the lipid membrane of the liposome.
  • the “lipid tube” includes not only a lipid tube having both ends opened, but also a lipid tube having at least one end closed. Liposomes may be supported on both ends or one end of the lipid tube.
  • nanoparticles are a preferred example of particles
  • nanoparticle-encapsulated liposomes are preferred examples of particle-encapsulated liposomes
  • lipid nanotube portions are preferred examples of lipid tube portions.
  • the same members may be denoted by the same reference numerals and description thereof may be omitted.
  • FIG. 1 is a schematic cross-sectional view schematically showing an example of a nanoparticle-encapsulating liposome prepared in the preparation step of the present invention.
  • the nanoparticle-encapsulating liposome 10 includes a liposome having a capsule-like lipid membrane 14 and nanoparticles 12 encapsulated in the liposome.
  • the lipid membrane 14 is a phospholipid bimolecular membrane.
  • the inside of the capsule-like lipid membrane 14 is filled with an inner aqueous phase (an aqueous medium such as water or a buffer solution).
  • the structure of the liposome excluding the nanoparticles 12 from the nanoparticle-encapsulating liposome 10 is the same as the structure of a known liposome.
  • the following method can be used. That is, a solution in which phospholipid is dissolved in an organic solvent is prepared, and the organic solvent is evaporated from the solution to produce a phospholipid membrane. By bringing the prepared phospholipid membrane into contact with a liquid containing nanoparticles and water, a nanoparticle-encapsulating liposome having a structure in which the nanoparticles are incorporated into the liposome can be obtained by self-organization of the phospholipid. .
  • the preparation step in the present invention may be a step of preparing a nanoparticle-encapsulating liposome prepared in advance, or a step of manufacturing a nanoparticle-encapsulating liposome.
  • the process of preparing a liposome is performed.
  • FIG. 2 is a schematic cross-sectional view schematically showing a lipid nanotube part forming step as an example of the lipid tube part forming step in the present invention.
  • the nanoparticle-encapsulating liposome 10 (FIG. 1) is arranged in the external field F
  • the nanoparticles 12 in the nanoparticle-encapsulating liposome 10 are directed from the inside of the liposome to the outside of the liposome by the external field F. Move in the direction.
  • a part of the lipid membrane 14 is pushed out by the nanoparticles 12 to form the lipid nanotube portion 20.
  • 10 A of lipid structures which have the lipid nanotube part 20 are obtained.
  • the lipid membrane (lipid membrane 14 in FIG. 1) after the formation of the lipid nanotube portion is defined as a lipid membrane 14A.
  • the external field F that moves the particles (for example, the nanoparticles 12) is, for example, an electric field, a magnetic field, or an inertial force. However, when the external field F is a fluid force, the particles (for example, the nanoparticles 12) are moved.
  • a part other than the lipid tube part (for example, the lipid nanotube part 20) in the lipid structure (for example, the lipid structure 10A) may be referred to as a liposome part. That is, the lipid structure in the present invention has a structure including a lipid tube part and a liposome part.
  • the inside of a lipid tube part and the inside of a liposome part are connected, and these insides are satisfy
  • the production method of the present invention utilizes the movement of particles (for example, nanoparticles) by an external field, the extrusion of lipid membranes by the movement of particles (for example, nanoparticles), and the elasticity of the lipid membrane.
  • a part of the lipid membrane is extruded to form a lipid tube part (for example, a lipid nanotube part).
  • it is a method of forming a lipid tube part (for example, a lipid nanotube part) by extending a part of the lipid membrane by an external fluid force.
  • the production method of the present invention is simpler and more efficient than a conventional method in which a part of liposome is sucked with a micropipette and physically stretched to form lipid nanotube parts one by one ( This is a manufacturing method with excellent productivity.
  • the size (length) of the lipid tube part (for example, lipid nanotube part) can be easily controlled by controlling the strength of the external field, the time for applying the external field, and the like.
  • the type of lipid used is not limited, and the range of lipid selection is wide. wide.
  • the flexibility required as the lipid nanotube may be impaired. Compared with the method, flexibility is not easily lost.
  • the lipid structure produced by the production method of the present invention is contained in a gel (eg, agarose gel), the obtained lipid structure-containing gel is frozen, and the lipid structure-containing gel is frozen to a lipid tube.
  • a lipid tube (for example, a lipid nanotube) can be easily obtained by cutting out a portion including a portion (for example, a lipid nanotube portion).
  • a part of the lipid membrane of the particle-encapsulated liposome for example, nanoparticle-encapsulated liposome
  • the two liposomes are lipid tubes (for example, lipid nanotubes).
  • a lipid structure having a structure in which the insides of the two liposomes are connected to each other can also be produced. Furthermore, a lipid structure having a structure in which two or more liposomes are bound in a three-dimensional network by lipid tubes (for example, lipid nanotubes) can be prepared.
