WO2011001869A1 - Method for producing nanospheres, nanospheres, external composition for skin containing same, and cosmetic preparation - Google Patents

Method for producing nanospheres, nanospheres, external composition for skin containing same, and cosmetic preparation Download PDF

Info

Publication number
WO2011001869A1
WO2011001869A1 PCT/JP2010/060630 JP2010060630W WO2011001869A1 WO 2011001869 A1 WO2011001869 A1 WO 2011001869A1 JP 2010060630 W JP2010060630 W JP 2010060630W WO 2011001869 A1 WO2011001869 A1 WO 2011001869A1
Authority
WO
WIPO (PCT)
Prior art keywords
emulsion
solution
dissolved
primary
polymer
Prior art date
Application number
PCT/JP2010/060630
Other languages
French (fr)
Japanese (ja)
Inventor
辰彦 金
裕一 大矢
哲 南柿
Original Assignee
株式会社シャローム
学校法人関西大学
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 株式会社シャローム, 学校法人関西大学 filed Critical 株式会社シャローム
Publication of WO2011001869A1 publication Critical patent/WO2011001869A1/en

Links

Images

Classifications

    • 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
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/513—Organic macromolecular compounds; Dendrimers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/34—Macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyesters, polyamino acids, polysiloxanes, polyphosphazines, copolymers of polyalkylene glycol or poloxamers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/02—Cosmetics or similar toiletry preparations characterised by special physical form
    • A61K8/11—Encapsulated compositions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/72—Cosmetics or similar toiletry preparations characterised by the composition containing organic macromolecular compounds
    • A61K8/90—Block copolymers
    • 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
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0014—Skin, i.e. galenical aspects of topical compositions
    • 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
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5192—Processes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
    • A61K2800/40—Chemical, physico-chemical or functional or structural properties of particular ingredients
    • A61K2800/41—Particular ingredients further characterized by their size
    • A61K2800/412—Microsized, i.e. having sizes between 0.1 and 100 microns

Definitions

  • the present invention includes, for example, a method for producing a nanosphere capable of enclosing one or both of a hydrophilic substance such as a protein, a nucleic acid, a drug and a contrast agent, or a hydrophobic substance such as a lipid and a drug.
  • a hydrophilic substance such as a protein, a nucleic acid, a drug and a contrast agent
  • a hydrophobic substance such as a lipid and a drug.
  • the present invention relates to an external composition for skin and a cosmetic.
  • DDS drug delivery system
  • sustained drug release type that can increase a patient's QOL (Quality Of Life) by administering a polymer microparticle encapsulating a drug with a biodegradable polymer or the like and releasing a predetermined amount of the drug over a long period of time. DDS is also in the spotlight.
  • QOL Quality Of Life
  • Patent Document 1 discloses microspheres containing leuprorelin acetate in a polylactic acid or polylactic acid copolymer, and the microspheres containing such leuprorelin acetate have been put into practical use as a preparation for subcutaneous injection. Yes.
  • the polylactic acid or polylactic acid copolymer (hereinafter simply referred to as a hydrophobic biodegradable polymer) is hydrophobic. Therefore, a W / O / W (Water in Oil Water) emulsion method or the like is used.
  • a W / O / W Water in Oil Water
  • microspheres encapsulating water-soluble polymers are produced using hydrophobic biodegradable polymers, the uniformity of the distribution of water-soluble polymers in the microspheres is reduced, and a drug sustained-release DDS preparation is obtained. In some cases, the sustainability (sustained release) of the water-soluble polymer was insufficient.
  • the water-soluble polymer is a protein or nucleic acid, in the process of producing the microsphere, the three-dimensional structure may not be maintained or may be denatured and may be deactivated.
  • Non-Patent Document 1 a technique is disclosed that can produce microspheres encapsulating water-soluble polymers by a W / O / W emulsion method by using amphiphilic polylactic acid grafted dextran as a biodegradable polymer.
  • the microsphere described in Non-Patent Document 1 includes bovine serum albumin (hereinafter simply referred to as BSA) as a water-soluble polymer.
  • BSA bovine serum albumin
  • BSA bovine serum albumin
  • Non-Patent Document 1 the polymer fine particles encapsulating the water-soluble polymer described in Non-Patent Document 1 are suitable for intravenous or arterial injection and percutaneous absorption because the sphere particle size is as large as micrometer order, that is, microspheres. Not.
  • Non-Patent Document 2 discloses a technique for producing nanospheres using polylactic acid-grafted dextran by the O / W emulsion method
  • Non-Patent Document 3 discloses an amphiphilic block co-polymer by the O / W emulsion method. Techniques for producing nanospheres using coalescence are disclosed. Japanese Patent Laid-Open No. 10-182496 Ouchi T, Saito T, Kontani T, Ohya Y, Macromol Biosci.
  • the polymer containing the water-soluble polymer and having a nanometer order Development of fine particles is desired.
  • the present invention is to produce polymer fine particles that contain either or both of a hydrophilic substance and a hydrophobic substance such as a water-soluble polymer and that have a particle size of nano-order. It is an object to provide a method for producing nanospheres, nanospheres, a composition for external use on skin containing the nanospheres, and a cosmetic.
  • a method for producing nanospheres using a W / O / W emulsion method as an example of the present invention includes an aqueous solvent in which a hydrophilic substance is dissolved, a water-immiscible organic solvent in which an amphiphilic polymer is dissolved, and a hydrophobic property.
  • a secondary solution generating step for adding a water-based solvent in which a hydrophilic polymer is dissolved to the primary emulsion to generate a secondary solution, and a secondary emulsion for generating a secondary emulsion by irradiating the secondary solution with ultrasonic waves Generating step.
  • the nanosphere contains a hydrophilic substance.
  • biodegradable polymers are used for amphiphilic polymers, hydrophobic polymers, and hydrophilic polymers, drugs that contain water-soluble (hydrophilic) high molecular weight drugs such as proteins, nucleic acids, and biologically active natural extracts.
  • Sustained release cosmetics can be produced that contain pharmaceuticals such as sustained release DDS preparations and hydrophilic active substances.
  • the polymer fine particles encapsulating the hydrophilic substance are on the nanometer order, it can be used for pharmaceutical preparations for intravenous or arterial injection, pharmaceutical preparations for percutaneous absorption, cosmetic compositions such as cosmetics, Systemic administration of drugs, targeting to affected areas and penetration into the skin are possible.
  • Another method for producing nanospheres using the W / O / W emulsion method is to mix an aqueous solvent in which a hydrophilic substance is dissolved with a water-immiscible organic solvent in which an amphiphilic polymer is dissolved.
  • a primary solution generation step for generating a primary emulsion a primary emulsion generation step for generating a primary emulsion by irradiating ultrasonic waves to the primary solution, and an aqueous solvent in which a hydrophilic polymer is dissolved in the primary emulsion
  • a secondary solution generating step for generating a secondary solution and a secondary emulsion generating step for generating a secondary emulsion by irradiating the secondary solution with ultrasonic waves.
  • the nanosphere contains a hydrophilic substance.
  • aggregation is performed by irradiating ultrasonic waves to a secondary solution obtained by adding a hydrophilic polymer to a primary emulsion formed by irradiating ultrasonic waves to a primary solution containing an amphiphilic polymer and a hydrophilic substance. While suppressing, it becomes possible to produce polymer fine particles of a W / O / W emulsion encapsulating a hydrophilic substance on the nanometer order.
  • a biodegradable polymer is used for an amphiphilic polymer and a hydrophilic polymer
  • a pharmaceutical or hydrophilic drug containing a water-soluble (hydrophilic) polymer such as a protein, a nucleic acid, or a physiologically active natural extract is included.
  • a sustained-release cosmetic that encapsulates an active substance can be produced.
  • the polymer fine particles encapsulating the hydrophilic substance are on the nanometer order, it can be used for pharmaceutical preparations for intravenous or arterial injection, pharmaceutical preparations for percutaneous absorption, cosmetic compositions such as cosmetics, Systemic administration of drugs, targeting to affected areas and penetration into the skin are possible.
  • the nanosphere manufacturing method using another O / W emulsion method includes a water-immiscible organic solvent in which a hydrophobic substance is dissolved, a water-immiscible organic solvent in which an amphiphilic polymer is dissolved, and a hydrophobic polymer.
  • a water-immiscible organic solvent in which water is dissolved is mixed to form a primary solution, and a primary solution is generated.
  • a water-based solvent in which a hydrophilic polymer is dissolved is added to the primary solution to form a secondary solution.
  • the nanosphere contains a hydrophobic substance.
  • a hydrophobic solution and a secondary solution obtained by adding a hydrophilic polymer to a primary solution obtained by adding an amphiphilic polymer to a hydrophobic substance are irradiated with ultrasonic waves, thereby suppressing aggregation and hydrophobicity. It becomes possible to produce polymer fine particles enclosing a substance on the nanometer order.
  • the drug sustained-release DDS encapsulating a lipid or a polymer of a hydrophobic polymer such as a natural extract having physiological activity Sustained-release cosmetics containing pharmaceuticals such as pharmaceutical preparations and hydrophobic active substances can be produced.
  • the polymer fine particles encapsulating the hydrophobic substance are on the order of nanometers, they can be used in pharmaceutical compositions for intravenous or arterial injection, pharmaceutical compositions for percutaneous absorption, cosmetic compositions such as cosmetics, Systemic administration of drugs, targeting to affected areas and penetration into the skin are possible.
  • the method for producing nanospheres using another O / W emulsion method comprises mixing a water-immiscible organic solvent in which a hydrophobic substance is dissolved and a water-immiscible organic solvent in which an amphiphilic polymer is dissolved, A primary solution generating step for generating a primary solution, an aqueous solvent in which a hydrophilic polymer is dissolved is added to the primary solution, a secondary solution generating step for generating a secondary solution, and an ultrasonic wave is applied to the secondary solution.
  • the nanosphere contains a hydrophobic substance.
  • a secondary solution in which a hydrophilic polymer is further added to a primary solution containing an amphiphilic polymer and a hydrophobic substance is irradiated with ultrasonic waves, so that aggregation is suppressed and a hydrophobic substance is included. It becomes possible to produce molecular fine particles on the nanometer order.
  • a biodegradable polymer is used for the amphiphilic polymer and the hydrophilic polymer
  • a drug containing a hydrophobic polymer such as a lipid or a physiologically active natural drug or a hydrophobic active substance is included.
  • a sustained-release cosmetic can be produced.
  • the polymer fine particles encapsulating the hydrophobic substance are on the order of nanometers, they can be used in pharmaceutical compositions for intravenous or arterial injection, pharmaceutical compositions for percutaneous absorption, cosmetic compositions such as cosmetics, Systemic administration of drugs, targeting to affected areas and penetration into the skin are possible.
  • the nanosphere manufacturing method using another W / O / W emulsion method involves dissolving an aqueous solvent in which a hydrophilic substance is dissolved, a water-immiscible organic solvent in which a hydrophobic substance is dissolved, and an amphiphilic polymer.
  • the water-immiscible organic solvent thus prepared and the water-immiscible organic solvent in which the hydrophobic polymer is dissolved are mixed to form a primary solution generating step, and the primary solution is irradiated with ultrasonic waves.
  • a primary emulsion generating step for generating a secondary emulsion, an aqueous solvent in which a hydrophilic polymer is dissolved is added to the primary emulsion, a secondary solution generating step for generating a secondary solution, and ultrasonic irradiation to the secondary solution And a secondary emulsion generating step of generating a secondary emulsion.
  • the nanosphere includes a hydrophilic substance and a hydrophobic substance.
  • biodegradable polymers are used for amphiphilic polymers, hydrophobic polymers, and hydrophilic polymers, water-soluble (hydrophilic) high molecular weight drugs such as proteins, nucleic acids, and biologically active natural extracts, and lipids And pharmaceuticals such as drug sustained-release DDS preparations that encapsulate hydrophobic polymers such as natural extracts with physiological activity, and sustained-release cosmetics that encapsulate hydrophilic and hydrophobic active substances Can do.
  • the fine polymer particles encapsulating hydrophilic substances and hydrophobic substances are on the order of nanometers, they are used for compositions for external use in skin, such as pharmaceuticals for intravenous or arterial injection, pharmaceuticals for percutaneous absorption, and cosmetics. And allows systemic administration of the drug, targeting the affected area and penetration into the skin.
  • the nanosphere manufacturing method using another W / O / W emulsion method involves dissolving an aqueous solvent in which a hydrophilic substance is dissolved, a water-immiscible organic solvent in which a hydrophobic substance is dissolved, and an amphiphilic polymer.
  • the nanosphere includes a hydrophilic substance and a hydrophobic substance.
  • biodegradable polymers are used for amphiphilic polymers and hydrophilic polymers, water-soluble (hydrophilic) high molecular weight drugs such as proteins, nucleic acids, and biologically active natural extracts, and lipids and physiological activities can be obtained. It is possible to produce pharmaceuticals that encapsulate hydrophobic polymers such as natural extracts, and sustained-release cosmetics that encapsulate hydrophilic and hydrophobic active substances.
  • the fine polymer particles encapsulating hydrophilic substances and hydrophobic substances are on the order of nanometers, they are used for compositions for external use in skin, such as pharmaceuticals for intravenous or arterial injection, pharmaceuticals for percutaneous absorption, and cosmetics. And allows systemic administration of the drug, targeting the affected area and penetration into the skin.
  • the amphiphilic polymer is a copolymer composed of a hydrophilic segment and a hydrophobic segment, and the hydrophilic segment is a polypeptide having three or more charged amino acids, polyethylene having a number average molecular weight of 500 to 100,000.
  • the hydrophobic segment may include a biodegradable polyester including one or more selected from the group of glycols and polysaccharides.
  • amphiphilic polymer one or more selected from the group consisting of a polypeptide having three or more charged amino acids, a polyethylene glycol having a number average molecular weight of 500 to 100,000, and a polysaccharide, and a biodegradable polyester
  • a copolymer By using such a copolymer, it is possible to produce biodegradable nanospheres that include either or both of a hydrophilic substance and a hydrophobic substance.
  • the amino acid constituting the polypeptide may be one or more amino acids selected from the group of lysine, arginine and histidine which are positively charged amino acids, and the group of aspartic acid and glutamic acid which are negatively charged amino acids. .
  • amino acids of a polypeptide constituting an amphiphilic polymer as amino acids constituting a protein such as one or a plurality of amino acids selected from the group of lysine, arginine, histidine, aspartic acid, and glutamic acid.
  • amino acids constituting a protein such as one or a plurality of amino acids selected from the group of lysine, arginine, histidine, aspartic acid, and glutamic acid.
  • an amino acid of a polypeptide that constitutes an amphiphilic polymer is an amino acid that constitutes a protein
  • nanospheres that enclose either or both of a hydrophilic substance and a hydrophobic substance are introduced into the blood. The possibility of being trapped as foreign matter by phagocytic cells such as macrophages can be reduced, and the circulation time in blood can be prolonged.
  • polyethylene glycol is included in the amphiphilic polymer, so that when nanospheres that contain either or both of hydrophilic and hydrophobic substances are introduced into the blood, they are trapped as foreign matter in phagocytic cells such as macrophages. It is possible to reduce the possibility that the blood is circulated and prolong the circulation time in the blood.
  • the polysaccharide constituting the amphiphilic polymer is selected from the group of hyaluronic acid, amylose, pullulan, chondroitin, chondroitin sulfate, dextran, dextran sulfate, dermatan sulfate, keratan sulfate, heparan sulfate, chitin, chitosan, and ⁇ -glucan. It may be one or more.
  • the content of polysaccharide in the amphiphilic polymer may be 1 to 50% by mass.
  • a biodegradable nanosphere containing either or both of a hydrophilic substance and a hydrophobic substance is produced. be able to.
  • the structural unit of the biodegradable polyester may be one or more selected from the group of lactic acid, glycolic acid, and amino acid.
  • biocompatibility can be improved when producing pharmaceuticals and cosmetics.
  • the nanosphere may include stevia fermented extract or sphingomyelin as one or both of a hydrophilic substance and a hydrophobic substance.
  • composition for external use on the skin containing nanospheres produced using the method for producing nanospheres is a substance that percutaneously absorbs a substance contained in the composition for external use of skin by bringing it into contact with human skin.
  • the composition for external application to the skin such as a coating agent is applied to the skin surface by a composition containing nanospheres containing either or both of a hydrophilic substance and a hydrophobic substance produced by using the above-mentioned nanosphere production method.
  • the nanospheres can be applied almost uniformly on the fine irregularities of the film.
  • a biodegradable polymer as a polymer constituting the nanosphere, either one or both of the hydrophilic substance and the hydrophobic substance encapsulated in the nanosphere can be suitably and slowly released on the skin surface. In comparison with the prior art, it is possible to reduce the number of times of application while maintaining the effect of the external composition for skin.
  • the nanospheres contained in the composition for external skin may contain stevia fermented extract or sphingomyelin as one or both of a hydrophilic substance and a hydrophobic substance.
  • Stevia fermented extract and sphingomyelin have a high moisturizing effect, improve rough skin, suppress itching, suppress inflammation, and have an antihistaminic action. Therefore, the composition for external use of skin containing nanospheres containing stevia fermented extract or sphingomyelin can distribute stevia fermented extract or sphingomyelin substantially evenly on fine irregularities on the skin surface.
  • a cosmetic containing nanospheres produced using the above-described method for producing nanospheres is provided.
  • the cosmetic is a composition for treating the human body for the purpose of beautification, cleanliness, protection or deodorization.
  • the composition containing nanospheres containing either or both of a hydrophilic substance and a hydrophobic substance produced by using the above-mentioned nanosphere production method the nanospheres are formed into fine irregularities on the skin surface. It can be applied substantially uniformly.
  • a biodegradable polymer as a polymer constituting the nanosphere, either one or both of the hydrophilic substance and the hydrophobic substance encapsulated in the nanosphere can be suitably and slowly released on the skin surface. It is possible to obtain the same effect even if the number of times of applying the cosmetic is reduced as compared with the conventional case.
  • the nanospheres contained in the cosmetic may contain stevia fermented extract or sphingomyelin as one or both of a hydrophilic substance and a hydrophobic substance.
  • Stevia fermented extract and sphingomyelin have a high moisturizing effect, improve rough skin, suppress itching, suppress inflammation, and have an antihistaminic action. Therefore, with the constitution of the cosmetic containing nanospheres encapsulating stevia fermented extract or sphingomyelin, stevia fermented extract or sphingomyelin can be distributed almost uniformly on the fine irregularities of the skin surface.