  • the three-dimensional network lipid structure produced in this way can be used as a three-dimensional artificial cell array or a biochip.
  • FIG. 1 and 2 schematically show an example in which the nanoparticle-encapsulating liposome 10 and the lipid structure 10A encapsulate five nanoparticles 12, and one of these nanoparticles moves.
  • the number of particles (for example, nanoparticles) encapsulated in particle-encapsulated liposomes (for example, nanoparticle-encapsulated liposomes) and the number of moving particles (for example, nanoparticles) are not particularly limited.
  • a plurality of particles (for example, nanoparticles) are included in the particle-encapsulated liposome (for example, nanoparticle-encapsulated liposome), at least a part of the plurality of particles (for example, nanoparticles) may be moved.
  • symbol "12" is attached
  • lipid tube part for example, a lipid nanotube part
  • particles for example, nanoparticles
  • lipid tube part for example, a lipid nanotube part
  • fluid force for a lipid membrane
  • particles for example, nanoparticles
  • a part of the lipid membrane is elongated, so that the lipid A tube part (for example, a lipid nanotube part) can be formed.
  • the particle-encapsulated liposome for example, the nanoparticle-encapsulated liposome
  • the particle-encapsulated liposome is retained on a substrate or in a gel. Is mentioned. These more specific forms will be described later as first to fourth embodiments.
  • a specific form in which the movement of liposomes not encapsulating particles (for example, nanoparticles) is restricted includes a form in which liposomes are held on a substrate. This more specific form will be described later as a fifth embodiment.
  • the structure of the liposome in the present invention (that is, the portion excluding the particles (for example, nanoparticles) in the particle-encapsulated liposomes (for example, nanoparticle-encapsulated liposomes) in the present invention) is not particularly limited, and known liposomes can be used. .
  • known liposomes having a capsule-like lipid membrane mainly composed of a phospholipid bilayer membrane and an inner aqueous phase present in the capsule-like lipid membrane can be used without particular limitation.
  • the liposome in the present invention may be a monolayer liposome as shown in FIGS. 1 and 2, or a multilamellar liposome.
  • the volume average particle diameter of the liposome is preferably 2 ⁇ m to 100 ⁇ m, more preferably 2 ⁇ m to 50 ⁇ m, and particularly preferably 5 ⁇ m to 20 ⁇ m from the viewpoint of the formation of the lipid nanotube portion.
  • the phospholipid is not particularly limited, and may be glycerophospholipid or sphingophospholipid.
  • Specific examples of the phospholipid include lecithin (phosphatidylcholine), cephalin (phosphatidylethanolamine), phosphatidylserine, sphingomyelin and the like.
  • one or more other substances such as biotin and rhodamine may be bound to the phospholipid.
  • the internal aqueous phase and the external aqueous phase of the liposome are water, various buffer solutions (4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid (“HEPES”), trishydroxymethylaminomethane (“TRIS”).
  • HEPES 4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid
  • TMS trishydroxymethylaminomethane
  • An aqueous medium such as “), a buffered aqueous solution of boric acid, phosphoric acid, etc.” can be used without particular limitation.
  • the “external field” is not particularly limited as long as it can move particles (nanoparticles), but is preferably an electric field, a magnetic field, or an inertial force, for example.
  • the “external field” is not particularly limited as long as it can move particles (nanoparticles), but is preferably an electric field, a magnetic field, or an inertial force, for example.
  • a part of the lipid membrane of the liposome is elongated instead of moving the particles (for example, nanoparticles), fluid force is used.
  • the strength of the electric field is preferably 2.0 kV / m to 10.0 kV / m, more preferably 4.0 kV / m to 7.0 kV / m, from the viewpoint of the formation of the lipid nanotube portion. That is, when the intensity of the electric field is 2.0 kV / m or more, the pushability of the lipid membrane by the movement of the nanoparticles can be further improved. In addition, when the electric field strength is 10.0 kV / m or less, in addition to being able to further suppress the temperature rise, the phenomenon that the nanoparticles break through the lipid membrane (that is, the phenomenon in which the formation of the lipid nanotube portion is impaired). It can be suppressed more.
  • the strength of the magnetic field is preferably from 100 KA / m to 1000 KA / m, more preferably from 200 KA / m to 500 KA / m, from the viewpoint of the formability of the lipid nanotube portion. That is, when the strength of the magnetic field is 100 KA / m or more, the pushability of the lipid membrane due to the movement of the nanoparticles can be further improved. Moreover, when the electric field strength is 1000 KA / m or less, the phenomenon that nanoparticles break through the lipid membrane (that is, the phenomenon in which the formation of the lipid nanotube portion is impaired) can be further suppressed.
  • the inertia force can be obtained by, for example, centrifugal force.
  • Strength of the inertial force from the viewpoint of formation of the lipid nanotube portion, preferably 10km / s 2 ⁇ 100km / s 2, and more preferably 30km / s 2 ⁇ 100km / s 2. That is, when the strength of the inertial force is 10 km / s 2 or more, the pushability of the lipid membrane due to the movement of the nanoparticles can be further improved.
  • the intensity of the inertial force is 100 km / s 2 or less, a phenomenon that nanoparticles break through the lipid membrane (that is, a phenomenon in which the formation of the lipid nanotube portion is impaired) can be further suppressed.
  • the fluid force can be obtained, for example, by circulating an aqueous medium or gas such as the above-described water or various buffer solutions as an external aqueous phase of the liposome using a syringe pump or the like.
  • the strength of the fluid force is preferably a high flow rate within a range in which the movement of the liposome in the fluid can be restricted (the liposome can be retained on the base material) from the viewpoint of the formation of the lipid nanotube portion. That is, by making the flow rate as fast as possible, the extensibility of the lipid membrane by fluid force can be further improved.
  • the particles refer to particles having a volume average particle diameter of 5 ⁇ m or less, for example.
  • the volume average particle diameter of the particles is, for example, from 10 nm to 500 nm, preferably from 10 nm to 300 nm, more preferably from 10 nm to 90 nm, and particularly preferably from 20 nm to 80 nm, from the viewpoint of the formability of the lipid nanotube portion. It is.
  • the particles may be organic particles (resin particles, etc.) or inorganic particles (metal particles, metal oxide particles, semiconductor particles, etc.).
  • the particles may be surface-modified with an anionic group or a cationic group described later.
  • silica nanoparticles As the inorganic particles, silica nanoparticles, alumina nanoparticles, and magnetic nanoparticles (iron oxide nanoparticles, etc.) are preferable.
  • the resin particles are preferably polystyrene nanoparticles, SBR (styrene-butadiene rubber) nanoparticles, and hydrogel nanoparticles.
  • the resin particles are preferably used in the form of a suspension.
  • the particles When an electric field is used as the external field, the particles preferably have a charge.
  • the charge may be a positive charge or a negative charge.
  • Specific forms of the charged particles include a form modified with an anionic group (or cationic group) and a form in which charged particles are charged positively or negatively.
  • the resin particles having an anionic group for example, (1) a monomer having an anionic group (or cationic group) is homopolymerized or copolymerized with other monomers. Resin obtained by addition polymerization of a monomer having an anionic group (or cationic group) to the obtained resin nanoparticle or (2) a resin nanoparticle having no anionic group (or cationic group) Nanoparticles, (3) nanoparticles obtained by surface-modifying nanoparticles not having an anionic group (or cationic group) with a polymer compound having an anionic group (or cationic group), and the like. .
  • anionic group carboxylate group (-COO - group), a sulfonate group (-SO 3 - group), a phosphate group (-PO 4 2-group), such as silanol groups.
  • cationic group include an unsubstituted ammonium group, an ammonium group substituted with an alkyl group (preferably an alkyl group having 1 to 6 carbon atoms), and a guanidinium group.
  • the particles having an anionic group, carboxylate group (COO - groups) at modified polystyrene nanoparticles include silica nanoparticles having a silanol group. Among them, polystyrene nanoparticles modified with a carboxylate group are preferable from the viewpoint of availability.
  • the particles having a cationic group include polystyrene nanoparticles modified with an ammonium group.
  • the charged particles have a zeta potential at pH 7.4 of ⁇ 60 mV to ⁇ 5 mV (more preferably ⁇ 50 mV to ⁇ ) in the case of particles having an anionic group from the viewpoint of the formation of the lipid nanotube portion by an electric field.
  • a cationic group it is preferably +60 mV to +5 mV (more preferably +50 mV to +10 mV).
  • a commercial item can also be used as particle
  • particles having a charge examples include particles made by Polysciences, particles made by Thermo Fisher, and particles made by Micromod.
  • the particles When a magnetic field is used as the external field, the particles preferably have magnetism (magnetism).
  • magnetism magnetism
  • Examples of a method for imparting magnetism (magnetism) to particles include using particles containing iron oxide (for example, iron oxide particles).
  • grains which have magnetism a commercial item can also be used.
  • examples of commercially available particles having magnetism include Merck particles and Micromod particles.
  • the lipid tube part in the present invention is a part obtained by deforming a part of the lipid membrane into a tube shape.
  • the inner diameter of the lipid tube portion is about the same as the volume average particle diameter of the particles (that is, preferably 10 nm to 5 ⁇ m, more preferably 10 nm to 1 ⁇ m, particularly preferably 20 nm to 100 nm).
  • FIG. 3 is a schematic perspective view schematically showing a lipid structure production apparatus 30 suitable for the production method of the present invention as the first embodiment of the present invention.