  • the constituent elements based on the technical idea of the method for producing nanospheres described above and the explanation thereof are also applicable to the nanospheres, compositions for external use on skin and cosmetics containing the nanospheres.
  • polymer fine particles having a nano-order particle size including either or both of a hydrophilic substance and a hydrophobic substance such as a water-soluble polymer.
  • FIG. 6 is an explanatory diagram for explaining a synthesis method of PLys + -b-PLLA according to Example 1.
  • FIG. 6 is an explanatory diagram for explaining the decomposability in the micelle state of PLys + -b-PLLA according to Example 1.
  • FIG. 3 is an explanatory diagram for explaining Example 1; It is explanatory drawing for demonstrating the result of having observed the biodegradable nanosphere which includes BSA with the scanning electron microscope. It is explanatory drawing for demonstrating the type
  • FIG. 6 is an explanatory diagram for explaining a method of synthesizing PEG3K-b-PLLA according to Example 2.
  • FIG. FIG. 6 is an explanatory diagram for explaining Example 2; It is explanatory drawing for demonstrating the result of having observed the biodegradable nanosphere which includes sphingomyelin with the scanning electron microscope.
  • FIG. 6 is an explanatory diagram for explaining a Dex-g-PLLA synthesis method according to Example 3; 6 is an explanatory diagram for explaining the degradability of Dex-g-PLLA according to Example 3.
  • FIG. 10 is an explanatory diagram for explaining Example 3; It is explanatory drawing for demonstrating the result of having observed the biodegradable nanosphere which includes sphingomyelin with the scanning electron microscope.
  • FIG. 10 is an explanatory diagram for explaining Example 4; It is explanatory drawing for demonstrating the result of having observed the biodegradable nanosphere which includes BSA with the scanning electron microscope.
  • Drawing 1 is an explanatory view for explaining the manufacturing method of the nanosphere concerning an embodiment.
  • a solvent in which an encapsulating substance is dissolved, a water-immiscible organic solvent in which an amphiphilic polymer is dissolved, and a water-immiscible organic solvent in which a hydrophobic polymer is dissolved are mixed to form a primary solution ( S100: primary solution generation step).
  • an aqueous solvent in which the hydrophilic substance is dissolved is used as a water-immiscible organic solvent in which the amphiphilic polymer is dissolved and water in which the hydrophobic polymer is dissolved. Mix with immiscible organic solvent to form primary solution.
  • the substance to be included is a hydrophilic substance and a hydrophobic substance
  • an aqueous solvent in which the hydrophilic substance is dissolved and a water-immiscible organic solvent in which the hydrophobic substance is dissolved are used as an amphiphile.
  • a water-immiscible organic solvent in which the soluble polymer is dissolved and a water-immiscible organic solvent in which the hydrophobic polymer is dissolved are mixed to form a primary solution.
  • the generated primary solution is irradiated with ultrasonic waves to generate a primary emulsion that is a reverse phase emulsion (W / O emulsion) (S102: primary emulsion generation step). Then, an aqueous solvent in which a hydrophilic polymer is dissolved is added to the primary emulsion to generate a secondary solution (S104: secondary solution generation step).
  • the produced secondary solution is irradiated with ultrasonic waves to produce a secondary emulsion (S106: secondary emulsion production step), and the water-immiscible organic solvent is removed from the secondary emulsion (S108: organic solvent removal step).
  • the primary solution generation step S100 when the encapsulating substance is a hydrophobic substance, in the primary solution generation step S100, the water-immiscible organic solvent in which the hydrophobic substance is dissolved is changed to the water-immiscible organic solvent in which the amphiphilic polymer is dissolved. The water-immiscible organic solvent is mixed with the soluble polymer to form a primary solution. Then, the primary emulsion generation step S102 is omitted, and an aqueous solvent in which the hydrophilic polymer is dissolved is added to the primary solution to generate a secondary solution (S104: secondary solution generation step).
  • the generated secondary solution is irradiated with ultrasonic waves to generate an emulsion (O / W emulsion) (S106: emulsion generation step (secondary emulsion generation step)), and the water-immiscible organic solvent is removed from the emulsion (S108). : Organic solvent removal step).
  • Primary solution generation step S100 A primary solution is produced by mixing a solvent in which the substance to be encapsulated is dissolved, a water-immiscible organic solvent in which the amphiphilic polymer is dissolved, and a water-immiscible organic solvent in which the hydrophobic polymer is dissolved.
  • amphiphilic polymer 1 in primary solution generation step S100 is a copolymer composed of a hydrophilic segment and a hydrophobic segment, and the hydrophilic segment is a polypeptide having three or more charged amino acids or a number average. Polyethylene glycol having a molecular weight of 500 to 100,000 is included, and the hydrophobic segment includes a biodegradable polyester.
  • the polymerized form of an amphiphilic polymer containing a polypeptide or an amphiphilic polymer containing polyethylene glycol is preferably a polypeptide having three or more charged amino acids or polyethylene glycol having a number average molecular weight of 500 to 100,000, Random copolymers, alternating copolymers, block copolymers, and graft copolymers with degradable polyesters, more preferably block copolymers or graft copolymers, and even more preferably block copolymers. It is a polymer.
  • the polypeptide constituting the amphiphilic polymer may be positively or negatively charged, and the amino acid constituting such a polypeptide is preferably selected from the group of lysine, arginine, histidine, aspartic acid, and glutamic acid.
  • One or more amino acids may be used, more preferably one or more amino acids selected from the group of lysine, arginine or histidine, more preferably lysine.
  • a drug such as a drug sustained-release DDS formulation or a sustained-release type using nanospheres using the amphiphilic polymer. Biocompatibility can be improved when producing a composition for external use on skin and a sustained release cosmetic.
  • an amino acid of a polypeptide that constitutes an amphiphilic polymer is an amino acid that constitutes a protein
  • nanospheres that enclose either or both of a hydrophilic substance and a hydrophobic substance are introduced into the blood. The possibility of being trapped as foreign matter by phagocytic cells such as macrophages can be reduced, and the circulation time in blood can be prolonged.
  • the number average molecular weight of the polyethylene glycol constituting the amphiphilic polymer is not particularly limited, but is preferably a number average molecular weight of 500 to 100,000, more preferably a number average molecular weight of 1,000 to 50,000, and still more preferably a number average.
  • the molecular weight is 2000-20000.
  • nanospheres that contain either or both of hydrophilic substances and hydrophobic substances are introduced into the blood, they are trapped by phagocytic cells such as macrophages as foreign substances. The possibility can be reduced and the circulation time in the blood can be extended.
  • the structural unit of the biodegradable polyester is a polymer that becomes a biodegradable polyester when polymerized and can form a copolymer with the above polypeptide or polyethylene glycol.
  • lactic acid L-lactic acid
  • succinic acid and butanediol 1,2-butanediol, 1,3-butanediol, 1,4-butanediol, 2,3-butanediol, etc.
  • These oligomers may be used, and more preferably lactic acid, glycolic acid and amino acid.
  • Biodegradable polyesters include, for example, polylactic acid (PLA: Poly Lactic Acid), polyglycolic acid (PGA), polylactic acid-glycolic acid copolymer, polydepsipeptide (copolymer of amino acid and hydroxy acid) Can be used.
  • PLA Poly Lactic Acid
  • PGA polyglycolic acid
  • PGA polylactic acid-glycolic acid copolymer
  • polydepsipeptide copolymer of amino acid and hydroxy acid
  • biocompatibility can be improved when producing pharmaceuticals, external compositions for skin, and cosmetics.
  • amphiphilic polymer 2 in primary solution generation step S100 is a copolymer composed of a hydrophilic segment and a hydrophobic segment, the hydrophilic segment contains a polysaccharide, and the hydrophobic segment is a biodegradable polyester. It may be comprised including.
  • the polymerization form of the amphiphilic polymer containing polysaccharide may be a random copolymer, alternating copolymer, block copolymer, or graft copolymer of polysaccharide and biodegradable polyester. It is a copolymer or a block copolymer, and more preferably a graft copolymer.
  • the amphiphilic polymer is more preferably a graft copolymer having a polysaccharide as a main chain and a biodegradable polyester as a side chain.
  • the content of the polysaccharide constituting the amphiphilic polymer is not particularly limited, but may be 1 to 50% by mass, preferably 1 to 30% by mass, and more preferably 4 to 25% by mass. It is.
  • the degradation of the polysaccharides that make up the amphiphilic polymer is within this range. It can be adjusted to a degradation rate that does not cause an inflammatory reaction or the like associated with, and has disappeared from the living body after a period required in the living body.
  • the period required in vivo is preferably 1 month to 10 months, more preferably 2 months to 10 months, and most preferably 2 months to 8 months.
  • the polysaccharide constituting the amphiphilic polymer may be stable in vivo, preferably hyaluronic acid, amylose, pullulan, chondroitin, chondroitin sulfate, dextran, dextran sulfate, dermatan sulfate, keratan sulfate, heparan sulfate, chitin, One or more selected from the group of chitosan and ⁇ -glucan, more preferably one or more selected from the group of dextran, pullulan and hyaluronic acid, more preferably dextran.
  • the structure in which the polysaccharide in the amphiphilic polymer is dextran allows nanospheres enclosing one or both of the hydrophilic substance and the hydrophobic substance to be stably present in the blood vessel.
  • the weight average molecular weight of the polysaccharide is not particularly limited, but may be 1000 to 100,000 g / mol, preferably 5000 to 50000 g / mol, and more preferably 10,000 to 30000 g / mol.
  • nanospheres containing either or both of hydrophilic substances and hydrophobic substances maintain high moldability and flexibility.
  • cell adhesion can be reduced.
  • the structural unit of the biodegradable polyester is that which becomes a biodegradable polyester when polymerized and can form a copolymer with the above-mentioned polysaccharide, preferably lactic acid (L-lactic acid, D- Lactic acid, DL-lactic acid), glycolic acid (hydroxyacetic acid), amino acids, caprolactone ( ⁇ -caprolactone, ⁇ -caprolactone, ⁇ -caprolactone, ⁇ -caprolactone, ⁇ -caprolactone, etc.), a mixture of succinic acid and ethylene glycol, succinic acid Or a mixture of butanediol (1,2-butanediol, 1,3-butanediol, 1,4-butanediol, 2,3-butanediol, etc.), or one or more thereof, or an oligomer thereof And more preferably one or more selected from the group of lactic acid, glycolic acid and amino acids
  • biodegradable polyester for example, polylactic acid, polyglycolic acid, polylactic acid-glycolic acid copolymer, and polydepsipeptide can be used.
  • biocompatibility can be improved when producing pharmaceuticals, external compositions for skin, and cosmetics.
  • amphiphilic polymer that is a copolymer of the polysaccharide and the biodegradable polyester described above is preferably a graft copolymer in which the main chain is dextran and the side chain is L-lactic acid.
  • an amphiphilic polymer which is a graft copolymer having dextran as a main chain and L-lactic acid as a side chain is referred to as Dex-g-PLLA (polylactic acid grafted dextran).
  • the number of lactic acid grafts in the above Dex-g-PLLA molecule may be 1 to 100, preferably 1 to 50, and more preferably 2 to 30. With the configuration in which the number of lactic acid grafts in one molecule of Dex-g-PLLA is within the above range, it becomes possible to reduce the cell adhesion of nanospheres enclosing one or both of hydrophilic substances and hydrophobic substances. .
  • the number average molecular weight of the Dex-g-PLLA is not particularly limited, but may be 1 ⁇ 10 4 to 100 ⁇ 10 4 / mol, and preferably 5 ⁇ 10 4 to 20 ⁇ 10 4 / mol. More preferably, it is 10 ⁇ 10 4 to 13 ⁇ 10 4 / mol.
  • nanospheres containing either or both of hydrophilic substances and hydrophobic substances can maintain cell strength while maintaining high strength and flexibility. Can be reduced.
  • the primary solution generation step S100 it is not necessary to add the water-immiscible organic solvent in which the hydrophobic polymer is dissolved, the solvent in which the substance to be included is dissolved, and the water-immiscible organic solvent in which the amphiphilic polymer is dissolved. May be mixed to form a primary solution.
  • the polymerization form of the hydrophobic polymer may be a random copolymer, an alternating copolymer, a block copolymer, or a graft copolymer.
  • the structural unit of the hydrophobic polymer is not particularly limited as long as it becomes a hydrophobic polymer when polymerized, preferably lactic acid (L-lactic acid, D-lactic acid, DL-lactic acid), glycolic acid (hydroxyacetic acid), amino acid, Caprolactone ( ⁇ -caprolactone, ⁇ -caprolactone, ⁇ -caprolactone, ⁇ -caprolactone, ⁇ -caprolactone, etc.), a mixture of succinic acid and ethylene glycol, succinic acid and butanediol (1,2-butanediol, 1,3-butane A mixture of diol, 1,4-butanediol, 2,3-butanediol, etc.), one or more, or an oligomer thereof, more preferably lactic acid, glycolic acid and amino acid, One or more selected from the group consisting of L-lactic acid and glycolic acid is there.
  • hydrophobic polymer for example, polylactic acid, polyglycolic acid, polylactic acid-glycolic acid copolymer, and polydepsipeptide can be used.
  • a random copolymer of L-lactic acid and glycolic acid is used as the hydrophobic polymer.
  • the hydrophobic polymer of the random copolymer of L-lactic acid and glycolic acid is referred to as PLGA (polylactic acid- Glycolic acid random copolymer).
  • the weight average molecular weight (Mw) of PLGA is not particularly limited, but is preferably 5000-75000.
  • the substance to be included in the primary solution generation step S100 may be either a hydrophilic substance or a hydrophobic substance, or both, and stevia fermented extract or parents as either a hydrophilic substance or a hydrophobic substance or both.
  • a medium substance may be used.
  • amphiphile for example, sphingomyelin can be used.
  • Stevia fermented extract a mixture of hydrophilic and hydrophobic substances, and sphingomyelin, an amphiphilic substance, are published in International Publication No. WO2008 / 126638A1 and “Study on Antihistamine Action of Stevia Fermented Extract” Pharmacology and Treatment vol.36 no As described in .8 2008, it has a high moisturizing effect, improves rough skin, suppresses itching, suppresses inflammation, and has antihistaminic activity.
  • the aqueous solvent in the primary solution generation step S100 is water or an aqueous solution containing inorganic salts, sugars, organic salts, amino acids, etc., and it is sufficient if the hydrophilic segment of the hydrophilic substance or amphiphilic substance can be dissolved.
  • the water-immiscible organic solvent in the primary solution generation step S100 is soluble in the hydrophobic polymer, the hydrophobic segment of the amphiphilic polymer, the hydrophobic substance, or the hydrophobic segment of the amphiphilic substance, and the amphiphile.
  • the hydrophilic segment of the hydrophilic polymer may be hardly soluble or insoluble, but both the hydrophobic segment and the hydrophilic segment of the amphiphilic polymer may be hardly soluble or soluble.
  • the solubility of the water-immiscible organic solvent in water is 10 g (water-immiscible organic solvent) / 100 ml (water) or less, preferably 1 g (water-immiscible organic solvent) / 100 ml (water) or less. More preferably, it is 0.1 g (water-immiscible organic solvent) / 100 ml (water) or less.
  • the water-immiscible organic solvent is not particularly limited, and ethyl acetate, isopropyl acetate, butyl acetate, dimethyl carbonate, diethyl carbonate, methylene chloride (dichloromethane), chloroform and the like can be suitably used.
  • the ratio of the water-immiscible organic solvent to the aqueous solvent may be 1: 1000 to 1: 1, preferably 1: 100 to 1: 3, more preferably 1:50 to 1:10. is there.
  • the amount of the water-immiscible organic solvent is the amount of the water-immiscible organic solvent that dissolves these amphiphilic polymers when using an amphiphilic polymer containing a polypeptide or an amphiphilic polymer containing polyethylene glycol.
  • the total amount of the water-immiscible organic solvent that dissolves the hydrophobic polymer is used.
  • an amphiphilic polymer containing a polysaccharide is used, the amount of the water-immiscible organic solvent that dissolves the amphiphilic polymer containing the polysaccharide is used.
  • the concentration of the amphiphilic polymer and the hydrophobic polymer in the water-immiscible organic solvent may be 1 to 500 mg / ml when using an amphiphilic polymer containing a polypeptide or an amphiphilic polymer containing polyethylene glycol. It is preferably 10 to 250 mg / ml, more preferably 50 to 100 mg / ml.
  • the ratio of the hydrophobic polymer to the amphiphilic polymer may be 1: 1000 to 1: 1, preferably 1: 100 to 1: 3, more preferably 1:50 to 1: 5. is there.
  • the concentration of the amphiphilic polymer in the water-immiscible organic solvent may be 1 to 500 mg / ml, preferably 10 to 250 mg / ml when an amphiphilic polymer containing a polysaccharide is used. More preferably, it is 50 to 100 mg / ml.
  • Primary emulsion generation step S102 The produced primary solution is irradiated with ultrasonic waves to produce a primary emulsion which is a reverse phase emulsion (W / O emulsion).
  • the ultrasonic irradiation time may be 0.5 to 15 minutes, preferably 1 to 10 minutes, and more preferably 3 to 5 minutes.
  • the ultrasonic irradiation may be performed using a bus sonicator, a probe sonicator, or a bus sonicator and a probe sonicator.
  • the primary solution which is a reverse phase emulsion is produced
  • Stirring apparatuses such as a magnetic stirrer, turbine type You may produce
  • the hydrophilic polymer may be chemically bonded or physically adsorbed with the hydrophobic segment or hydrophobic polymer of the above-described amphiphilic polymer. Further, the hydrophilic polymer may be one that prevents aggregation of the primary emulsion in the solvent or delays aggregation.
  • the hydrophilic polymer may be chemically bonded or physically adsorbed with the hydrophobic segment or the hydrophobic polymer of the amphiphilic polymer in the secondary solution generation step S104. In the removal step S108, it may be chemically bonded or physically adsorbed with the hydrophobic segment or the hydrophobic polymer of the amphiphilic polymer when the solvent is removed.
  • the hydrophilic polymer may be one or more selected from the group of polyvinyl alcohol, polyethylene glycol, polypeptide, protein, or polysaccharide, and is preferably polyvinyl alcohol.
  • the hydrophilic polymer may be one or more analogs selected from the group of polyvinyl alcohol, polyethylene glycol, polypeptide, protein or polysaccharide.