  • the lipid structure production apparatus 30 includes a cover glass 32 (base material), a gel 34 (for example, agarose gel, polyacrylamide gel, etc.) disposed on the cover glass 32, and the gel 34.
  • a voltage applying means power source
  • the nanoparticle-encapsulating liposome 31 has a configuration in which charged nanoparticles are encapsulated in the liposome. Preferred forms of the nanoparticles and liposomes are as described above.
  • symbol (31) is attached
  • the nanoparticle-encapsulating liposome 31 is held by the gel 34 and disposed in the electric field generated by the anode 36 ⁇ / b> A and the cathode 36 ⁇ / b> B. Since the nanoparticle-encapsulating liposome 31 is held by the gel 34, movement of the nanoparticle-encapsulating liposome 31 excluding a part that becomes the lipid nanotube portion is restricted by the electric field. On the other hand, part of the lipid nanotube part is pushed out by the movement of the nanoparticles by the electric field to become the lipid nanotube part.
  • FIG. 4 is a schematic perspective view schematically showing a lipid structure production apparatus 40 suitable for the production method of the present invention as the second embodiment of the present invention.
  • the lipid structure manufacturing apparatus 40 includes a cover glass chamber 42 (first base material), a cover glass 49 (second base material), a cover glass chamber 42, and a cover glass 49.
  • the cover glass chamber 42 and the cover glass 49 are fixed by a sealant 48 (for example, a silicone seal).
  • the nanoparticle-encapsulating liposome-containing gel 44 is a gel containing the nanoparticle-encapsulating liposome in the present invention.
  • the gel the same gel as the gel exemplified as the gel 34 in the first embodiment can be used.
  • inner_cover liposome the thing similar to the nanoparticle inclusion
  • the nanoparticle-encapsulating liposome is held by a gel and is disposed in an electric field generated by the anode 46A and the cathode 46B.
  • a portion excluding a portion that becomes a lipid nanotube portion is restricted from moving by an electric field, while a portion that becomes a lipid nanotube portion is pushed out by the movement of the nanoparticle and becomes a lipid. It becomes a nanotube part.
  • FIG. 5 is a schematic perspective view which shows typically the lipid structure manufacturing apparatus 50 suitable for the manufacturing method of this invention as the 3rd Embodiment of this invention.
  • the lipid structure production apparatus 50 includes a cell 52 having an anode tank and a cathode tank that are in communication with each other, and a gel 54 that is disposed in a communication portion between the anode tank and the cathode tank of the cell 52.
  • anolyte 55A for example, an aqueous medium such as water or buffer
  • a catholyte 55B for example, an aqueous medium such as water or buffer
  • the nanoparticle-encapsulating liposome 51 present (for example, dispersed) in the catholyte 55B, the anode 56A immersed in the anolyte 55A, the cathode 56B immersed in the catholyte 55B, and the anode 56A-cathode Voltage applying means (power supply) for applying a DC voltage between 56B.
  • the gel 54 the thing similar to the gel 34 in 1st Embodiment can be used.
  • the nanoparticle-encapsulating liposome 51 a nanoparticle-encapsulating liposome having a structure in which nanoparticles having a negative charge are encapsulated in the liposome is used.
  • the nanoparticle-encapsulating liposome 51 can freely move in the catholyte 55B, but cannot move in the gel 54.
  • the nanoparticle-encapsulating liposome 51 moves in the catholyte 55B and moves to the interface with the gel 54 by the electric field.
  • a part of the lipid membrane of the nanoparticle-encapsulating liposome 51 reaching the interface is pushed out into the gel 54 by the nanoparticles, and a lipid nanotube portion is formed in the gel 54.
  • nanoparticle-encapsulating liposomes having a structure in which positively charged nanoparticles are encapsulated are added to the anolyte 55A. Even in this case, a part of the lipid membrane of the nanoparticle-encapsulating liposome that has reached the interface between the anolyte 55A and the gel 54 is pushed out into the gel 54 by the nanoparticles based on the same principle as that of the lipid structure production apparatus 50. The lipid nanotube portion is formed in the gel 54.
  • FIG. 6 is a schematic perspective view which shows typically the lipid structure manufacturing apparatus 60 suitable for the manufacturing method of this invention as the 4th Embodiment of this invention.
  • the lipid structure production apparatus 60 includes a slide glass 62 (base material), nanoparticle-encapsulated liposomes 61 held on the slide glass 62 via a linker 65, and an electric field applied to the nanoparticle-encapsulated liposomes 61.
  • FIG. 6 is the surface of the slide glass 62 that holds the nanoparticle-encapsulating liposome 61 and the aqueous medium (water, buffer solution, etc.) in which the nanoparticle-encapsulating liposome 61 is immersed.
  • the aqueous medium is indicated by a broken line in order to make it easier to see the surface holding the nanoparticle-encapsulating liposome 61.