  • the aqueous solvent in the secondary solution generation step S100 only needs to dissolve the hydrophilic polymer, and is, for example, water or an aqueous solution containing inorganic salts, saccharides, organic salts, amino acids, and the like.
  • the concentration of the hydrophilic polymer in the aqueous solvent may be 0.1 to 500 mg / ml, preferably 10 to 250 mg / ml, and more preferably 50 to 100 mg / ml.
  • the ratio of the aqueous solvent in which the hydrophilic polymer in the secondary solution generation step S100 is dissolved to the primary solution in the primary solution generation step S100 may be 1: 500 to 1:10, and preferably 1: 300. To 1:50, more preferably 1: 200 to 1: 100.
  • the ultrasonic irradiation time may be 0.5 to 20 minutes, preferably 1 to 15 minutes, and more preferably 3 to 7 minutes.
  • the ultrasonic irradiation may be performed using a bus sonicator, a probe sonicator, or a bus sonicator and a probe sonicator.
  • a secondary emulsion is produced
  • Stirring apparatuses such as a magnetic stirrer, a turbine type stirring apparatus, a homogenizer, etc. You may produce
  • Organic solvent removal step S108 Remove the water immiscible organic solvent from the secondary emulsion.
  • in-liquid drying dialysis, centrifugation, freeze-drying, filtration, reprecipitation, etc. may be used, preferably in-liquid drying, centrifugation, or freeze-drying. To do.
  • the secondary emulsion (in the case of including a hydrophobic substance, an emulsion) is introduced into an aqueous solvent, and further the water-immiscible organic solvent is removed to thereby remove the hydrophilic substance. Or it is good also as the aqueous solvent dispersion of the nanosphere which encloses any one or both of hydrophobic substances.
  • a hydrophilic substance or a hydrophobic substance or An aqueous solvent dispersion of nanospheres enclosing both can be obtained.
  • a biodegradable polymer is used for the amphiphilic polymer, hydrophobic polymer and hydrophilic polymer, a sustained release type skin external composition and active ingredient containing a drug or an active substance such as a drug sustained release type DDS formulation can be obtained. It is possible to obtain an aqueous solvent dispersion of biodegradable nanospheres that enclose either or both of a hydrophilic substance and a hydrophobic substance, which is suitable for a cosmetic to be encapsulated.
  • the dispersion medium by removing the dispersion medium from the aqueous dispersion of nanospheres enclosing one or both of the hydrophilic substance and the hydrophobic substance obtained by the present embodiment, either the hydrophilic substance or the hydrophobic substance is obtained. It is also possible to form nanospheres that include either or both.
  • the method for removing the dispersion medium is not particularly limited, and submerged drying, dialysis, centrifugation, lyophilization, filtration, reprecipitation, and the like can be used. Preferably, submerged drying, centrifuging, and lyophilization are performed. Is used.
  • the primary emulsion is generated in the primary emulsion generation step S102, either one or both of the hydrophilic substance and the hydrophobic substance is added to the hydrophobic polymer by the configuration in which the amphiphilic polymer is added. Can be included.
  • the primary emulsion produced by irradiating the hydrophobic polymer and the primary solution obtained by adding the amphiphilic polymer to one or both of the hydrophilic substance and the hydrophobic substance under the above-described conditions is hydrophilic.
  • the polymer solution containing the hydrophilic polymer and / or the hydrophobic substance is incorporated into the secondary solution to which the hydrophilic polymer is added under the above-described conditions while the aggregation is suppressed. It becomes possible to manufacture by order.
  • the primary solution containing the amphiphilic polymer and either one or both of the hydrophilic substance and the hydrophobic substance is used under the above-described conditions.
  • a hydrophilic substance or a hydrophobic substance is suppressed while agglomeration is suppressed by a structure in which ultrasonic waves are applied to the secondary solution in which a hydrophilic polymer is added to a primary emulsion formed by irradiating sound waves under the above-described conditions. It is possible to produce polymer fine particles enclosing one or both on the nanometer order.
  • a biodegradable polymer as an amphiphilic polymer, a hydrophobic polymer, and a hydrophilic polymer
  • a water-soluble (hydrophilic) polymer drug such as a protein, a nucleic acid, or a natural extract having physiological activity
  • Drugs encapsulating lipids and hydrophobic polymer drugs such as natural extracts having physiological activity and sustained-release cosmetics encapsulating hydrophilic and hydrophobic active substances
  • polymer fine particles encapsulating either or both of hydrophilic substances and hydrophobic substances are on the order of nanometers, so skin for pharmaceuticals for intravenous or arterial injection, pharmaceuticals for percutaneous absorption, cosmetics, etc. It can be used as an external composition, and enables systemic administration of drugs, targeting of affected areas, and penetration into the skin.
  • the composition for external use such as a coating agent can be applied to the fine irregularities on the skin surface substantially uniformly.
  • a biodegradable polymer as a polymer constituting the nanosphere, either one or both of the hydrophilic substance and the hydrophobic substance encapsulated in the nanosphere can be suitably and slowly released on the skin surface. In comparison with the prior art, it is possible to reduce the number of times of application while maintaining the effect of the external composition for skin.
  • nanospheres encapsulating either one or both of the hydrophilic substance and the hydrophobic substance produced by using the method for producing nanospheres encapsulating one or both of the hydrophilic substance and the hydrophobic substance With the constitution containing the nanosphere, the nanosphere can be applied substantially uniformly on the fine irregularities of the skin surface.
  • a biodegradable polymer as a polymer constituting the nanosphere, either one or both of the hydrophilic substance and the hydrophobic substance encapsulated in the nanosphere can be suitably and slowly released on the skin surface. It is possible to obtain the same effect even if the number of times of applying the cosmetic is reduced as compared with the conventional case.
  • stevia fermented extract which is a mixture of hydrophilic and hydrophobic substances, and nanospheres encapsulating sphingomyelin as an amphiphilic substance having the function of one or both of hydrophilic substances and hydrophobic substances It is also possible to produce a skin external composition and a cosmetic.
  • Stevia fermented extract and sphingomyelin have high moisturizing effect and rough skin as described in International Publication No. WO2008 / 126638A1 and “Research on Antihistamine Action of Stevia Fermented Extract” Pharmacology and Treatment vol.36 no.8 2008 Improves it, suppresses itching, suppresses inflammation, and has an antihistamine effect. Therefore, it is possible to distribute the stevia fermented extract or sphingomyelin substantially evenly on the fine irregularities on the skin surface by using a composition for external skin or cosmetic containing nanospheres containing stevia fermented extract or sphingomyelin. it can.
  • nanospheres containing either or both of stevia fermented extract and sphingomyelin are cosmetics for hair, hair styling, hair nourishing, scalp, hair coloring, hair washing, Hair rinse, skin cosmetics / skin, cosmetic liquid, cream, milky lotion, tanning, sunscreen, cleaning agent, shaving, dead hair shave, facial rinse, pack, cosmetic oil, body rinse, massage, finishing cosmetics and foundation , Makeup base, funny, lipstick, eye makeup, cheek cosmetics, body makeup, eau de cologne / perfume, bath cosmetics, nail cosmetics, body powders, etc., and in pharmaceuticals, powders / fine granules, granules, Tablets, capsules, pills, glazes, pencils, liquid contents, external liquids, extracts, plasters, suppositories Aerosol, gas chemistry, chemical adsorbents, ophthalmic agents, injections, bandages and the like are widely available.
  • Example 1 a diblock copolymer of polylysine and L-lactic acid (hereinafter simply referred to as PLys + -b-PLLA) is used as an amphiphilic polymer, PLGA is used as a hydrophobic polymer, and polyvinyl is used as a hydrophilic polymer.
  • BSA was used as a hydrophilic substance containing alcohol (hereinafter, simply referred to as PVA).
  • FIG. 2 is an explanatory diagram for explaining the synthesis method of PLys + -b-PLLA according to the first example.
  • the amino group of 2-aminoethanol was protected with a t-butoxycarbonyl (hereinafter simply referred to as Boc) group.
  • Boc t-butoxycarbonyl
  • the terminal hydroxyl group of Boc-aminoethanol was alkoxided with potassium naphthalene, and solution based anionic ring-opening polymerization of lactide was performed using this as an initiator.
  • the protecting group (Boc group) of the terminal amino group of the obtained polymer was removed with 25% hydrobromic acetic acid (25% HBr / AcOH), desalting was performed using triethylamine (TEA).
  • TEA triethylamine
  • the PLys + -b-PLLA according to this example had a polymerization degree of lysine of 16 and a polymerization degree of lactic acid of 39.
  • PLys + -b-PLLA according to this example has amphipathic properties and thus self-associates in water to form micelles.
  • FIG. 3 is an explanatory diagram for explaining the decomposability in the micelle state of PLys + -b-PLLA according to the first example.
  • PBS phosphate buffered saline
  • FIG. 4 is an explanatory diagram for explaining the first embodiment.
  • 20 mg of PLys + -b-PLLA and 180 mg of PLGA having a weight average molecular weight of 5000-75000 were placed in a test tube and dissolved in 2.5 ml of chloroform (CHCl 3 ).
  • 100 ⁇ l of PBS in which 5 mg of BSA was dissolved as a hydrophilic substance was added to the test tube to produce a primary solution.
  • ultrasonic irradiation was performed with a probe sonicator for 3 minutes to prepare a primary emulsion.
  • UD-200 manufactured by Tommy Seiko Co., Ltd.
  • UD-200 manufactured by Tommy Seiko Co., Ltd. and irradiating ultrasonic waves at levels 5 to 10 is simply referred to as irradiating ultrasonic waves with a probe sonicator.
  • the primary emulsion was added to 300 ml of PBS containing 0.1% PVA to prepare a secondary solution.
  • the secondary solution was irradiated with ultrasonic waves for 3 minutes with a bath sonicator, stirred vigorously with a spatula for 90 seconds, and further irradiated with ultrasonic waves for 90 seconds with a probe sonicator to prepare a secondary emulsion.
  • US-2 manufactured by SND Co., Ltd. was used as a bath sonicator, and ultrasonic waves were applied with the level set to 5.
  • the irradiation with ultrasonic waves set to level 5 using US-2 manufactured by SND Corporation is simply referred to as irradiating ultrasonic waves with a bath-type sonicator.
  • the secondary emulsion was further added to 300 ml of PBS containing 0.1% PVA and dried in liquid to obtain a suspension from which chloroform was removed. Thereafter, the suspension was centrifuged at 20000 rpm for 10 minutes to obtain a precipitate containing biodegradable nanospheres containing BSA. The precipitate was washed with ultrapure water. After repeating this operation consisting of centrifugation and washing several times, the precipitate was freeze-dried to remove water, thereby obtaining biodegradable nanospheres containing BSA.
  • the shape and particle size of the biodegradable nanosphere encapsulating BSA were examined using a scanning electron microscope (SEM). A carbon double-sided tape is affixed to a scanning electron microscope sample stage, and biodegradable nanospheres containing BSA after freeze-drying are placed on the sample. Created. The particle size of the biodegradable nanosphere encapsulating BSA was determined by measuring the particle size of a biodegradable nanosphere encapsulating randomly selected BSA.
  • FIG. 5 is an explanatory diagram for explaining the results of observation of biodegradable nanospheres containing BSA with a scanning electron microscope.
  • the particle size of the biodegradable nanosphere encapsulating BSA was about 150 nm to about 800 nm. Further, aggregation was relatively suppressed, and a fine particle surface was obtained.
  • biodegradable nanospheres containing either or both of hydrophilic substances and hydrophobic substances are used as pharmaceuticals for intravenous or arterial injection, pharmaceuticals for transdermal absorption And can be used for compositions for external use of skin such as cosmetics and the like, and enables systemic administration of drugs, targeting to affected areas and penetration into skin.
  • the BSA encapsulation rate and BSA recovery rate of the biodegradable nanospheres were measured.
  • six sample tubes were prepared, of which five sample tubes contained empty biodegradable nanospheres without inclusions, and the remaining one sample tube contained BSA whose inclusion rate was unknown. 20 mg of each biodegradable nanosphere was added.
  • sample tubes containing empty biodegradable nanospheres contain 1N water containing BSA so that the BSA concentrations are 0, 0.3, 0.5, 0.7, and 0.9 mg / ml, respectively. 3 ml each of an aqueous sodium oxide solution was placed, and 3 ml of a 1N aqueous sodium hydroxide solution was placed in a sample tube containing biodegradable nanospheres containing BSA.
  • a calibration curve was prepared by measuring absorbance at a wavelength of 290 nm using a solution obtained by decomposing empty biodegradable nanospheres having a BSA concentration of 0 mg / ml as a background. And the light absorbency of the solution which decomposed
  • FIG. 6 is an explanatory diagram for explaining formulas for calculating the inclusion rate, recovery rate, yield, and inclusion efficiency in the examples.
  • the encapsulation rate is the ratio (%) of the weight of the encapsulated substance (in this embodiment, hydrophilic substance) to the nanospheres per unit weight obtained
  • the recovery rate is the encapsulated substance used. It is the ratio (%) of the collected encapsulated substance (in this example, hydrophilic substance) to the (in this example, hydrophilic substance), and the yield is the encapsulated used as a component of the nanosphere.
  • the ratio of the recovered nanosphere weight to the weight of the substance (hydrophilic substance in this example), amphiphilic polymer, hydrophobic polymer and hydrophilic polymer, and the encapsulation efficiency is the encapsulated efficiency used.
  • Nanospheres recovered with either or both of the included hydrophilic substance and / or hydrophobic substance with respect to the ratio of the substance to be used (hydrophilic substance in this example) and the total of the polymers used Shows the proportion of the polymer, the ratio of a percentage (%).
  • the BSA encapsulation rate of the biodegradable nanosphere of Example 1 was 2.9%.
  • the BSA recovery rate in Example 1 was calculated using the formula for calculating the recovery rate shown in FIG. 6 from the BSA concentration in the obtained supernatant. Asked. As a result, the recovery rate of BSA in Example 1 was 91.0%.
  • the recovery rate of BSA is as high as 90% or more, even when expensive substances such as pharmaceuticals are included, it is possible to suitably recover expensive substances such as pharmaceuticals. Therefore, the collected substance can be reused, and an unnecessary increase in drug price can be suppressed.
  • Example 2 a diblock copolymer of polyethylene glycol having a number average molecular weight of 3000 and L-lactic acid (hereinafter simply referred to as PEG3K-b-PLLA) as an amphiphilic polymer, PLGA as a hydrophobic polymer, Sphingomyelin was used as an amphiphilic substance having functions of a hydrophilic substance and a hydrophobic substance as a substance encapsulating PVA as a hydrophilic polymer.
  • PEG3K-b-PLLA polyethylene glycol having a number average molecular weight of 3000 and L-lactic acid
  • FIG. 7 is an explanatory diagram for explaining a method of synthesizing PEG3K-b-PLLA according to Example 2.
  • FIG. 8 is an explanatory diagram for explaining the second embodiment.
  • 20 mg of PEG3K-b-PLLA and 180 mg of PLGA having a weight average molecular weight of 5000-75000 were placed in a test tube and dissolved in 2.5 ml of chloroform.
  • 5 mg of sphingomyelin was added to the test tube to produce a primary solution.
  • the primary solution was vigorously stirred with a spatula, ultrasonic irradiation was performed with a probe sonicator for 3 minutes to prepare a primary emulsion.
  • the primary emulsion was added to 300 ml of PBS containing 0.1% PVA to prepare a secondary solution.
  • the secondary solution was irradiated with ultrasonic waves for 3 minutes with a bath sonicator, stirred vigorously with a spatula for 90 seconds, and further irradiated with ultrasonic waves for 90 seconds with a probe sonicator to prepare a secondary emulsion.
  • the secondary emulsion was further added to 300 ml of PBS containing 0.1% PVA and dried in liquid to obtain a suspension from which chloroform was removed. Thereafter, the suspension was centrifuged at 20000 rpm for 10 minutes to obtain a precipitate containing biodegradable nanospheres encapsulating sphingomyelin as an amphiphilic substance. The precipitate was washed with ultrapure water. After repeating this operation consisting of centrifugation and washing several times, the precipitate was freeze-dried to remove water, thereby obtaining biodegradable nanospheres containing sphingomyelin.
  • FIG. 9 is an explanatory diagram for explaining the results of observing biodegradable nanospheres containing sphingomyelin with a scanning electron microscope. As shown in FIG. 9, the particle size of the biodegradable nanosphere encapsulating sphingomyelin was relatively suppressed from aggregation and a fine particle surface was obtained.
  • the concentration of sphingomyelin in Example 2 was calculated from the concentration of sphingomyelin in the obtained supernatant using the formula for calculating the yield shown in FIG. The rate was determined. As a result, the yield of sphingomyelin in Example 2 was 53.7%.
  • nanospheres including not only hydrophilic substances but also amphiphile sphingomyelin having functions of hydrophilic substances and hydrophobic substances could be produced. Therefore, it has become possible to produce nanospheres that contain not only hydrophilic substances but also hydrophilic substances and hydrophobic substances.
  • the fine polymer particles encapsulating sphingomyelin are on the order of nanometers, they can be used for pharmaceutical preparations for intravenous or arterial injection, pharmaceutical preparations for percutaneous absorption, cosmetic preparations, etc. Can be administered systemically, targeted to the affected area, and penetrated into the skin.
  • a skin external composition or cosmetic containing nanospheres containing sphingomyelin is produced by the moisturizing effect based on ceramide of sphingomyelin
  • the skin external composition or cosmetic can be applied to fine irregularities on the skin surface. Since it can be applied substantially uniformly, the moisturizing effect of the external composition for skin or cosmetic can be evenly applied to the skin.
  • a biodegradable polymer as the polymer constituting the nanosphere, sphingomyelin can be suitably and slowly released on the skin surface, and the application of a composition for external skin or cosmetic compared to the conventional case Even if the number of times is reduced, a similar moisturizing effect can be obtained.
  • Example 3 In this example, Dex-g-PLLA is used as an amphiphilic polymer, PVA is used as a hydrophilic polymer, and a stevia ferment extract is used as a mixture of a hydrophilic substance and a hydrophobic substance as an encapsulating substance, Hydrophobic polymer was not used.
  • FIG. 10 is an explanatory diagram for explaining the Dex-g-PLLA synthesis method according to the third embodiment.
  • TMS trimethylsilyl
  • TMSDex trimethylsilyl dextran
  • L-lactide and TMSDex obtained in FIG. 10 (a) were first dried under reduced pressure on the day before the synthesis.