  • charged nanoparticles are encapsulated in the liposome.
  • the nanoparticle-encapsulating liposome 61 those similar to the nanoparticle-encapsulating liposome 31 in the first embodiment can be used.
  • the nanoparticle-encapsulating liposome 61 is held on the slide glass 62 (base material) by the linker 65, and is disposed in the electric field generated by the anode 66A and the cathode 66B.
  • a portion excluding a portion that becomes the lipid nanotube portion is restricted from moving by the electric field, while a portion that becomes the lipid nanotube portion is in the aqueous medium due to the movement of the nanoparticle. Extruded into the lipid nanotube part.
  • FIG. 7 is a conceptual diagram showing an example of a nanoparticle-encapsulating liposome held on a glass substrate via a linker.
  • DSPE-biotin of nanoparticle-encapsulating liposome 10 modified with DSPE-biotin (DSPE-Biotin) and BSA of glass substrate modified with BSA-biotin (BSA-Biotin) -Biotin is bound via streptavidin.
  • the nanoparticle-encapsulating liposome 10 is held on the glass substrate via a linker composed of DSPE-biotin, streptavidin, and BSA-biotin.
  • linker in the present invention a linker containing biotin and avidin (for example, DSPE-biotin, streptavidin, BSA-biotin, DOPE-biotin, etc.) can be used.
  • the linker in the present invention is not limited to biotin and avidin, and a known linker (for example, linker DNA, disulfide bond, etc.) for connecting a biomolecule and a substrate can be used without particular limitation.
  • FIG. 8 is a schematic perspective view which shows typically the lipid structure manufacturing apparatus 70 suitable for the manufacturing method of this invention as one example of the 5th Embodiment of this invention.
  • one example of the fifth embodiment is a lipid structure manufacturing apparatus 70 in which an inlet 76 ⁇ / b> A and an outlet 76 ⁇ / b> B are provided at both ends in order to control the fluid force (shear flow) applied to the liposome 71.
  • a flow chamber m-Slides VI, ibidi-GmbH, Kunststoff, Germany
  • a lower slide glass 72 base material
  • an upper slide glass 73 is used.
  • the lipid structure production apparatus 70 includes a lower slide glass 72 (base material), liposomes 71 held on the lower slide glass 72 via a linker 75, and an aqueous medium provided on the upper slide glass 73.
  • An inlet 76A and an outlet 76B for (water, buffer solution, etc.) and a syringe pump (not shown) for flowing an aqueous medium between the inlet 76A and the outlet 76B are provided.
  • the liposome 71 of 5th Embodiment becomes a structure by which the nanoparticle in the nanoparticle inclusion
  • the liposome 71 is held on the lower slide glass 72 (base material) via a streptavidin-biotin bond, similarly to the nanoparticle-encapsulating liposome 10 shown in FIG.
  • Such liposomes can be prepared by the following method. That is, 1,2-disolearoyl-sn-glycero-3-phosphoethanolamine- [biotinyl (poly-ethylene glycol) -2000] (DSPE-Biotin) against 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC) ) was added in an amount of 1 mol% to 4 mol%, and giant liposomes were prepared by the stationary hydration method.
  • HEPES buffer (10 mM, pH 7.4) was used for hydration.
  • Injected in order of albumin bovine biotinamidocaproyl (BSA-Biotin) solution (2 mg / mL) and streptavidin solution (1 mg / ml) into a flow chamber washed with HEPES buffer, and fixed streptavidin on the surface of lower slide glass 72 Obtained Next, the chamber was filled with the previously prepared giant liposome solution, and the liposome 71 was immobilized on the lower slide glass 72.
  • BSA-Biotin albumin bovine biotinamidocaproyl
  • the fluid force is increased by injecting the HEPES buffer at a flow rate of 0.05 mL / min to 1.0 mL / min into the liposome 71 held on the lower slide glass 72 by the streptavidin-biotin interaction.
  • a shear flow By acting and applying a shear flow, a lipid nanotube portion 71A whose elongation direction was controlled was obtained.
  • most of the liposomes 71 are restricted from moving by fluid force, but a part of the lipid membrane is deformed by the shear flow and is elongated along with the flow of the HEPES buffer solution to obtain the lipid nanotube portion 71A.
  • FIG. 9 is a graph showing the relationship between the retention of liposomes and the number of lipid nanotubes formed in the fifth embodiment of the present invention. That is, the tube formation behavior when the concentration of DSPE-Biotin for immobilizing liposomes was varied was quantitatively evaluated by counting the number of tubes in a specific region in the chamber. As shown in FIG. 9, the tube formation behavior was examined by changing the concentration of DSPE-Biotin contained in the lipid from 0 ⁇ M to 40 ⁇ M. When the concentration was 2 ⁇ M to 10 ⁇ M, the number of lipid nanotubes formed dramatically increased. is increasing. In addition, the number of tubes formed decreased with increasing DSPE-Biotin concentration, and almost no tubes were formed when the concentration was 40 ⁇ M.