  • L-lactide was weighed into a Claisen flask and directly drawn under reduced pressure at room temperature.
  • the hydroxyl group was alkoxided with potassium naphthalene, and this was used as an initiator for solution-based anionic ring-opening polymerization of lactide.
  • TMSDex-g-PLLA was dissolved by adding DMSO (Dimethylsulfoxide) and stirring with a stirrer. After confirming that TMSDex-g-PLLA was completely dissolved, methanol was added. Then, acetic acid was added and stirred for 4 hours. After 4 hours, reprecipitation using methanol as a poor solvent was performed, ultracentrifugation was performed, and the precipitate was collected and dried under reduced pressure in a desiccator. Further purification was performed by reprecipitation using DMSO or chloroform as a good solvent and methanol as a poor solvent, depending on the sugar content. This was repeated several times to obtain Dex-g-PLLA.
  • DMSO Dimethylsulfoxide
  • FIG. 11 is an explanatory diagram for explaining the degradability of Dex-g-PLLA according to Example 3
  • FIG. 12 is an explanatory diagram for explaining Dex-g-PLLA used in the decomposability experiment. is there.
  • the mass of the Dex-g-PLLA film and PLLA film prepared by the casting method using chloroform was weighed with an electronic balance, immersed in PBS, incubated at 37 ° C., 1 day, 2 days, 4 days, 7 days After 14 days and after 28 days, the mass was measured after removing moisture by drying under reduced pressure. From the weight of the film before decomposition and the weight of the film after decomposition, the weight reduction rate of the film was determined using the formula shown in FIG.
  • the molecular weight reduction rate of Dex-g-PLLA was determined using the formula shown in FIG. .
  • Dex-g-PLLA has a higher weight reduction rate than the PLLA.
  • the lower the degree of polymerization of L-lactide in Dex-g-PLLA the higher the weight reduction rate of the film.
  • Dex-g-PLLA has a lower molecular weight reduction rate than PLLA. Further, the higher the sugar content in Dex-g-PLLA, the higher the molecular weight reduction rate. Therefore, by adjusting the polymerization degree or sugar content of L-lactide in Dex-g-PLLA as an amphiphilic polymer, when biodegradable nanospheres are placed in vivo, the inflammatory reaction associated with the degradation can be reduced. It can be adjusted to a biodegradation rate that does not occur and disappears from the living body after a lapse of a period required in the living body.
  • FIG. 13 is an explanatory diagram for explaining the third embodiment.
  • 200 mg of G45-12-20 or G80-9-16 which is Dex-g-PLLA
  • G45-12-20 or G80-9-16 which is Dex-g-PLLA
  • CHCl 3 chloroform
  • 5 mg of stevia fermented extract was added to the test tube to produce a primary solution.
  • a primary emulsion was prepared by irradiating ultrasonic waves with a bath sonicator for 5 minutes while stirring the primary solution vigorously with a spatula.
  • the primary emulsion was added to 300 ml of PBS containing 0.1% PVA to prepare a secondary solution.
  • a secondary emulsion was prepared by irradiating the secondary solution with ultrasonic waves for 3 minutes using a probe sonicator.
  • the secondary emulsion was further added to 300 ml of PBS containing 0.1% PVA and dried in liquid to obtain a suspension from which chloroform was removed. Thereafter, the suspension was centrifuged at 20000 rpm for 10 minutes to obtain a precipitate containing biodegradable nanospheres containing stevia fermentation extract. The precipitate was washed with ultrapure water. After repeating this operation consisting of centrifugation and washing several times, the precipitate was freeze-dried and water was removed to obtain biodegradable nanospheres containing stevia fermentation extract.
  • FIG. 14 is an explanatory diagram for explaining the result of observing the biodegradable nanosphere containing the stevia fermented extract with a scanning electron microscope. As shown in FIG. 14, the particle size of the biodegradable nanosphere encapsulating the stevia fermented extract was relatively suppressed from aggregation and a fine particle surface was obtained.
  • the stevia fermented extract in Example 3 was calculated from the concentration of stevia fermented extract in the obtained supernatant using the formula for calculating the yield shown in FIG. The yield of was determined. As a result, the yield of stevia fermented extract in Example 3 was 65.8% for G45-12-20 and 63.7% for G80-9-16.
  • Example 3 it was possible to produce nanospheres containing not only hydrophilic substances but also stevia fermented extract, which is a mixture of hydrophilic substances and hydrophobic substances. Therefore, it has become possible to produce nanospheres that include not only hydrophilic substances but also mixtures of hydrophilic substances and hydrophobic substances.
  • the fine polymer particles encapsulating the stevia fermented extract are on the order of nanometers, and therefore can be used for pharmaceutical preparations for intravenous or arterial injection, pharmaceutical preparations for transdermal absorption, cosmetic compositions such as cosmetics, Systemic administration of drugs, targeting to affected areas and penetration into the skin are possible.
  • the recovery rate of stevia fermented extract is as high as 60% or more, it is suitable even when encapsulating expensive substances such as effective substances used in pharmaceuticals, compositions for external use of skin, and cosmetics. Expensive substances such as active ingredients can be recovered. Therefore, the collected substance can be reused, and an unnecessary increase in drug price can be suppressed.
  • Stevia fermented extract has a high moisturizing effect, improves rough skin, suppresses itching, suppresses inflammation, and has an antihistaminic action. Therefore, if a composition for external skin or cosmetics containing nanospheres containing stevia fermented extract is manufactured, stevia fermented extract can be distributed almost evenly on the fine irregularities of the skin surface, and the moisturizing effect of stevia fermented extract It is possible to uniformly apply rough skin improving effect, itching inhibiting effect, inflammation inhibiting effect and antihistamine effect to the skin surface.
  • Example 4 In this example, Dex-g-PLLA was used as the amphiphilic polymer, EtO-PLLA was used as the hydrophobic polymer, PVA was used as the hydrophilic polymer, and BSA was used as the hydrophilic substance to be included.
  • FIG. 15 is an explanatory diagram for explaining the fourth embodiment.
  • 170 mg of EtO-PLLA and G71-13-13 which is Dex-g-PLLA (see FIG. 13B) were placed in a test tube, and methylene chloride (CH 2 Cl 2 ) It was dissolved in 2.4 ml.
  • 100 ⁇ l of PBS in which 5 mg of BSA was dissolved was added to the test tube to form a primary solution.
  • the primary solution was vigorously stirred with a spatula, ultrasonic waves were irradiated for 5 minutes with a bath sonicator and 1.5 minutes with a probe sonicator to prepare a primary emulsion.
  • a secondary emulsion was prepared by irradiating the secondary solution with ultrasonic waves with a probe-type sonicator for 1.5 minutes.
  • the secondary emulsion was added to 200 ml of PBS containing 0.1% PVA, stirred for 1 hour, and dried in liquid at about 40 ° C. for 2 hours to obtain a suspension from which methylene chloride was removed. Thereafter, the suspension was centrifuged at 20000 rpm for 10 minutes to obtain a precipitate containing biodegradable nanospheres containing BSA. The precipitate was washed with ultrapure water. After repeating this operation consisting of centrifugation and washing several times, the precipitate was freeze-dried to remove water, thereby obtaining biodegradable nanospheres containing BSA.
  • FIG. 16 is an explanatory diagram for explaining the results of observation of biodegradable nanospheres containing BSA with a scanning electron microscope.
  • the particle size of the biodegradable nanosphere encapsulating BSA was about 400 nm to about 600 nm. Further, aggregation was relatively suppressed, and a fine particle surface was obtained.
  • the biodegradable nanosphere encapsulating a hydrophilic substance is used as a skin for pharmaceuticals for intravenous or arterial injection, pharmaceuticals for transdermal absorption, cosmetics, etc. It can be used as an external composition, and enables systemic administration of drugs, targeting of affected areas, and penetration into the skin.
  • the BSA encapsulation rate and BSA recovery rate of the biodegradable nanospheres were measured.
  • six sample tubes were prepared, of which five sample tubes contained empty biodegradable nanospheres without inclusions, and the remaining one sample tube contained BSA whose inclusion rate was unknown. 20 mg of each biodegradable nanosphere was added.
  • sample tubes containing empty biodegradable nanospheres contain 1N water containing BSA so that the BSA concentrations are 0, 0.1, 0.15, 0.23, and 0.3 mg / ml, respectively. 3 ml each of an aqueous sodium oxide solution was placed, and 3 ml of a 1N aqueous sodium hydroxide solution was placed in a sample tube containing biodegradable nanospheres containing BSA.
  • a calibration curve was prepared by measuring the absorbance at a wavelength of 291 nm using a solution obtained by decomposing empty biodegradable nanospheres having a BSA concentration of 0 mg / ml as a background. And the light absorbency of the solution which decomposed
  • the BSA encapsulation efficiency of the biodegradable nanosphere of Example 4 was calculated to be 52.9%.
  • the BSA recovery rate in Example 4 was calculated using the formula for calculating the recovery rate shown in FIG. 6 from the BSA concentration in the obtained supernatant. Asked. As a result, the recovery rate of BSA in Example 4 was 82.7%.
  • BSA recovery rate of BSA is as high as 80% or more, even when expensive substances such as active ingredients used for pharmaceuticals, compositions for external use of skin, and cosmetics are encapsulated, the active ingredients etc. Expensive material can be recovered. Therefore, the collected substance can be reused, and an unnecessary increase in drug price can be suppressed.
  • each process in the manufacturing method of the nanosphere of this specification does not necessarily need to process in time series along the order described as a flowchart, and may include the process by parallel or a subroutine.
  • the present invention includes, for example, a method for producing a nanosphere capable of enclosing one or both of a hydrophilic substance such as a protein, a nucleic acid, a drug and a contrast agent, or a hydrophobic substance such as a lipid and a drug. It can be used for an external composition for skin and cosmetics.
  • a hydrophilic substance such as a protein, a nucleic acid, a drug and a contrast agent
  • a hydrophobic substance such as a lipid and a drug. It can be used for an external composition for skin and cosmetics.

Abstract

Disclosed is a method for producing nanospheres, by which fine polymer particles that internally contain either a hydrophilic substance and/or a hydrophobic substance and have a particle diameter of nano order can be produced. In the method for producing nanospheres, a solvent in which either a hydrophilic substance and/or a hydrophobic substance is dissolved, a water-immiscible organic solvent in which an amphiphilic polymer is dissolved, and a water-immiscible organic solvent in which a hydrophobic polymer is dissolved are mixed together, then the thus-obtained primary solution is irradiated with an ultrasonic wave, thereby forming a primary emulsion, then an aqueous solvent in which a hydrophilic polymer is dissolved is added into the primary emulsion, thereby forming a secondary solution, and then the secondary solution is irradiated with an ultrasonic wave, thereby forming a secondary emulsion.

Description

ナノスフェアの製造方法、ナノスフェア、これを含有する皮膚外用組成物および化粧料Method for producing nanosphere, nanosphere, external composition for skin containing the same, and cosmetic
　本発明は、例えば、タンパク質、核酸、薬物、造影剤等の親水性物質、もしくは、脂質、薬物等の疎水性物質のいずれか一方または両方を内包できるナノスフェアの製造方法、ナノスフェア、これを含有する皮膚外用組成物および化粧料に関する。 The present invention includes, for example, a method for producing a nanosphere capable of enclosing one or both of a hydrophilic substance such as a protein, a nucleic acid, a drug and a contrast agent, or a hydrophobic substance such as a lipid and a drug. The present invention relates to an external composition for skin and a cosmetic.
発明の背景Background of the Invention
　一般に病気の治療においては、「必要なときに、必要な量の薬物を、必要な病巣に選択的に送り届ける」ことが理想的であると言われている。従来の飲み薬等は、体内の目的場所（病巣）に到達する過程において、ほとんどの成分が変化してしまい、投与量と比較して、目的場所に到達する量はわずかであるという課題があった。 In general, in the treatment of diseases, it is said that it is ideal to "selectively deliver a necessary amount of a drug to a necessary lesion when necessary". With conventional swallows, most of the components change in the process of reaching the target location (lesion) in the body, and there is a problem that the amount reaching the target location is small compared to the dose. It was.
　そこで、生分解性ポリマー等で薬物を内包した高分子微粒子を投与することにより、薬物を変化させることなく目的場所に到達させる、所謂ドラッグ・デリバリー・システム（Drug Delivery System:以下単にＤＤＳと称する）が注目を集めている。 Therefore, a so-called drug delivery system (hereinafter simply referred to as DDS) that administers polymer microparticles encapsulating a drug with a biodegradable polymer or the like to reach the target location without changing the drug. Has attracted attention.
　さらに、生分解性ポリマー等で薬物を内包した高分子微粒子を投与して、所定量の薬物を長時間にわたって放出させることで、患者のＱＯＬ（Quality Of Life）を高めることができる薬物徐放型ＤＤＳも脚光を浴びている。 Furthermore, a sustained drug release type that can increase a patient's QOL (Quality Of Life) by administering a polymer microparticle encapsulating a drug with a biodegradable polymer or the like and releasing a predetermined amount of the drug over a long period of time. DDS is also in the spotlight.
　薬物徐放型ＤＤＳ製剤に利用される、生分解性ポリマーを用いた高分子微粒子として、粒子径０．１μｍ程度～数１００μｍと幅広い範囲の粒子径の高分子微粒子（マイクロスフェア、またはマイクロパーティクル）が報告されている。例えば、特許文献１には、ポリ乳酸やポリ乳酸共重合体で酢酸リュープロレリンを内包したマイクロスフェアが開示され、かかる酢酸リュープロレリンを内包したマイクロスフェアは皮下注射用製剤として実用化されている。 Polymeric microparticles (microspheres or microparticles) with a wide range of particle sizes ranging from about 0.1 μm to several hundreds of μm as polymer microparticles using biodegradable polymers used in drug sustained-release DDS formulations Has been reported. For example, Patent Document 1 discloses microspheres containing leuprorelin acetate in a polylactic acid or polylactic acid copolymer, and the microspheres containing such leuprorelin acetate have been put into practical use as a preparation for subcutaneous injection. Yes.
　一方、近年では、ホルモンや成長因子等のタンパク質やＤＮＡ、ＲＮＡ等の核酸、生理活性を有する天然抽出物等の水溶性高分子の薬物としての利用が、ますます重要になっている。そこで、水溶性高分子の薬物を内包する高分子微粒子を利用した薬物徐放型ＤＤＳ製剤が要求されている。 On the other hand, in recent years, the use of water-soluble polymers such as proteins such as hormones and growth factors, nucleic acids such as DNA and RNA, and natural extracts having physiological activity has become increasingly important. Therefore, there is a demand for a drug sustained-release DDS preparation using polymer fine particles encapsulating a water-soluble polymer drug.
　特許文献１に記載のポリ乳酸やポリ乳酸共重合体を用いて水溶性高分子を内包する場合、ポリ乳酸やポリ乳酸共重合体（以下、単に疎水性生分解性ポリマーと称する）は疎水性であるため、Ｗ／Ｏ／Ｗ（Water in Oil in Water）エマルション法等が利用される。しかし、疎水性生分解性ポリマーを用いて水溶性高分子を内包するマイクロスフェアを製造すると、マイクロスフェアの内部における水溶性高分子の分布の均一性が低くなり、薬物徐放型ＤＤＳ製剤とした場合における水溶性高分子の効力持続性（徐放化）が不十分であった。また、水溶性高分子がタンパク質や核酸である場合、マイクロスフェアを製造する過程で、立体構造が維持できなくなったり、変性してしまったりして、失活してしまうこともあった。 When the water-soluble polymer is encapsulated using the polylactic acid or polylactic acid copolymer described in Patent Document 1, the polylactic acid or polylactic acid copolymer (hereinafter simply referred to as a hydrophobic biodegradable polymer) is hydrophobic. Therefore, a W / O / W (Water in Oil Water) emulsion method or the like is used. However, when microspheres encapsulating water-soluble polymers are produced using hydrophobic biodegradable polymers, the uniformity of the distribution of water-soluble polymers in the microspheres is reduced, and a drug sustained-release DDS preparation is obtained. In some cases, the sustainability (sustained release) of the water-soluble polymer was insufficient. In addition, when the water-soluble polymer is a protein or nucleic acid, in the process of producing the microsphere, the three-dimensional structure may not be maintained or may be denatured and may be deactivated.
　そこで、生分解性ポリマーとして両親媒性を有するポリ乳酸グラフト化デキストランを用いることにより、Ｗ／Ｏ／Ｗエマルション法で、水溶性高分子を内包するマイクロスフェアが製造可能な技術が開示されている（非特許文献１）。非特許文献１に記載のマイクロスフェアは、水溶性高分子としてウシ血清アルブミン（Bovine Serum Albumin:以下、単にＢＳＡと称する）を内包している。かかるマイクロスフェアは、ＢＳＡがマイクロスフェアの内部で略均一に分布しており、ＢＳＡの高次構造および活性も維持されているため、薬物徐放型ＤＤＳ製剤として十分な徐放化を達成することが可能となる。 Thus, a technique is disclosed that can produce microspheres encapsulating water-soluble polymers by a W / O / W emulsion method by using amphiphilic polylactic acid grafted dextran as a biodegradable polymer. (Non-Patent Document 1). The microsphere described in Non-Patent Document 1 includes bovine serum albumin (hereinafter simply referred to as BSA) as a water-soluble polymer. In such a microsphere, BSA is distributed almost uniformly inside the microsphere, and the higher-order structure and activity of BSA are maintained, so that sufficient sustained release as a drug sustained-release DDS preparation is achieved. Is possible.
　しかし、非特許文献１に記載の水溶性高分子を内包する高分子微粒子は、スフェア粒径がマイクロメートルオーダーと大きいため、すなわちマイクロスフェアとなるため、静脈または動脈注射、経皮吸収には適していない。 However, the polymer fine particles encapsulating the water-soluble polymer described in Non-Patent Document 1 are suitable for intravenous or arterial injection and percutaneous absorption because the sphere particle size is as large as micrometer order, that is, microspheres. Not.
　静脈または動脈注射や経皮吸収に適したナノメートルオーダーの高分子微粒子（ナノスフェア）を製造する技術として、Ｏ／Ｗ（Oil in Water）エマルション法を利用する技術が開示されている。例えば、非特許文献２には、Ｏ／Ｗエマルション法でポリ乳酸グラフト化デキストランを用いてナノスフェアを製造する技術が、非特許文献３には、Ｏ／Ｗエマルション法で両親媒性のブロック共重合体を用いてナノスフェアを製造する技術が開示されている。
特開平１０－１８２４９６号公報 Ouchi T, Saito T, Kontani T, Ohya Y, Macromol Biosci. 2004 Apr 19;4(4):458-463 Nouvel C, Raynaud J, Marie E, Dellacherie E, Six JL, Durand A, J Colloid Interface Sci. 2009 Feb 15; 330(2):337-343 Ouchi T, Toyohara M, Arimura H, Ohya Y,Biomacromolecules. 2002 Sep-Oct; 3(5):885-888 As a technique for producing nanometer order polymer fine particles (nanospheres) suitable for intravenous or arterial injection or percutaneous absorption, a technique using an O / W (Oil in Water) emulsion method is disclosed. For example, Non-Patent Document 2 discloses a technique for producing nanospheres using polylactic acid-grafted dextran by the O / W emulsion method, and Non-Patent Document 3 discloses an amphiphilic block co-polymer by the O / W emulsion method. Techniques for producing nanospheres using coalescence are disclosed.
Japanese Patent Laid-Open No. 10-182496 Ouchi T, Saito T, Kontani T, Ohya Y, Macromol Biosci. 2004 Apr 19; 4 (4): 458-463 Nouvel C, Raynaud J, Marie E, Dellacherie E, Six JL, Durand A, J Colloid Interface Sci. 2009 Feb 15; 330 (2): 337-343 Ouchi T, Toyohara M, Arimura H, Ohya Y, Biomacromolecules. 2002 Sep-Oct; 3 (5): 885-888
　しかし、上述した非特許文献２および３に記載された、Ｏ／Ｗエマルション法を利用した場合、ナノスフェアを製造できるものの、疎水性物質しか内包できない、すなわち水溶性高分子を内包することができないといった問題点がある。 However, when the O / W emulsion method described in Non-Patent Documents 2 and 3 described above is used, nanospheres can be produced, but only hydrophobic substances can be included, that is, water-soluble polymers cannot be included. There is a problem.
　上述したように、水溶性高分子の内包と、高分子微粒子の粒径の微細化との両立を成し得た例はなく、水溶性高分子を内包し、かつ、ナノメートルオーダーの高分子微粒子の開発が望まれている。 As mentioned above, there is no example that can achieve both the inclusion of the water-soluble polymer and the refinement of the particle size of the polymer fine particle, the polymer containing the water-soluble polymer and having a nanometer order Development of fine particles is desired.
　そこで本発明は、このような課題に鑑み、水溶性高分子等の親水性物質もしくは疎水性物質のいずれか一方または両方を内包し、かつ粒径がナノオーダーである高分子微粒子を製造することができるナノスフェアの製造方法、ナノスフェア、これを含有する皮膚外用組成物および化粧料を提供することを目的とする。 Therefore, in view of such problems, the present invention is to produce polymer fine particles that contain either or both of a hydrophilic substance and a hydrophobic substance such as a water-soluble polymer and that have a particle size of nano-order. It is an object to provide a method for producing nanospheres, nanospheres, a composition for external use on skin containing the nanospheres, and a cosmetic.
　本発明の一例であるＷ／Ｏ／Ｗエマルション法を用いた、ナノスフェアの製造方法は、親水性物質を溶解した水系溶媒と、両親媒性ポリマーを溶解した水非混和性有機溶媒と、疎水性ポリマーを溶解した水非混和性有機溶媒とを混合し、１次溶液を生成する１次溶液生成ステップと、１次溶液に超音波を照射して１次エマルションを生成する１次エマルション生成ステップと、１次エマルションに、親水性ポリマーを溶解した水系溶媒を添加し、２次溶液を生成する２次溶液生成ステップと、２次溶液に超音波を照射して２次エマルションを生成する２次エマルション生成ステップと、を含む。当該ナノスフェアは親水性物質を内包する。 A method for producing nanospheres using a W / O / W emulsion method as an example of the present invention includes an aqueous solvent in which a hydrophilic substance is dissolved, a water-immiscible organic solvent in which an amphiphilic polymer is dissolved, and a hydrophobic property. A primary solution generation step of mixing a water-immiscible organic solvent in which a polymer is dissolved to generate a primary solution; and a primary emulsion generation step of generating a primary emulsion by irradiating the primary solution with ultrasonic waves. A secondary solution generating step for adding a water-based solvent in which a hydrophilic polymer is dissolved to the primary emulsion to generate a secondary solution, and a secondary emulsion for generating a secondary emulsion by irradiating the secondary solution with ultrasonic waves Generating step. The nanosphere contains a hydrophilic substance.
　疎水性ポリマーおよび親水性物質に両親媒性ポリマーを添加した１次溶液に超音波を照射して生成した１次エマルションに親水性ポリマーを添加した２次溶液に、超音波を照射する構成により、凝集を抑制しつつ、親水性物質を内包するＷ／Ｏ／Ｗエマルションの高分子微粒子をナノメートルオーダーで製造することが可能となる。 By irradiating the secondary solution in which the hydrophilic polymer is added to the primary emulsion formed by irradiating the primary solution in which the amphiphilic polymer is added to the hydrophobic polymer and the hydrophilic substance with ultrasonic waves, It becomes possible to produce polymer fine particles of a W / O / W emulsion containing a hydrophilic substance on the nanometer order while suppressing aggregation.
　したがって、両親媒性ポリマー、疎水性ポリマーおよび親水性ポリマーに生分解性ポリマーを用いれば、タンパク質や核酸、生理活性を有する天然抽出物等の水溶性（親水性）高分子の薬物を内包する薬物徐放型ＤＤＳ製剤等の医薬品や親水性の有効物質を内包する徐放型の化粧料を製造することができる。また、親水性物質を内包する高分子微粒子は、ナノメートルオーダーであるため、静脈または動脈注射用の医薬品、経皮吸収用の医薬品や化粧料等の皮膚外用組成物に利用することができ、薬物の全身投与、患部へのターゲッティングおよび皮膚への浸透が可能となる。 Therefore, if biodegradable polymers are used for amphiphilic polymers, hydrophobic polymers, and hydrophilic polymers, drugs that contain water-soluble (hydrophilic) high molecular weight drugs such as proteins, nucleic acids, and biologically active natural extracts. Sustained release cosmetics can be produced that contain pharmaceuticals such as sustained release DDS preparations and hydrophilic active substances. In addition, since the polymer fine particles encapsulating the hydrophilic substance are on the nanometer order, it can be used for pharmaceutical preparations for intravenous or arterial injection, pharmaceutical preparations for percutaneous absorption, cosmetic compositions such as cosmetics, Systemic administration of drugs, targeting to affected areas and penetration into the skin are possible.
　他のＷ／Ｏ／Ｗエマルション法を用いた、ナノスフェアの製造方法は、親水性物質を溶解した水系溶媒と、両親媒性ポリマーを溶解した水非混和性有機溶媒とを混合し、１次溶液を生成する１次溶液生成ステップと、１次溶液に超音波を照射して１次エマルションを生成する１次エマルション生成ステップと、１次エマルションに、親水性ポリマーを溶解した水系溶媒を添加し、２次溶液を生成する２次溶液生成ステップと、２次溶液に超音波を照射して２次エマルションを生成する２次エマルション生成ステップと、を含む。当該ナノスフェアは、親水性物質を内包する。 Another method for producing nanospheres using the W / O / W emulsion method is to mix an aqueous solvent in which a hydrophilic substance is dissolved with a water-immiscible organic solvent in which an amphiphilic polymer is dissolved. A primary solution generation step for generating a primary emulsion, a primary emulsion generation step for generating a primary emulsion by irradiating ultrasonic waves to the primary solution, and an aqueous solvent in which a hydrophilic polymer is dissolved in the primary emulsion, A secondary solution generating step for generating a secondary solution; and a secondary emulsion generating step for generating a secondary emulsion by irradiating the secondary solution with ultrasonic waves. The nanosphere contains a hydrophilic substance.
　ここでは、両親媒性ポリマーと親水性物質を含む１次溶液に超音波を照射して生成した１次エマルションに親水性ポリマーを添加した２次溶液に、超音波を照射する構成により、凝集を抑制しつつ、親水性物質を内包するＷ／Ｏ／Ｗエマルションの高分子微粒子をナノメートルオーダーで製造することが可能となる。 Here, aggregation is performed by irradiating ultrasonic waves to a secondary solution obtained by adding a hydrophilic polymer to a primary emulsion formed by irradiating ultrasonic waves to a primary solution containing an amphiphilic polymer and a hydrophilic substance. While suppressing, it becomes possible to produce polymer fine particles of a W / O / W emulsion encapsulating a hydrophilic substance on the nanometer order.
　したがって、両親媒性ポリマーおよび親水性ポリマーに生分解性ポリマーを用いれば、タンパク質や核酸、生理活性を有する天然抽出物等の水溶性（親水性）高分子の薬物を内包する医薬品や親水性の有効物質を内包する徐放型の化粧料を製造することができる。また、親水性物質を内包する高分子微粒子は、ナノメートルオーダーであるため、静脈または動脈注射用の医薬品、経皮吸収用の医薬品や化粧料等の皮膚外用組成物に利用することができ、薬物の全身投与、患部へのターゲッティングおよび皮膚への浸透が可能となる。 Therefore, if a biodegradable polymer is used for an amphiphilic polymer and a hydrophilic polymer, a pharmaceutical or hydrophilic drug containing a water-soluble (hydrophilic) polymer such as a protein, a nucleic acid, or a physiologically active natural extract is included. A sustained-release cosmetic that encapsulates an active substance can be produced. In addition, since the polymer fine particles encapsulating the hydrophilic substance are on the nanometer order, it can be used for pharmaceutical preparations for intravenous or arterial injection, pharmaceutical preparations for percutaneous absorption, cosmetic compositions such as cosmetics, Systemic administration of drugs, targeting to affected areas and penetration into the skin are possible.
　さらに他のＯ／Ｗエマルション法を用いた、ナノスフェアの製造方法は、疎水性物質を溶解した水非混和性有機溶媒と、両親媒性ポリマーを溶解した水非混和性有機溶媒と、疎水性ポリマーを溶解した水非混和性有機溶媒とを混合し、１次溶液を生成する１次溶液生成ステップと、１次溶液に、親水性ポリマーを溶解した水系溶媒を添加し、２次溶液を生成する２次溶液生成ステップと、２次溶液に超音波を照射してエマルションを生成するエマルション生成ステップと、を含む。当該ナノスフェアは、疎水性物質を内包する。 Furthermore, the nanosphere manufacturing method using another O / W emulsion method includes a water-immiscible organic solvent in which a hydrophobic substance is dissolved, a water-immiscible organic solvent in which an amphiphilic polymer is dissolved, and a hydrophobic polymer. A water-immiscible organic solvent in which water is dissolved is mixed to form a primary solution, and a primary solution is generated. A water-based solvent in which a hydrophilic polymer is dissolved is added to the primary solution to form a secondary solution. A secondary solution generation step, and an emulsion generation step of generating an emulsion by irradiating the secondary solution with ultrasonic waves. The nanosphere contains a hydrophobic substance.
　ここでは、疎水性ポリマーおよび疎水性物質に両親媒性ポリマーを添加した１次溶液に親水性ポリマーをさらに添加した２次溶液に、超音波を照射する構成により、凝集を抑制しつつ、疎水性物質を内包する高分子微粒子をナノメートルオーダーで製造することが可能となる。 Here, a hydrophobic solution and a secondary solution obtained by adding a hydrophilic polymer to a primary solution obtained by adding an amphiphilic polymer to a hydrophobic substance are irradiated with ultrasonic waves, thereby suppressing aggregation and hydrophobicity. It becomes possible to produce polymer fine particles enclosing a substance on the nanometer order.
　したがって、両親媒性ポリマー、疎水性ポリマーおよび親水性ポリマーに生分解性ポリマーを用いれば、脂質や、生理活性を有する天然抽出物等の疎水性の高分子の薬物を内包する薬物徐放型ＤＤＳ製剤等の医薬品や疎水性の有効物質を内包する徐放型の化粧料を製造することができる。また、疎水性物質を内包する高分子微粒子は、ナノメートルオーダーであるため、静脈または動脈注射用の医薬品、経皮吸収用の医薬品や化粧料等の皮膚外用組成物に利用することができ、薬物の全身投与、患部へのターゲッティングおよび皮膚への浸透が可能となる。 Therefore, if a biodegradable polymer is used for the amphiphilic polymer, the hydrophobic polymer and the hydrophilic polymer, the drug sustained-release DDS encapsulating a lipid or a polymer of a hydrophobic polymer such as a natural extract having physiological activity Sustained-release cosmetics containing pharmaceuticals such as pharmaceutical preparations and hydrophobic active substances can be produced. In addition, since the polymer fine particles encapsulating the hydrophobic substance are on the order of nanometers, they can be used in pharmaceutical compositions for intravenous or arterial injection, pharmaceutical compositions for percutaneous absorption, cosmetic compositions such as cosmetics, Systemic administration of drugs, targeting to affected areas and penetration into the skin are possible.
　さらに他のＯ／Ｗエマルション法を用いた、ナノスフェアの製造方法は、疎水性物質を溶解した水非混和性有機溶媒と、両親媒性ポリマーを溶解した水非混和性有機溶媒とを混合し、１次溶液を生成する１次溶液生成ステップと、１次溶液に、親水性ポリマーを溶解した水系溶媒を添加し、２次溶液を生成する２次溶液生成ステップと、２次溶液に超音波を照射してエマルションを生成するエマルション生成ステップと、を含む。当該ナノスフェアは、疎水性物質を内包する。 Furthermore, the method for producing nanospheres using another O / W emulsion method comprises mixing a water-immiscible organic solvent in which a hydrophobic substance is dissolved and a water-immiscible organic solvent in which an amphiphilic polymer is dissolved, A primary solution generating step for generating a primary solution, an aqueous solvent in which a hydrophilic polymer is dissolved is added to the primary solution, a secondary solution generating step for generating a secondary solution, and an ultrasonic wave is applied to the secondary solution. An emulsion generating step of generating an emulsion upon irradiation. The nanosphere contains a hydrophobic substance.
　ここでは、両親媒性ポリマーと疎水性物質を含む１次溶液に親水性ポリマーをさらに添加した２次溶液に、超音波を照射する構成により、凝集を抑制しつつ、疎水性物質を内包する高分子微粒子をナノメートルオーダーで製造することが可能となる。 Here, a secondary solution in which a hydrophilic polymer is further added to a primary solution containing an amphiphilic polymer and a hydrophobic substance is irradiated with ultrasonic waves, so that aggregation is suppressed and a hydrophobic substance is included. It becomes possible to produce molecular fine particles on the nanometer order.
　したがって、両親媒性ポリマーおよび親水性ポリマーに生分解性ポリマーを用いれば、脂質や、生理活性を有する天然抽出物等の疎水性の高分子の薬物を内包する医薬品や疎水性の有効物質を内包する徐放型の化粧料を製造することができる。また、疎水性物質を内包する高分子微粒子は、ナノメートルオーダーであるため、静脈または動脈注射用の医薬品、経皮吸収用の医薬品や化粧料等の皮膚外用組成物に利用することができ、薬物の全身投与、患部へのターゲッティングおよび皮膚への浸透が可能となる。 Therefore, if a biodegradable polymer is used for the amphiphilic polymer and the hydrophilic polymer, a drug containing a hydrophobic polymer such as a lipid or a physiologically active natural drug or a hydrophobic active substance is included. A sustained-release cosmetic can be produced. In addition, since the polymer fine particles encapsulating the hydrophobic substance are on the order of nanometers, they can be used in pharmaceutical compositions for intravenous or arterial injection, pharmaceutical compositions for percutaneous absorption, cosmetic compositions such as cosmetics, Systemic administration of drugs, targeting to affected areas and penetration into the skin are possible.
　さらに他のＷ／Ｏ／Ｗエマルション法を用いた、ナノスフェアの製造方法は、親水性物質を溶解した水系溶媒と、疎水性物質を溶解した水非混和性有機溶媒と、両親媒性ポリマーを溶解した水非混和性有機溶媒と、疎水性ポリマーを溶解した水非混和性有機溶媒とを混合し、１次溶液を生成する１次溶液生成ステップと、１次溶液に超音波を照射して１次エマルションを生成する１次エマルション生成ステップと、１次エマルションに、親水性ポリマーを溶解した水系溶媒を添加し、２次溶液を生成する２次溶液生成ステップと、２次溶液に超音波を照射して２次エマルションを生成する２次エマルション生成ステップと、を含む。当該ナノスフェアは、親水性物質および疎水性物質を内包する。 Furthermore, the nanosphere manufacturing method using another W / O / W emulsion method involves dissolving an aqueous solvent in which a hydrophilic substance is dissolved, a water-immiscible organic solvent in which a hydrophobic substance is dissolved, and an amphiphilic polymer. The water-immiscible organic solvent thus prepared and the water-immiscible organic solvent in which the hydrophobic polymer is dissolved are mixed to form a primary solution generating step, and the primary solution is irradiated with ultrasonic waves. A primary emulsion generating step for generating a secondary emulsion, an aqueous solvent in which a hydrophilic polymer is dissolved is added to the primary emulsion, a secondary solution generating step for generating a secondary solution, and ultrasonic irradiation to the secondary solution And a secondary emulsion generating step of generating a secondary emulsion. The nanosphere includes a hydrophilic substance and a hydrophobic substance.
　疎水性ポリマー、親水性物質および疎水性物質に両親媒性ポリマーを添加した１次溶液に超音波を照射して生成した１次エマルションに親水性ポリマーを添加した２次溶液に、超音波を照射する構成により、凝集を抑制しつつ、親水性物質および疎水性物質を内包する高分子微粒子をナノメートルオーダーで製造することが可能となる。 Irradiate the secondary solution in which the hydrophilic polymer is added to the primary emulsion produced by irradiating the primary solution in which the hydrophobic polymer, the hydrophilic substance and the amphiphilic polymer are added to the hydrophobic substance. With this configuration, it is possible to produce polymer fine particles containing a hydrophilic substance and a hydrophobic substance on the nanometer order while suppressing aggregation.