  • the liposome is strongly immobilized on the substrate due to the increase in the DSPE-Biotin concentration, so that it is less susceptible to shear flow. Therefore, the liposome is held on the substrate in such a range that the movement in the fluid is limited and a shear flow acts on the lipid membrane of the liposome.
  • the relationship between the flow rate and the number of formations is preferable.
  • a particle-encapsulating liposome for example, a nanoparticle-encapsulating liposome
  • magnetic particles for example, a nanoparticle
  • a lipid structure using a magnetic field can be produced by applying a magnetic field to particle-encapsulated liposomes (for example, nanoparticle-encapsulated liposomes) using a magnetic field-applying means such as.
  • the inertial force was used by applying an inertial force (centrifugal force) to the lipid structure production apparatus according to the first, second, and fourth embodiments by a centrifuge.
  • Lipid structures can be produced.
  • a lipid structure can be produced by causing a fluid force to act on the liposome by flowing a liquid or gas other than the aqueous medium.
  • room temperature refers to 25 ° C.
  • HEPES buffer is a 10 mM, pH 7.4 HEPES buffer unless otherwise specified.
  • Example 1 ⁇ Formation of lipid nanotube part 1 using electric field> A lipid structure production apparatus having the same configuration as the lipid structure production apparatus 30 shown in FIG. 3 was used to form a lipid nanotube portion using an electric field. Specific operations are shown below.
  • nanoparticle-containing liposome dispersion A First, 1,2-dioleoyl-sn-glycero-3-phosphocholine (phospholipid) and Rhodamine-DMPE (fluorescent dye) were dissolved in chloroform to prepare a solution A. Next, chloroform was dried and removed from the solution A to obtain a fluorescent dye-containing phospholipid thin film. Next, polystyrene nanoparticles modified with a carboxylate group (Fluoresbrite Carboxylate Microspheres manufactured by Polysciences; volume average particle size 50 nm) were added to a HEPES buffer to prepare a nanoparticle-containing solution B.
  • carboxylate group Frute particle size 50 nm
  • the zeta potential of the polystyrene nanoparticles at pH 7.4 is ⁇ 40 mV.
  • the obtained nanoparticle-containing liquid B was added to the phospholipid thin film, allowed to stand at 27 ° C. for 1 hour or longer, and the thin film was hydrated, whereby the nanoparticle-containing liposome dispersion A (concentration of nanoparticle-containing liposomes was 1). 0.0 mM).
  • liposomes containing nanoparticles having a particle size of about 10 ⁇ m to 20 ⁇ m were formed.
  • agarose gel A preparation of agarose gel A
  • the dispersion A and an agarose aqueous solution are mixed at 45 ° C., and cooled to room temperature and gelled to prepare an agarose gel containing nanoparticle-containing liposomes (herein referred to as “agarose gel A”).
  • the amount of each component is such that the composition in agarose gel A is 2.0 mass% agarose gel, liposome 0.05 mM, Rhodamine-DMPE 0.25 ⁇ M, and nanoparticles 0.32 mg / mL (4.62 ⁇ 10 12 pieces / mL).
  • lipid structure production apparatus having the same configuration as that of the lipid structure production apparatus 30 shown in FIG. 3 was produced. The following were used as each member.
  • the distance between the anode 36A and the cathode 36B was 10 mm.
  • -Components of lipid structure manufacturing equipment- Cover glass 32 Cover glass of 24 mm ⁇ 60 mm ⁇ 0.12 mmt Gel 34 and nanoparticle-encapsulating liposome 31: Agarose gel A (5.0 g)
  • Anode 36A and cathode 36B 1 mm ⁇ platinum (Pt) electrode
  • the nanoparticle-containing liposome itself (the liposome part) itself maintained a spherical shape and was retained in the gel even when voltage was applied. It was done. Since the outer shape of the lipid nanotube is smaller than the resolution of the optical microscope, the lipid nanotube is not observed in the optical micrograph.
  • Example 2 ⁇ Formation of lipid nanotube part 2 using electric field> A lipid structure production apparatus having the same configuration as the lipid structure production apparatus 40 shown in FIG. 4 was used to form a lipid nanotube portion using an electric field. Specific operations are shown below.
  • a lipid structure production apparatus having the same configuration as the lipid structure production apparatus 40 shown in FIG. 4 was produced. The following were used as each member.