　したがって、両親媒性ポリマー、疎水性ポリマーおよび親水性ポリマーに生分解性ポリマーを用いれば、タンパク質や核酸、生理活性を有する天然抽出物等の水溶性（親水性）高分子の薬物、および、脂質や生理活性を有する天然抽出物等の疎水性の高分子を内包する薬物徐放型ＤＤＳ製剤等の医薬品や、親水性および疎水性の有効物質を内包する徐放型の化粧料を製造することができる。また、親水性物質および疎水性物質を内包する高分子微粒子は、ナノメートルオーダーであるため、静脈または動脈注射用の医薬品、経皮吸収用の医薬品や化粧料等の皮膚外用組成物に利用することができ、薬物の全身投与、患部へのターゲッティングおよび皮膚への浸透が可能となる。 Therefore, if biodegradable polymers are used for amphiphilic polymers, hydrophobic polymers, and hydrophilic polymers, water-soluble (hydrophilic) high molecular weight drugs such as proteins, nucleic acids, and biologically active natural extracts, and lipids And pharmaceuticals such as drug sustained-release DDS preparations that encapsulate hydrophobic polymers such as natural extracts with physiological activity, and sustained-release cosmetics that encapsulate hydrophilic and hydrophobic active substances Can do. In addition, since the fine polymer particles encapsulating hydrophilic substances and hydrophobic substances are on the order of nanometers, they are used for compositions for external use in skin, such as pharmaceuticals for intravenous or arterial injection, pharmaceuticals for percutaneous absorption, and cosmetics. And allows systemic administration of the drug, targeting the affected area and penetration into the skin.
　さらに他のＷ／Ｏ／Ｗエマルション法を用いた、ナノスフェアの製造方法は、親水性物質を溶解した水系溶媒と、疎水性物質を溶解した水非混和性有機溶媒と、両親媒性ポリマーを溶解した水非混和性有機溶媒とを混合し、１次溶液を生成する１次溶液生成ステップと、１次溶液に超音波を照射して１次エマルションを生成する１次エマルション生成ステップと、１次エマルションに、親水性ポリマーを溶解した水系溶媒を添加し、２次溶液を生成する２次溶液生成ステップと、２次溶液に超音波を照射して２次エマルションを生成する２次エマルション生成ステップと、を含む。当該ナノスフェアは、親水性物質および疎水性物質を内包する。 Furthermore, the nanosphere manufacturing method using another W / O / W emulsion method involves dissolving an aqueous solvent in which a hydrophilic substance is dissolved, a water-immiscible organic solvent in which a hydrophobic substance is dissolved, and an amphiphilic polymer. A primary solution generating step of mixing the water-immiscible organic solvent thus formed to generate a primary solution, a primary emulsion generating step of generating a primary emulsion by irradiating the primary solution with ultrasonic waves, and a primary A secondary solution generation step of adding a water-based solvent in which a hydrophilic polymer is dissolved to the emulsion to generate a secondary solution; and a secondary emulsion generation step of generating a secondary emulsion by irradiating the secondary solution with ultrasonic waves; ,including. The nanosphere includes a hydrophilic substance and a hydrophobic substance.
　ここでは、両親媒性ポリマーと親水性物質および疎水性物質を含む１次溶液に超音波を照射して生成した１次エマルションに親水性ポリマーを添加した２次溶液に、超音波を照射する構成により、凝集を抑制しつつ、親水性物質および疎水性物質を内包する高分子微粒子をナノメートルオーダーで製造することが可能となる。 Here, a configuration in which an ultrasonic wave is irradiated to a secondary solution in which a hydrophilic polymer is added to a primary emulsion formed by irradiating an ultrasonic wave to a primary solution containing an amphiphilic polymer, a hydrophilic substance, and a hydrophobic substance. As a result, it is possible to produce polymer fine particles enclosing a hydrophilic substance and a hydrophobic substance on the nanometer order while suppressing aggregation.
　したがって、両親媒性ポリマーおよび親水性ポリマーに生分解性ポリマーを用いれば、タンパク質や核酸、生理活性を有する天然抽出物等の水溶性（親水性）高分子の薬物、および、脂質や生理活性を有する天然抽出物等の疎水性の高分子を内包する医薬品や、親水性および疎水性の有効物質を内包する徐放型の化粧料を製造することができる。また、親水性物質および疎水性物質を内包する高分子微粒子は、ナノメートルオーダーであるため、静脈または動脈注射用の医薬品、経皮吸収用の医薬品や化粧料等の皮膚外用組成物に利用することができ、薬物の全身投与、患部へのターゲッティングおよび皮膚への浸透が可能となる。 Therefore, if biodegradable polymers are used for amphiphilic polymers and hydrophilic polymers, water-soluble (hydrophilic) high molecular weight drugs such as proteins, nucleic acids, and biologically active natural extracts, and lipids and physiological activities can be obtained. It is possible to produce pharmaceuticals that encapsulate hydrophobic polymers such as natural extracts, and sustained-release cosmetics that encapsulate hydrophilic and hydrophobic active substances. In addition, since the fine polymer particles encapsulating hydrophilic substances and hydrophobic substances are on the order of nanometers, they are used for compositions for external use in skin, such as pharmaceuticals for intravenous or arterial injection, pharmaceuticals for percutaneous absorption, and cosmetics. And allows systemic administration of the drug, targeting the affected area and penetration into the skin.
　両親媒性ポリマーは、親水性セグメントと疎水性セグメントとで構成される共重合体であり、親水性セグメントは、電荷を有するアミノ酸を３個以上有するポリペプチド、数平均分子量５００～１０００００であるポリエチレングリコール、および、多糖、の群から選択される１または複数を含み、疎水性セグメントは、生分解性ポリエステルを含んでもよい。 The amphiphilic polymer is a copolymer composed of a hydrophilic segment and a hydrophobic segment, and the hydrophilic segment is a polypeptide having three or more charged amino acids, polyethylene having a number average molecular weight of 500 to 100,000. The hydrophobic segment may include a biodegradable polyester including one or more selected from the group of glycols and polysaccharides.
　両親媒性ポリマーとして、電荷を有するアミノ酸を３個以上有するポリペプチド、数平均分子量５００～１０００００であるポリエチレングリコール、および多糖、の群から選択される１または複数と、生分解性ポリエステルとで構成される共重合体を用いることにより、親水性物質もしくは疎水性物質のいずれか一方または両方を内包する生分解性のナノスフェアを製造することができる。 As an amphiphilic polymer, one or more selected from the group consisting of a polypeptide having three or more charged amino acids, a polyethylene glycol having a number average molecular weight of 500 to 100,000, and a polysaccharide, and a biodegradable polyester By using such a copolymer, it is possible to produce biodegradable nanospheres that include either or both of a hydrophilic substance and a hydrophobic substance.
　ポリペプチドを構成するアミノ酸は、正電荷を有するアミノ酸であるリジン、アルギニンおよびヒスチジンの群および負電荷を有するアミノ酸であるアスパラギン酸およびグルタミン酸の群から選択される１または複数のアミノ酸であってもよい。 The amino acid constituting the polypeptide may be one or more amino acids selected from the group of lysine, arginine and histidine which are positively charged amino acids, and the group of aspartic acid and glutamic acid which are negatively charged amino acids. .
　両親媒性ポリマーを構成するポリペプチドのアミノ酸を、リジン、アルギニン、ヒスチジン、アスパラギン酸、グルタミン酸の群から選択される１または複数のアミノ酸といったタンパク質を構成するアミノ酸とする構成により、医薬品や化粧料を製造する際に、生体適合性を向上させることができる。また、両親媒性ポリマーを構成するポリペプチドのアミノ酸を、タンパク質を構成するアミノ酸とすることにより、親水性物質もしくは疎水性物質のいずれか一方または両方を内包するナノスフェアを血中に導入した場合に、マクロファージなどの貪食細胞に異物として捕捉される可能性を低減させることができ、血中の循環時間を延長させることが可能となる。 By using amino acids of a polypeptide constituting an amphiphilic polymer as amino acids constituting a protein such as one or a plurality of amino acids selected from the group of lysine, arginine, histidine, aspartic acid, and glutamic acid. When manufactured, biocompatibility can be improved. In addition, when an amino acid of a polypeptide that constitutes an amphiphilic polymer is an amino acid that constitutes a protein, nanospheres that enclose either or both of a hydrophilic substance and a hydrophobic substance are introduced into the blood. The possibility of being trapped as foreign matter by phagocytic cells such as macrophages can be reduced, and the circulation time in blood can be prolonged.
　また、両親媒性ポリマーにポリエチレングリコールを含ませる構成により、親水性物質もしくは疎水性物質のいずれか一方または両方を内包するナノスフェアを血中に導入した場合に、マクロファージなどの貪食細胞に異物として捕捉される可能性を低減させることができ、血中の循環時間を延長させることが可能となる。 In addition, polyethylene glycol is included in the amphiphilic polymer, so that when nanospheres that contain either or both of hydrophilic and hydrophobic substances are introduced into the blood, they are trapped as foreign matter in phagocytic cells such as macrophages. It is possible to reduce the possibility that the blood is circulated and prolong the circulation time in the blood.
　両親媒性ポリマーを構成する多糖は、ヒアルロン酸、アミロース、プルラン、コンドロイチン、コンドロイチン硫酸、デキストラン、デキストラン硫酸、デルマタン硫酸、ケラタン硫酸、ヘパラン硫酸、キチン、キトサン、および、βグルカン、の群から選択される１または複数であってもよい。また、両親媒性ポリマーにおける多糖の含有量が１～５０質量％であってもよい。 The polysaccharide constituting the amphiphilic polymer is selected from the group of hyaluronic acid, amylose, pullulan, chondroitin, chondroitin sulfate, dextran, dextran sulfate, dermatan sulfate, keratan sulfate, heparan sulfate, chitin, chitosan, and β-glucan. It may be one or more. The content of polysaccharide in the amphiphilic polymer may be 1 to 50% by mass.
　両親媒性ポリマーとして、多糖と、生分解性ポリエステルとで構成される共重合体を用いることにより、親水性物質もしくは疎水性物質のいずれか一方または両方を内包する生分解性のナノスフェアを製造することができる。 By using a copolymer composed of a polysaccharide and a biodegradable polyester as an amphiphilic polymer, a biodegradable nanosphere containing either or both of a hydrophilic substance and a hydrophobic substance is produced. be able to.
　生分解性ポリエステルの構成単位は、乳酸、グリコール酸、および、アミノ酸、の群から選択される１または複数であってもよい。 The structural unit of the biodegradable polyester may be one or more selected from the group of lactic acid, glycolic acid, and amino acid.
　生分解性ポリエステルを生体由来材料である乳酸やアミノ酸、またはグリコール酸で構成することにより、医薬品や化粧料を製造する際に、生体適合性を向上させることができる。 By configuring the biodegradable polyester with lactic acid, amino acid, or glycolic acid, which is a bio-derived material, biocompatibility can be improved when producing pharmaceuticals and cosmetics.
　上記ナノスフェアの製造方法を用いて製造されるナノスフェアが提供される。ここで、ナノスフェアは、親水性物質もしくは疎水性物質のいずれか一方または両方として、ステビア発酵エキスまたはスフィンゴミエリンを内包してもよい。 Provided is a nanosphere manufactured using the above-described method of manufacturing a nanosphere. Here, the nanosphere may include stevia fermented extract or sphingomyelin as one or both of a hydrophilic substance and a hydrophobic substance.
　上記ナノスフェアの製造方法を用いて製造されたナノスフェアを含有する皮膚外用組成物が提供される。ここで、皮膚外用組成物は、ヒトの皮膚に接触させることにより、皮膚外用組成物に含まれる物質を経皮吸収させるものである。 Provided is a composition for external use on the skin containing nanospheres produced using the method for producing nanospheres. Here, the composition for external use of skin is a substance that percutaneously absorbs a substance contained in the composition for external use of skin by bringing it into contact with human skin.
　皮膚外用組成物として、上記ナノスフェアの製造方法を用いて製造された親水性物質もしくは疎水性物質のいずれか一方または両方を内包するナノスフェアを含有する構成により、塗り薬等の皮膚外用組成物を皮膚表面の微細な凹凸にナノスフェアを略均一に塗布することができる。また、上述したようにナノスフェアを構成するポリマーに生分解性ポリマーを用いる構成により、ナノスフェアに内包させた親水性物質もしくは疎水性物質のいずれか一方または両方を皮膚表面で好適に徐放させることができ、従来と比較して、皮膚外用組成物の効果を維持しつつ、塗布する回数を減少させることが可能となる。 As a composition for external use on the skin, the composition for external application to the skin such as a coating agent is applied to the skin surface by a composition containing nanospheres containing either or both of a hydrophilic substance and a hydrophobic substance produced by using the above-mentioned nanosphere production method. The nanospheres can be applied almost uniformly on the fine irregularities of the film. In addition, as described above, by using a biodegradable polymer as a polymer constituting the nanosphere, either one or both of the hydrophilic substance and the hydrophobic substance encapsulated in the nanosphere can be suitably and slowly released on the skin surface. In comparison with the prior art, it is possible to reduce the number of times of application while maintaining the effect of the external composition for skin.
　上記皮膚外用組成物に含有されるナノスフェアは、親水性物質もしくは疎水性物質のいずれか一方または両方として、ステビア発酵エキスまたはスフィンゴミエリンを内包してもよい。ステビア発酵エキスおよびスフィンゴミエリンは、保湿効果が高く、荒れ肌を改善したり、かゆみを抑止したり、炎症を抑えたり、抗ヒスタミン作用を有したりする。したがって、ステビア発酵エキスまたはスフィンゴミエリンを内包させたナノスフェアを含有させた皮膚外用組成物とする構成により、皮膚表面の微細な凹凸にステビア発酵エキスまたはスフィンゴミエリンを略均一に行き渡らせることができる。 The nanospheres contained in the composition for external skin may contain stevia fermented extract or sphingomyelin as one or both of a hydrophilic substance and a hydrophobic substance. Stevia fermented extract and sphingomyelin have a high moisturizing effect, improve rough skin, suppress itching, suppress inflammation, and have an antihistaminic action. Therefore, the composition for external use of skin containing nanospheres containing stevia fermented extract or sphingomyelin can distribute stevia fermented extract or sphingomyelin substantially evenly on fine irregularities on the skin surface.
　上記ナノスフェアの製造方法を用いて製造されたナノスフェアを含有する化粧料が提供される。ここで、化粧料は、美化、清潔、保護あるいは、防臭を目的として人体を処置するための組成物である。 A cosmetic containing nanospheres produced using the above-described method for producing nanospheres is provided. Here, the cosmetic is a composition for treating the human body for the purpose of beautification, cleanliness, protection or deodorization.
　化粧料として、上記ナノスフェアの製造方法を用いて製造された親水性物質もしくは疎水性物質のいずれか一方または両方を内包するナノスフェアを含有する構成により、化粧料を皮膚表面の微細な凹凸にナノスフェアを略均一に塗布することができる。また、上述したようにナノスフェアを構成するポリマーに生分解性ポリマーを用いる構成により、ナノスフェアに内包させた親水性物質もしくは疎水性物質のいずれか一方または両方を皮膚表面で好適に徐放させることができ、従来と比較して化粧料の塗布回数を減らしても同程度の効果を得ることが可能となる。 As a cosmetic, the composition containing nanospheres containing either or both of a hydrophilic substance and a hydrophobic substance produced by using the above-mentioned nanosphere production method, the nanospheres are formed into fine irregularities on the skin surface. It can be applied substantially uniformly. In addition, as described above, by using a biodegradable polymer as a polymer constituting the nanosphere, either one or both of the hydrophilic substance and the hydrophobic substance encapsulated in the nanosphere can be suitably and slowly released on the skin surface. It is possible to obtain the same effect even if the number of times of applying the cosmetic is reduced as compared with the conventional case.
　上記化粧料に含有されるナノスフェアは、親水性物質もしくは疎水性物質のいずれか一方または両方として、ステビア発酵エキスまたはスフィンゴミエリンを内包してもよい。ステビア発酵エキスおよびスフィンゴミエリンは、保湿効果が高く、荒れ肌を改善したり、かゆみを抑止したり、炎症を抑えたり、抗ヒスタミン作用を有したりする。したがって、ステビア発酵エキスまたはスフィンゴミエリンを内包させたナノスフェアを含有させた化粧料とする構成により、皮膚表面の微細な凹凸にステビア発酵エキスまたはスフィンゴミエリンを略均一に行き渡らせることができる。 The nanospheres contained in the cosmetic may contain stevia fermented extract or sphingomyelin as one or both of a hydrophilic substance and a hydrophobic substance. Stevia fermented extract and sphingomyelin have a high moisturizing effect, improve rough skin, suppress itching, suppress inflammation, and have an antihistaminic action. Therefore, with the constitution of the cosmetic containing nanospheres encapsulating stevia fermented extract or sphingomyelin, stevia fermented extract or sphingomyelin can be distributed almost uniformly on the fine irregularities of the skin surface.
　上述したナノスフェアの製造方法の技術的思想に基づく構成要素やその説明は、当該ナノスフェア、これを含有する皮膚外用組成物および化粧料にも適用可能である。 The constituent elements based on the technical idea of the method for producing nanospheres described above and the explanation thereof are also applicable to the nanospheres, compositions for external use on skin and cosmetics containing the nanospheres.
　本発明によれば、水溶性高分子等の親水性物質もしくは疎水性物質のいずれか一方または両方を内包し、かつ粒径がナノオーダーである高分子微粒子を製造することが可能となる。 According to the present invention, it is possible to produce polymer fine particles having a nano-order particle size including either or both of a hydrophilic substance and a hydrophobic substance such as a water-soluble polymer.