  • -Components of lipid structure manufacturing equipment- Cover glass chamber 42 A cover glass chamber having a slide glass of 20 mm ⁇ 45 mm ⁇ 0.12 mmt on the bottom (made by IWAKI Glass, 5202-001)
  • Cover glass 49 10 mm ⁇ 10 mm ⁇ 0.12 mmt cover glass
  • Nanoparticle-encapsulating liposome-containing gel 44 The agarose gel B (0.01 g)
  • Anode 46A and cathode 46B 1 mm ⁇ platinum (Pt) electrode
  • the nanoparticle-encapsulating liposome-containing gel 44 is immersed in a HEPES buffer, the temperature is adjusted to 25 ° C., and the electric field strength is 3.0 kV / m between the anode 46A and the cathode 46B. Observation with a fluorescence microscope was performed in a state where a voltage of (current 2 to 3 mA) was applied. A fluorescence micrograph (60 seconds after the start of voltage application) is shown in FIG. As shown in FIG. 12, in Example 2, as in Example 1, it was confirmed that a lipid nanotube portion was formed by applying a voltage.
  • Example 3 ⁇ Formation of lipid nanotube part 3 using electric field> A lipid structure production apparatus having the same configuration as the lipid structure production apparatus 50 shown in FIG. 5 was used to form a lipid nanotube portion using an electric field. Specific operations are shown below.
  • agarose gel C (Preparation of agarose gel C) In the preparation of agarose gel A in Example 1, agarose gel C, which is a 2.0% by mass agarose gel, was obtained in the same manner as the preparation of agarose gel A except that the nanoparticle-containing liposome dispersion liquid A was not used. .
  • lipid structure production apparatus having the same configuration as that of the lipid structure production apparatus 50 shown in FIG. 5 was produced. The following were used as each member.
  • -Components of lipid structure manufacturing equipment- -Cell 52 It has an anode tank with a volume of 0.1 mL and a cathode tank with a volume of 0.1 mL, and the size of the communicating part between the anode tank and the cathode tank is 17 mm long ⁇ 4.8 mm wide ⁇ 0.4 mm high
  • a plastic cell was used.
  • the length of the communication portion corresponds to the closest distance between the anode tank and the cathode tank.
  • Gel 54 The agarose gel C accommodated in the communication part -Anolyte 55A-0.06 mL HEPES buffer-Catholyte 55B-0.06 mL catholyte
  • Anode 56A and cathode 56B 1 mm ⁇ platinum (Pt) electrode
  • Example 3 a device was prepared in which the nanoparticles containing liposomes were replaced with nanoparticles obtained by coloring nanoparticles modified with carboxylate groups, and the same as described above. A voltage was applied to. Then, it was confirmed by visual observation that the colored nanoparticles advance into the gel. From this result and the formation of the lipid nanotube part, it was confirmed that the micro-sized liposome part cannot move in the gel, but the nano-sized substance can move in the gel. Was proved.
  • Example 4 ⁇ Formation of lipid nanotube part using electric field 4> A lipid structure production apparatus having the same configuration as the lipid structure production apparatus 60 shown in FIG. 6 was used to form a lipid nanotube portion using an electric field.
  • the liposome dispersion liquid A-1 containing nanoparticles introduced with DSPE-biotin prepared as described below was dropped and the operation of standing for 10 minutes was repeated twice, followed by washing with a HEPES buffer solution. Substrate (slide glass holding nanoparticle-containing liposomes) was obtained.
  • polystyrene nanoparticles modified with a carboxylate group (Fluoresbrite Carboxylate Microspheres manufactured by Polysciences; volume average particle diameter 50 nm) were added to a HEPES buffer to prepare a nanoparticle-containing solution B.
  • the zeta potential of the polystyrene nanoparticles at pH 7.4 is ⁇ 40 mV.
  • the obtained nanoparticle-containing liquid B was added to the phospholipid thin film, allowed to stand at 27 ° C. for 1 hour or longer, and the thin film was hydrated, whereby the nanoparticle-containing liposome dispersion A-1 (nanoparticle-containing liposome A concentration of 1.0 mM) was obtained.
  • the nanoparticle-containing liposome dispersion A-1 nanoparticle-containing liposome A concentration of 1.0 mM
  • a lipid structure production apparatus having the same configuration as that of the lipid structure production apparatus 60 shown in FIG. 6 was produced. The following were used as each member. -Components of lipid structure manufacturing equipment- -Slide glass 62, linker 65, and nanoparticle-encapsulating liposome 61 ... The slide glass holding the nanoparticle-containing liposome prepared above was used.
  • Anode 66A and cathode 66B 1 mm ⁇ platinum (Pt) electrode. The distance between the electrodes was 10 mm.
  • Aqueous medium (broken line in FIG. 6): HEPES buffer
  • the shooting order is (b), (c), (d), (e), (f), (g), (h).
  • FIG. 14 it was confirmed that the lipid nanotube portion grew as time elapsed from the start of voltage application. From this result, it was suggested that the length of the lipid nanotube part can be controlled by changing the voltage application time. Furthermore, since the lipid nanotube part maintained its structure even after the voltage application was stopped, it was shown that the liposome and the lipid nanotube part were connected via fusion between lipid membranes.
  • Example 5 ⁇ Formation of lipid nanotube part using magnetic field>
  • the DSPE-biotin-introduced magnetic nanoparticle-containing liposome dispersion A-2 was prepared as follows.
  • a slide glass holding liposomes was prepared in the same manner as in Example 4 except that was used.
  • a magnetic field of 200 KA / m to 500 KA / m was applied to the prepared slide glass nanoparticle-containing liposomes holding the magnetic nanoparticle-containing liposomes using an electromagnet (manufactured by Gigateco, Inc., TMN electromagnet).
  • magnetic nanoparticles modified with a carboxylate group (Nanomag D COOH manufactured by Funakoshi Co., Ltd .; volume average particle size 130 nm) were added to a HEPES buffer to prepare a magnetic nanoparticle-containing solution B-1.
  • the obtained magnetic nanoparticle-containing liquid B-1 was added to the phospholipid thin film, and allowed to stand at 27 ° C. for 1 hour or longer to hydrate the thin film, whereby the magnetic nanoparticle-containing liposome dispersion A-2 (magnetic Nanoparticle-containing liposome concentration 1.0 mM) was obtained.
  • the magnetic nanoparticle-containing liposome dispersion A-2 magnetic Nanoparticle-containing liposome concentration 1.0 mM
  • Pictures (a) to (d) of FIG. 15 show the state 60 seconds after the start of application of the magnetic field. As shown in the parts surrounded by the broken lines in the photographs (a) to (d) in FIG. 15, the lipid nanotube part was formed by the application of the magnetic field as in the case of applying the electric field.
  • Example 6 ⁇ Formation of lipid nanotube part using inertial force>
  • the inertial force (centrifugal force) of 20 km / s 2 was applied to the nanoparticle-containing liposome of the slide glass holding the nanoparticle-containing liposome prepared in Example 4 using a centrifuge (GS-15R manufactured by Beckman). Applied.
  • FIG. 16 shows a state after 3 minutes from the start of application of inertial force (centrifugal force). As shown in a portion surrounded by a broken line in FIG. 16, a lipid nanotube portion was formed by applying an inertial force (centrifugal force) as in the case of applying an electric field.
  • Example 7 ⁇ Formation of lipid nanotube part using fluid force>
  • DSPE-biotin was introduced as described below, but it did not contain nanoparticles.
  • a slide glass holding liposomes was prepared in the same manner as in Example 4 except that the liposome dispersion A-3 was used.
  • the slide glass liposomes thus prepared (liposome not containing nanoparticles) were subjected to 4- (2-hydroxy) in a flow chamber having a length of 17 mm ⁇ width of 3.8 mm ⁇ height of 0.4 mm.
  • Ethyl) -1-piperazineethanesulfonic acid (“HEPES”) was allowed to flow at a rate of 300 ⁇ l / min to apply fluid force to the liposomes.
  • the liposome can be deformed by shear flow to extend a part of the lipid membrane, and the same lipid nanotube part as in the other examples Formed.
  • dextran labeled with rhodamine is encapsulated in giant liposomes that are fluorescently labeled with NBD (appearing green in the color photograph; white portions in the upper and lower parts in the monochrome photograph of FIG. 17).
  • NBD fluorescently labeled with NBD
  • an external field for example, an electric field, a magnetic field, or an inertial force
  • applying an external field for example, an electric field, a magnetic field, or an inertial force
  • applying a fluid force to the liposome As a result, a lipid structure having a lipid nanotube portion could be produced easily, efficiently and with good controllability.

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Abstract

L'invention concerne un procédé de fabrication d'une structure lipidique, lequel consiste à : préparer un liposome encapsulant une particule contenant un liposome et une particule encapsulée par le liposome ; et former une section de tube lipidique par allongement d'une partie de la membrane lipidique du liposome au moyen de la particule, en provoquant le mouvement de la particule dans le liposome encapsulant une particule au moyen d'un champ externe. L'invention concerne également un procédé de fabrication d'une structure lipidique, lequel consiste à : préparer un liposome ; et former une section de tube lipidique par allongement d'une partie de la membrane lipidique du liposome par déformation du liposome au moyen d'une force fluide externe.
PCT/JP2011/069337 2011-02-28 2011-08-26 Procédé de fabrication d'une structure lipidique WO2012117587A1 (fr)

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EP3537098B1 (fr) 2016-11-04 2021-07-21 Fujidenolo Co., Ltd. Dispositif de mesure
EP3808344A4 (fr) * 2018-06-01 2022-03-30 Sogang University Research Foundation Composite de nanoparticules présentant une efficacité améliorée d'endocytose par modification de surface en utilisant un lipide et son procédé de fabrication

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