実施形態にかかるナノスフェアの製造方法を説明するための説明図である。It is explanatory drawing for demonstrating the manufacturing method of the nanosphere concerning embodiment. 実施例１にかかるＰＬｙｓ＋－ｂ－ＰＬＬＡの合成方法を説明するための説明図である。6 is an explanatory diagram for explaining a synthesis method of PLys + -b-PLLA according to Example 1. FIG. 実施例１にかかるＰＬｙｓ＋－ｂ－ＰＬＬＡのミセル状態における分解性を説明するための説明図である。6 is an explanatory diagram for explaining the decomposability in the micelle state of PLys + -b-PLLA according to Example 1. FIG. 実施例１を説明するための説明図である。FIG. 3 is an explanatory diagram for explaining Example 1; ＢＳＡを内包する生分解性ナノスフェアを、走査型電子顕微鏡で観察した結果を説明するための説明図である。It is explanatory drawing for demonstrating the result of having observed the biodegradable nanosphere which includes BSA with the scanning electron microscope. 実施例における内包率、回収率、収率および内包効率を算出する式を説明するための説明図である。It is explanatory drawing for demonstrating the type | formula which calculates the inclusion rate, recovery rate, yield, and inclusion efficiency in an Example. 実施例２にかかるＰＥＧ３Ｋ－ｂ－ＰＬＬＡの合成方法を説明するための説明図である。6 is an explanatory diagram for explaining a method of synthesizing PEG3K-b-PLLA according to Example 2. FIG. 実施例２を説明するための説明図である。FIG. 6 is an explanatory diagram for explaining Example 2; スフィンゴミエリンを内包する生分解性ナノスフェアを、走査型電子顕微鏡で観察した結果を説明するための説明図である。It is explanatory drawing for demonstrating the result of having observed the biodegradable nanosphere which includes sphingomyelin with the scanning electron microscope. 実施例３にかかるＤｅｘ－ｇ－ＰＬＬＡの合成方法を説明するための説明図である。FIG. 6 is an explanatory diagram for explaining a Dex-g-PLLA synthesis method according to Example 3; 実施例３にかかるＤｅｘ－ｇ－ＰＬＬＡの分解性を説明するための説明図である。6 is an explanatory diagram for explaining the degradability of Dex-g-PLLA according to Example 3. FIG. 分解性実験に用いたＤｅｘ－ｇ－ＰＬＬＡを説明するための説明図である。It is explanatory drawing for demonstrating Dex-g-PLLA used for degradability experiment. 、実施例３を説明するための説明図である。FIG. 10 is an explanatory diagram for explaining Example 3; スフィンゴミエリンを内包する生分解性ナノスフェアを、走査型電子顕微鏡で観察した結果を説明するための説明図である。It is explanatory drawing for demonstrating the result of having observed the biodegradable nanosphere which includes sphingomyelin with the scanning electron microscope. 実施例４を説明するための説明図である。FIG. 10 is an explanatory diagram for explaining Example 4; ＢＳＡを内包する生分解性ナノスフェアを、走査型電子顕微鏡で観察した結果を説明するための説明図である。It is explanatory drawing for demonstrating the result of having observed the biodegradable nanosphere which includes BSA with the scanning electron microscope.
Ｓ１００  …１次溶液生成ステップ
Ｓ１０２  …１次エマルション生成ステップ
Ｓ１０４  …２次溶液生成ステップ
Ｓ１０６  …２次エマルション生成ステップ
Ｓ１０８  …有機溶媒除去ステップ S100 ... primary solution generation step S102 ... primary emulsion generation step S104 ... secondary solution generation step S106 ... secondary emulsion generation step S108 ... organic solvent removal step
好適な実施形態の説明DESCRIPTION OF PREFERRED EMBODIMENTS
　以下に添付図面を参照しながら、本発明の好適な実施形態について詳細に説明する。かかる実施形態に示す寸法、材料、その他具体的な数値などは、発明の理解を容易とするための例示にすぎず、特に断る場合を除き、本発明を限定するものではない。なお、本明細書および図面において、実質的に同一の機能、構成を有する要素については、同一の符号を付することにより重複説明を省略し、また本発明に直接関係のない要素は図示を省略する。 Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the accompanying drawings. The dimensions, materials, and other specific numerical values shown in the embodiment are merely examples for facilitating understanding of the invention, and do not limit the present invention unless otherwise specified. In the present specification and drawings, elements having substantially the same function and configuration are denoted by the same reference numerals, and redundant description is omitted, and elements not directly related to the present invention are not illustrated. To do.
（実施形態）
　図１は、実施形態にかかるナノスフェアの製造方法を説明するための説明図である。 (Embodiment)
Drawing 1 is an explanatory view for explaining the manufacturing method of the nanosphere concerning an embodiment.
　まず、内包する物質を溶解した溶媒と、両親媒性ポリマーを溶解した水非混和性有機溶媒と、疎水性ポリマーを溶解した水非混和性有機溶媒とを混合し、１次溶液を生成する（Ｓ１００：１次溶液生成ステップ）。 First, a solvent in which an encapsulating substance is dissolved, a water-immiscible organic solvent in which an amphiphilic polymer is dissolved, and a water-immiscible organic solvent in which a hydrophobic polymer is dissolved are mixed to form a primary solution ( S100: primary solution generation step).
　１次溶液生成ステップＳ１００において、内包する物質を親水性物質とする場合、親水性物質を溶解した水系溶媒を、両親媒性ポリマーを溶解した水非混和性有機溶媒と疎水性ポリマーを溶解した水非混和性有機溶媒とに混合し、１次溶液を生成する。 In the primary solution generation step S100, when the substance to be included is a hydrophilic substance, an aqueous solvent in which the hydrophilic substance is dissolved is used as a water-immiscible organic solvent in which the amphiphilic polymer is dissolved and water in which the hydrophobic polymer is dissolved. Mix with immiscible organic solvent to form primary solution.
　また、１次溶液生成ステップＳ１００において、内包する物質を親水性物質および疎水性物質とする場合、親水性物質を溶解した水系溶媒および疎水性物質を溶解した水非混和性有機溶媒を、両親媒性ポリマーを溶解した水非混和性有機溶媒と疎水性ポリマーを溶解した水非混和性有機溶媒とに混合し、１次溶液を生成する。 In the primary solution generation step S100, when the substance to be included is a hydrophilic substance and a hydrophobic substance, an aqueous solvent in which the hydrophilic substance is dissolved and a water-immiscible organic solvent in which the hydrophobic substance is dissolved are used as an amphiphile. A water-immiscible organic solvent in which the soluble polymer is dissolved and a water-immiscible organic solvent in which the hydrophobic polymer is dissolved are mixed to form a primary solution.
　生成した１次溶液に超音波を照射し、逆相エマルション（Ｗ／Ｏエマルション）である１次エマルションを生成する（Ｓ１０２：１次エマルション生成ステップ）。そして、１次エマルションに、親水性ポリマーを溶解した水系溶媒を添加し、２次溶液を生成する（Ｓ１０４：２次溶液生成ステップ）。生成した２次溶液に超音波を照射して２次エマルションを生成し（Ｓ１０６：２次エマルション生成ステップ）、２次エマルションから水非混和性有機溶媒を除去する（Ｓ１０８：有機溶媒除去ステップ）。 The generated primary solution is irradiated with ultrasonic waves to generate a primary emulsion that is a reverse phase emulsion (W / O emulsion) (S102: primary emulsion generation step). Then, an aqueous solvent in which a hydrophilic polymer is dissolved is added to the primary emulsion to generate a secondary solution (S104: secondary solution generation step). The produced secondary solution is irradiated with ultrasonic waves to produce a secondary emulsion (S106: secondary emulsion production step), and the water-immiscible organic solvent is removed from the secondary emulsion (S108: organic solvent removal step).
　また、内包する物質が疎水性物質である場合、１次溶液生成ステップＳ１００において、疎水性物質を溶解した水非混和性有機溶媒を、両親媒性ポリマーを溶解した水非混和性有機溶媒と疎水性ポリマーを溶解した水非混和性有機溶媒とに混合し、１次溶液を生成する。そして、１次エマルション生成ステップＳ１０２を省略し、１次溶液に、親水性ポリマーを溶解した水系溶媒を添加し、２次溶液を生成する（Ｓ１０４：２次溶液生成ステップ）。生成した２次溶液に超音波を照射してエマルション（Ｏ／Ｗエマルション）を生成し（Ｓ１０６：エマルション生成ステップ（２次エマルション生成ステップ））、エマルションから水非混和性有機溶媒を除去する（Ｓ１０８：有機溶媒除去ステップ）。 Further, when the encapsulating substance is a hydrophobic substance, in the primary solution generation step S100, the water-immiscible organic solvent in which the hydrophobic substance is dissolved is changed to the water-immiscible organic solvent in which the amphiphilic polymer is dissolved. The water-immiscible organic solvent is mixed with the soluble polymer to form a primary solution. Then, the primary emulsion generation step S102 is omitted, and an aqueous solvent in which the hydrophilic polymer is dissolved is added to the primary solution to generate a secondary solution (S104: secondary solution generation step). The generated secondary solution is irradiated with ultrasonic waves to generate an emulsion (O / W emulsion) (S106: emulsion generation step (secondary emulsion generation step)), and the water-immiscible organic solvent is removed from the emulsion (S108). : Organic solvent removal step).
　以下に、各ステップの構成を詳細に説明する。 The configuration of each step will be described in detail below.
（１次溶液生成ステップＳ１００）
　内包する物質を溶解した溶媒と、両親媒性ポリマーを溶解した水非混和性有機溶媒と、疎水性ポリマーを溶解した水非混和性有機溶媒とを混合し、１次溶液を生成する。 (Primary solution generation step S100)
A primary solution is produced by mixing a solvent in which the substance to be encapsulated is dissolved, a water-immiscible organic solvent in which the amphiphilic polymer is dissolved, and a water-immiscible organic solvent in which the hydrophobic polymer is dissolved.
[１次溶液生成ステップＳ１００における両親媒性ポリマー１]
　１次溶液生成ステップＳ１００における両親媒性ポリマーは、親水性セグメントと疎水性セグメントとで構成される共重合体であり、親水性セグメントは、電荷を有するアミノ酸を３個以上有するポリペプチドまたは数平均分子量５００～１０００００であるポリエチレングリコールを含み、疎水性セグメントは生分解性ポリエステルを含んで構成される。 [Amphiphilic polymer 1 in primary solution generation step S100]
The amphiphilic polymer in the primary solution generation step S100 is a copolymer composed of a hydrophilic segment and a hydrophobic segment, and the hydrophilic segment is a polypeptide having three or more charged amino acids or a number average. Polyethylene glycol having a molecular weight of 500 to 100,000 is included, and the hydrophobic segment includes a biodegradable polyester.
＜両親媒性ポリマーの重合形態＞
　ポリペプチドを含む両親媒性ポリマーまたはポリエチレングリコールを含む両親媒性ポリマーの重合形態は、好ましくは、電荷を有するアミノ酸を３個以上有するポリペプチドまたは数平均分子量５００～１０００００であるポリエチレングリコールと、生分解性ポリエステルとの、ランダム共重合体、交互共重合体、ブロック共重合体、グラフト共重合体であり、より好ましくは、ブロック共重合体またはグラフト共重合体であり、さらに好ましくは、ブロック共重合体である。 <Polymerization form of amphiphilic polymer>
The polymerized form of an amphiphilic polymer containing a polypeptide or an amphiphilic polymer containing polyethylene glycol is preferably a polypeptide having three or more charged amino acids or polyethylene glycol having a number average molecular weight of 500 to 100,000, Random copolymers, alternating copolymers, block copolymers, and graft copolymers with degradable polyesters, more preferably block copolymers or graft copolymers, and even more preferably block copolymers. It is a polymer.
＜電荷を有するアミノ酸を３個以上有するポリペプチド＞
　両親媒性ポリマーを構成するポリペプチドは、正電荷または負電荷を有していればよく、かかるポリペプチドを構成するアミノ酸は、好ましくは、リジン、アルギニン、ヒスチジン、アスパラギン酸、グルタミン酸の群から選択される１または複数のアミノ酸であればよく、より好ましくは、リジン、アルギニンまたはヒスチジンの群から選択される１または複数のアミノ酸であり、さらに好ましくはリジンである。 <Polypeptide having 3 or more charged amino acids>
The polypeptide constituting the amphiphilic polymer may be positively or negatively charged, and the amino acid constituting such a polypeptide is preferably selected from the group of lysine, arginine, histidine, aspartic acid, and glutamic acid. One or more amino acids may be used, more preferably one or more amino acids selected from the group of lysine, arginine or histidine, more preferably lysine.
　両親媒性ポリマーを構成するポリペプチドのアミノ酸を、タンパク質を構成するアミノ酸とする構成により、かかる両親媒性ポリマーを用いたナノスフェアを利用して薬物徐放型ＤＤＳ製剤等の医薬品や徐放型の皮膚外用組成物、徐放型の化粧料を製造する際に、生体適合性を向上させることができる。また、両親媒性ポリマーを構成するポリペプチドのアミノ酸を、タンパク質を構成するアミノ酸とすることにより、親水性物質もしくは疎水性物質のいずれか一方または両方を内包するナノスフェアを血中に導入した場合に、マクロファージなどの貪食細胞に異物として捕捉される可能性を低減させることができ、血中の循環時間を延長させることが可能となる。 By configuring the amino acid of the polypeptide constituting the amphiphilic polymer as the amino acid constituting the protein, a drug such as a drug sustained-release DDS formulation or a sustained-release type using nanospheres using the amphiphilic polymer. Biocompatibility can be improved when producing a composition for external use on skin and a sustained release cosmetic. In addition, when an amino acid of a polypeptide that constitutes an amphiphilic polymer is an amino acid that constitutes a protein, nanospheres that enclose either or both of a hydrophilic substance and a hydrophobic substance are introduced into the blood. The possibility of being trapped as foreign matter by phagocytic cells such as macrophages can be reduced, and the circulation time in blood can be prolonged.
＜ポリエチレングリコール＞
　両親媒性ポリマーを構成するポリエチレングリコールの数平均分子量は、特に限定されないが、好ましくは、数平均分子量５００～１０００００であり、より好ましくは、数平均分子量１０００～５００００であり、さらに好ましくは数平均分子量２０００～２００００である。 <Polyethylene glycol>
The number average molecular weight of the polyethylene glycol constituting the amphiphilic polymer is not particularly limited, but is preferably a number average molecular weight of 500 to 100,000, more preferably a number average molecular weight of 1,000 to 50,000, and still more preferably a number average. The molecular weight is 2000-20000.
　両親媒性ポリマーにポリエチレングリコールを含ませる構成により、親水性物質もしくは疎水性物質のいずれか一方または両方を内包するナノスフェアを血中に導入した場合に、マクロファージなどの貪食細胞に異物として捕捉される可能性を低減させることができ、血中の循環時間を延長させることが可能となる。 By including polyethylene glycol in the amphiphilic polymer, when nanospheres that contain either or both of hydrophilic substances and hydrophobic substances are introduced into the blood, they are trapped by phagocytic cells such as macrophages as foreign substances. The possibility can be reduced and the circulation time in the blood can be extended.
＜生分解性ポリエステルの構成単位＞
　生分解性ポリエステルの構成単位は、重合した場合に生分解性ポリエステルとなるものであり、上記ポリペプチドまたはポリエチレングリコールと共重合体を構成できるものであればよく、好ましくは、乳酸（Ｌ－乳酸、Ｄ－乳酸、ＤＬ－乳酸）、グリコール酸（ヒドロキシ酢酸）、アミノ酸、カプロラクトン（α－カプロラクトン、β－カプロラクトン、γ－カプロラクトン、δ－カプロラクトン、ε－カプロラクトン等）、コハク酸とエチレングリコールの混合物、コハク酸とブタンジオール（１，２－ブタンジオール、１，３－ブタンジオール、１，４－ブタンジオール、２，３－ブタンジオール等）の混合物、からなる群のうち、１または複数、またはこれらのオリゴマーであればよく、より好ましくは、乳酸、グリコール酸およびアミノ酸、の群から選択される１または複数であり、さらに好ましくはＬ－乳酸である。 <Constitutional unit of biodegradable polyester>
The structural unit of the biodegradable polyester is a polymer that becomes a biodegradable polyester when polymerized and can form a copolymer with the above polypeptide or polyethylene glycol. Preferably, lactic acid (L-lactic acid) is used. , D-lactic acid, DL-lactic acid), glycolic acid (hydroxyacetic acid), amino acids, caprolactone (α-caprolactone, β-caprolactone, γ-caprolactone, δ-caprolactone, ε-caprolactone, etc.), a mixture of succinic acid and ethylene glycol One or more of the group consisting of succinic acid and butanediol (1,2-butanediol, 1,3-butanediol, 1,4-butanediol, 2,3-butanediol, etc.), or These oligomers may be used, and more preferably lactic acid, glycolic acid and amino acid. One or more selected from the group of noic acid, more preferably L-lactic acid.
　生分解性ポリエステルは、例えば、ポリ乳酸（ＰＬＡ：Poly Lactic Acid）、ポリグリコール酸（ＰＧＡ：Poly Glycolic Acid）、ポリ乳酸－グリコール酸共重合体、ポリデプシペプチド（アミノ酸とヒドロキシ酸の共重合体）を用いることができる。 Biodegradable polyesters include, for example, polylactic acid (PLA: Poly Lactic Acid), polyglycolic acid (PGA), polylactic acid-glycolic acid copolymer, polydepsipeptide (copolymer of amino acid and hydroxy acid) Can be used.
　生分解性ポリエステルを生体由来材料である乳酸やアミノ酸、またはグリコール酸で構成することにより、医薬品や、皮膚外用組成物、化粧料を製造する際に、生体適合性を向上させることができる。 By configuring the biodegradable polyester with lactic acid, amino acid, or glycolic acid, which is a bio-derived material, biocompatibility can be improved when producing pharmaceuticals, external compositions for skin, and cosmetics.
[１次溶液生成ステップＳ１００における両親媒性ポリマー２]
　また、１次溶液生成ステップＳ１００における両親媒性ポリマーは、親水性セグメントと疎水性セグメントとで構成される共重合体であり、親水性セグメントは、多糖を含み、疎水性セグメントは生分解性ポリエステルを含んで構成されてもよい。 [Amphiphilic polymer 2 in primary solution generation step S100]
The amphiphilic polymer in the primary solution generation step S100 is a copolymer composed of a hydrophilic segment and a hydrophobic segment, the hydrophilic segment contains a polysaccharide, and the hydrophobic segment is a biodegradable polyester. It may be comprised including.
＜両親媒性ポリマーの重合形態＞
　多糖を含む両親媒性ポリマーの重合形態は、多糖と、生分解性ポリエステルとの、ランダム共重合体、交互共重合体、ブロック共重合体、グラフト共重合体であればよく、好ましくは、グラフト共重合体またはブロック共重合体であり、より好ましくは、グラフト共重合体である。また、両親媒性ポリマーは、さらに好ましくは、多糖を主鎖とし、生分解性ポリエステルを側鎖としたグラフト共重合体である。 <Polymerization form of amphiphilic polymer>
The polymerization form of the amphiphilic polymer containing polysaccharide may be a random copolymer, alternating copolymer, block copolymer, or graft copolymer of polysaccharide and biodegradable polyester. It is a copolymer or a block copolymer, and more preferably a graft copolymer. The amphiphilic polymer is more preferably a graft copolymer having a polysaccharide as a main chain and a biodegradable polyester as a side chain.
＜多糖の含有量＞
　また、両親媒性ポリマーを構成する多糖の含有量は、特に限定されないが、１～５０質量％であればよく、好ましくは、１～３０質量％であり、さらに好ましくは、４～２５質量％である。 <Polysaccharide content>
The content of the polysaccharide constituting the amphiphilic polymer is not particularly limited, but may be 1 to 50% by mass, preferably 1 to 30% by mass, and more preferably 4 to 25% by mass. It is.
　両親媒性ポリマーを構成する多糖の含有量をかかる範囲の含有量とする構成により、親水性物質もしくは疎水性物質のいずれか一方または両方を内包するナノスフェアを生体内に留置した場合においても、分解に伴う炎症反応等を生じさせない分解速度であり、かつ、生体内で必要とされる期間の経過後には生体内から消失している分解速度に調節することができる。かかる生体内で必要とされる期間とは、好ましくは１ヶ月～１０ヶ月、さらに好ましくは、２ヶ月～１０ヶ月、最も好ましくは２ヶ月～８ヶ月である。 Even if nanospheres that contain either or both of hydrophilic substances and hydrophobic substances are placed in the living body, the degradation of the polysaccharides that make up the amphiphilic polymer is within this range. It can be adjusted to a degradation rate that does not cause an inflammatory reaction or the like associated with, and has disappeared from the living body after a period required in the living body. The period required in vivo is preferably 1 month to 10 months, more preferably 2 months to 10 months, and most preferably 2 months to 8 months.
＜多糖＞
　両親媒性ポリマーを構成する多糖は、生体内で安定であればよく、好ましくは、ヒアルロン酸、アミロース、プルラン、コンドロイチン、コンドロイチン硫酸、デキストラン、デキストラン硫酸、デルマタン硫酸、ケラタン硫酸、ヘパラン硫酸、キチン、キトサンおよびβグルカンの群から選択される１または複数であり、より好ましくは、デキストラン、プルランおよびヒアルロン酸の群から選択される１または複数であり、さらに好ましくは、デキストランである。 <Polysaccharide>
The polysaccharide constituting the amphiphilic polymer may be stable in vivo, preferably hyaluronic acid, amylose, pullulan, chondroitin, chondroitin sulfate, dextran, dextran sulfate, dermatan sulfate, keratan sulfate, heparan sulfate, chitin, One or more selected from the group of chitosan and β-glucan, more preferably one or more selected from the group of dextran, pullulan and hyaluronic acid, more preferably dextran.
　両親媒性ポリマーにおける多糖をデキストランとする構成により、親水性物質もしくは疎水性物質のいずれか一方または両方を内包するナノスフェアを安定して血管内に存在させることができる。 The structure in which the polysaccharide in the amphiphilic polymer is dextran allows nanospheres enclosing one or both of the hydrophilic substance and the hydrophobic substance to be stably present in the blood vessel.
　かかる多糖の重量平均分子量は、特に限定されないが、１０００～１０００００ｇ／ｍｏｌであればよく、好ましくは、５０００～５００００ｇ／ｍｏｌであり、さらに好ましくは、１００００～３００００ｇ／ｍｏｌである。 The weight average molecular weight of the polysaccharide is not particularly limited, but may be 1000 to 100,000 g / mol, preferably 5000 to 50000 g / mol, and more preferably 10,000 to 30000 g / mol.
　両親媒性ポリマーにおける多糖をかかる範囲の数平均分子量である多糖とする構成により、親水性物質もしくは疎水性物質のいずれか一方または両方を内包するナノスフェアが、高い成型性と、柔軟性とを維持しつつ細胞接着性を低下させることができる。 By making the polysaccharide in the amphiphilic polymer into a polysaccharide with a number average molecular weight within this range, nanospheres containing either or both of hydrophilic substances and hydrophobic substances maintain high moldability and flexibility. However, cell adhesion can be reduced.
＜生分解性ポリエステルの構成単位＞
　生分解性ポリエステルの構成単位は、重合した場合に、生分解性ポリエステルとなるものであり、上記多糖と共重合体を構成できるものであればよく、好ましくは、乳酸（Ｌ－乳酸、Ｄ－乳酸、ＤＬ－乳酸）、グリコール酸（ヒドロキシ酢酸）、アミノ酸、カプロラクトン（α－カプロラクトン、β－カプロラクトン、γ－カプロラクトン、δ－カプロラクトン、ε－カプロラクトン等）、コハク酸とエチレングリコールの混合物、コハク酸とブタンジオール（１，２－ブタンジオール、１，３－ブタンジオール、１，４－ブタンジオール、２，３－ブタンジオール等）の混合物、からなる群のうち、１または複数、またはこれらのオリゴマーであり、より好ましくは、乳酸、グリコール酸およびアミノ酸、の群から選択される１または複数であり、さらに好ましくはＬ－乳酸である。 <Constitutional unit of biodegradable polyester>
The structural unit of the biodegradable polyester is that which becomes a biodegradable polyester when polymerized and can form a copolymer with the above-mentioned polysaccharide, preferably lactic acid (L-lactic acid, D- Lactic acid, DL-lactic acid), glycolic acid (hydroxyacetic acid), amino acids, caprolactone (α-caprolactone, β-caprolactone, γ-caprolactone, δ-caprolactone, ε-caprolactone, etc.), a mixture of succinic acid and ethylene glycol, succinic acid Or a mixture of butanediol (1,2-butanediol, 1,3-butanediol, 1,4-butanediol, 2,3-butanediol, etc.), or one or more thereof, or an oligomer thereof And more preferably one or more selected from the group of lactic acid, glycolic acid and amino acids. More preferred is L-lactic acid.
　生分解性ポリエステルは、例えば、ポリ乳酸、ポリグリコール酸、ポリ乳酸－グリコール酸共重合体、ポリデプシペプチドを用いることができる。 As the biodegradable polyester, for example, polylactic acid, polyglycolic acid, polylactic acid-glycolic acid copolymer, and polydepsipeptide can be used.
　生分解性ポリエステルを生体由来材料である乳酸やアミノ酸、またはグリコール酸で構成することにより、医薬品や、皮膚外用組成物、化粧料を製造する際に、生体適合性を向上させることができる。 By configuring the biodegradable polyester with lactic acid, amino acid, or glycolic acid, which is a bio-derived material, biocompatibility can be improved when producing pharmaceuticals, external compositions for skin, and cosmetics.
　上述した多糖と生分解性ポリエステルとの共重合体である両親媒性ポリマーは、好ましくは、主鎖をデキストランとし、側鎖をＬ－乳酸としたグラフト共重合体である。以下、デキストランを主鎖としＬ－乳酸を側鎖としたグラフト共重合体である両親媒性ポリマーをＤｅｘ－ｇ－ＰＬＬＡ（ポリ乳酸グラフト化デキストラン）と称する。 The amphiphilic polymer that is a copolymer of the polysaccharide and the biodegradable polyester described above is preferably a graft copolymer in which the main chain is dextran and the side chain is L-lactic acid. Hereinafter, an amphiphilic polymer which is a graft copolymer having dextran as a main chain and L-lactic acid as a side chain is referred to as Dex-g-PLLA (polylactic acid grafted dextran).
　上記Ｄｅｘ－ｇ－ＰＬＬＡ１分子における乳酸のグラフト本数は、１～１００本であればよく、好ましくは、１～５０本であり、さらに好ましくは２～３０本である。Ｄｅｘ－ｇ－ＰＬＬＡ１分子における乳酸のグラフト本数を上記範囲の本数とする構成により、親水性物質もしくは疎水性物質のいずれか一方または両方を内包するナノスフェアの細胞接着性を低減させることが可能となる。 The number of lactic acid grafts in the above Dex-g-PLLA molecule may be 1 to 100, preferably 1 to 50, and more preferably 2 to 30. With the configuration in which the number of lactic acid grafts in one molecule of Dex-g-PLLA is within the above range, it becomes possible to reduce the cell adhesion of nanospheres enclosing one or both of hydrophilic substances and hydrophobic substances. .
　また、上記Ｄｅｘ－ｇ－ＰＬＬＡの数平均分子量は、特に限定されないが、１×１０４～１００×１０４／ｍｏｌであればよく、好ましくは、５×１０４～２０×１０４／ｍｏｌであり、さらに好ましくは、１０×１０４～１３×１０４／ｍｏｌである。 The number average molecular weight of the Dex-g-PLLA is not particularly limited, but may be 1 × 10 4 to 100 × 10 4 / mol, and preferably 5 × 10 4 to 20 × 10 4 / mol. More preferably, it is 10 × 10 4 to 13 × 10 4 / mol.
　Ｄｅｘ－ｇ－ＰＬＬＡをかかる範囲の数平均分子量とする構成により、親水性物質もしくは疎水性物質のいずれか一方または両方を内包するナノスフェアが、高い強度と、柔軟性を維持しつつ細胞接着性を低下させることができる。 Due to the composition of dex-g-PLLA in such a number average molecular weight, nanospheres containing either or both of hydrophilic substances and hydrophobic substances can maintain cell strength while maintaining high strength and flexibility. Can be reduced.
　１次溶液生成ステップＳ１００において、上記疎水性ポリマーを溶解した水非混和性有機溶媒を添加せずともよく、内包する物質を溶解した溶媒と、両親媒性ポリマーを溶解した水非混和性有機溶媒とを混合し、１次溶液を生成してもよい。 In the primary solution generation step S100, it is not necessary to add the water-immiscible organic solvent in which the hydrophobic polymer is dissolved, the solvent in which the substance to be included is dissolved, and the water-immiscible organic solvent in which the amphiphilic polymer is dissolved. May be mixed to form a primary solution.
＜疎水性ポリマー＞
　疎水性ポリマーの重合形態は、ランダム共重合体、交互共重合体、ブロック共重合体、グラフト共重合体であってもよい。 <Hydrophobic polymer>
The polymerization form of the hydrophobic polymer may be a random copolymer, an alternating copolymer, a block copolymer, or a graft copolymer.
　疎水性ポリマーの構成単位は、重合した場合に、疎水性ポリマーとなるものであればよく、好ましくは乳酸（Ｌ－乳酸、Ｄ－乳酸、ＤＬ－乳酸）、グリコール酸（ヒドロキシ酢酸）、アミノ酸、カプロラクトン（α－カプロラクトン、β－カプロラクトン、γ－カプロラクトン、δ－カプロラクトン、ε－カプロラクトン等）、コハク酸とエチレングリコールの混合物、コハク酸とブタンジオール（１，２－ブタンジオール、１，３－ブタンジオール、１，４－ブタンジオール、２，３－ブタンジオール等）の混合物、からなる群のうち、１または複数、またはこれらのオリゴマーであればよく、より好ましくは、乳酸、グリコール酸およびアミノ酸、の群から選択される１または複数であり、さらに好ましくはＬ－乳酸およびグリコール酸である。 The structural unit of the hydrophobic polymer is not particularly limited as long as it becomes a hydrophobic polymer when polymerized, preferably lactic acid (L-lactic acid, D-lactic acid, DL-lactic acid), glycolic acid (hydroxyacetic acid), amino acid, Caprolactone (α-caprolactone, β-caprolactone, γ-caprolactone, δ-caprolactone, ε-caprolactone, etc.), a mixture of succinic acid and ethylene glycol, succinic acid and butanediol (1,2-butanediol, 1,3-butane A mixture of diol, 1,4-butanediol, 2,3-butanediol, etc.), one or more, or an oligomer thereof, more preferably lactic acid, glycolic acid and amino acid, One or more selected from the group consisting of L-lactic acid and glycolic acid is there.
　疎水性ポリマーは、例えば、ポリ乳酸、ポリグリコール酸、ポリ乳酸－グリコール酸共重合体、ポリデプシペプチドを用いることができる。 As the hydrophobic polymer, for example, polylactic acid, polyglycolic acid, polylactic acid-glycolic acid copolymer, and polydepsipeptide can be used.
　本実施形態では、疎水性ポリマーとして、Ｌ－乳酸とグリコール酸とのランダム共重合体を用い、以下、かかるＬ－乳酸とグリコール酸とのランダム共重合体による疎水性ポリマーをＰＬＧＡ（ポリ乳酸－グリコール酸ランダム共重合体）と称する。 In the present embodiment, a random copolymer of L-lactic acid and glycolic acid is used as the hydrophobic polymer. Hereinafter, the hydrophobic polymer of the random copolymer of L-lactic acid and glycolic acid is referred to as PLGA (polylactic acid- Glycolic acid random copolymer).
　ＰＬＧＡの重量平均分子量（Ｍｗ）は、特に限定されないが、５０００～７５０００であるとよい。 The weight average molecular weight (Mw) of PLGA is not particularly limited, but is preferably 5000-75000.
[１次溶液生成ステップＳ１００における内包する物質]
　１次溶液生成ステップＳ１００における内包する物質は、親水性物質もしくは疎水性物質のいずれか一方または両方であってもよく、親水性物質もしくは疎水性物質のいずれか一方または両方としてステビア発酵エキスまたは両親媒性物質であってもよい。また両親媒性物質として、例えば、スフィンゴミエリンを用いることができる。 [Substances included in the primary solution generation step S100]
The substance to be included in the primary solution generation step S100 may be either a hydrophilic substance or a hydrophobic substance, or both, and stevia fermented extract or parents as either a hydrophilic substance or a hydrophobic substance or both. A medium substance may be used. As the amphiphile, for example, sphingomyelin can be used.
　親水性物質と疎水性物質の混合物であるステビア発酵エキスおよび両親媒性物質であるスフィンゴミエリンは、国際公開番号WO2008/126638A1や「ステビア発酵エキスの抗ヒスタミン作用に関する研究」薬理と治療 vol.36 no.8 2008に記載されているように、保湿効果が高く、荒れ肌を改善したり、かゆみを抑止したり、炎症を抑えたり、抗ヒスタミン作用を有したりする。したがって、ステビア発酵エキスまたはスフィンゴミエリンを内包させたナノスフェアを含有させた皮膚外用組成物や化粧料とする構成により、皮膚表面の微細な凹凸にステビア発酵エキスまたはスフィンゴミエリンを略均一に行き渡らせることができる。 Stevia fermented extract, a mixture of hydrophilic and hydrophobic substances, and sphingomyelin, an amphiphilic substance, are published in International Publication No. WO2008 / 126638A1 and “Study on Antihistamine Action of Stevia Fermented Extract” Pharmacology and Treatment vol.36 no As described in .8 2008, it has a high moisturizing effect, improves rough skin, suppresses itching, suppresses inflammation, and has antihistaminic activity. Therefore, it is possible to distribute the stevia fermented extract or sphingomyelin substantially evenly on the fine irregularities on the skin surface by using a composition for external skin or cosmetic containing nanospheres containing stevia fermented extract or sphingomyelin. it can.
[１次溶液生成ステップＳ１００における水系溶媒]
　１次溶液生成ステップＳ１００における水系溶媒は、水または無機塩類、糖類、有機塩類、アミノ酸等を含む水溶液であり、親水性物質または両親媒性物質の親水セグメントが溶解できれば足りる。 [Aqueous solvent in primary solution generation step S100]
The aqueous solvent in the primary solution generation step S100 is water or an aqueous solution containing inorganic salts, sugars, organic salts, amino acids, etc., and it is sufficient if the hydrophilic segment of the hydrophilic substance or amphiphilic substance can be dissolved.
[１次溶液生成ステップＳ１００における水非混和性有機溶媒]
　１次溶液生成ステップＳ１００における水非混和性有機溶媒は、疎水性ポリマー、両親媒性ポリマーの疎水性セグメント、疎水性物質、または両親媒性物質の疎水性セグメントが可溶であり、かつ両親媒性ポリマーの親水性セグメントが難溶または不溶であるとよいが、両親媒性ポリマーの疎水性セグメントおよび親水性セグメントの両者が難溶または可溶であってもよい。かかる水非混和性有機溶媒の水への溶解度は、１０ｇ（水非混和性有機溶媒）／１００ｍｌ（水）以下であり、好ましくは、１ｇ（水非混和性有機溶媒）／１００ｍｌ（水）以下であり、さらに好ましくは、０．１ｇ（水非混和性有機溶媒）／１００ｍｌ（水）以下である。水非混和性有機溶媒は、特に限定されないが、酢酸エチル、酢酸イソプロピル、酢酸ブチル、炭酸ジメチル、炭酸ジエチル、塩化メチレン（ジクロロメタン）、クロロホルム等を好適に利用できる。 [Water-immiscible organic solvent in primary solution generation step S100]
The water-immiscible organic solvent in the primary solution generation step S100 is soluble in the hydrophobic polymer, the hydrophobic segment of the amphiphilic polymer, the hydrophobic substance, or the hydrophobic segment of the amphiphilic substance, and the amphiphile. The hydrophilic segment of the hydrophilic polymer may be hardly soluble or insoluble, but both the hydrophobic segment and the hydrophilic segment of the amphiphilic polymer may be hardly soluble or soluble. The solubility of the water-immiscible organic solvent in water is 10 g (water-immiscible organic solvent) / 100 ml (water) or less, preferably 1 g (water-immiscible organic solvent) / 100 ml (water) or less. More preferably, it is 0.1 g (water-immiscible organic solvent) / 100 ml (water) or less. The water-immiscible organic solvent is not particularly limited, and ethyl acetate, isopropyl acetate, butyl acetate, dimethyl carbonate, diethyl carbonate, methylene chloride (dichloromethane), chloroform and the like can be suitably used.
[１次溶液生成ステップＳ１００における水系溶媒と水非混和性有機溶媒の比]
　上記水系溶媒に対する水非混和性有機溶媒の比は、１：１０００～１：１であってもよく、好ましくは、１：１００～１：３、より好ましくは、１：５０～１：１０である。ここで、水非混和性有機溶媒の量は、ポリペプチドを含む両親媒性ポリマーまたはポリエチレングリコールを含む両親媒性ポリマーを用いる場合、これらの両親媒性ポリマーを溶解する水非混和性有機溶媒と疎水性ポリマーを溶解する水非混和性有機溶媒の合計の量とし、多糖を含む両親媒性ポリマーを用いる場合、多糖を含む両親媒性ポリマーを溶解する水非混和性有機溶媒の量とする。 [Ratio of water-based solvent and water-immiscible organic solvent in primary solution generation step S100]
The ratio of the water-immiscible organic solvent to the aqueous solvent may be 1: 1000 to 1: 1, preferably 1: 100 to 1: 3, more preferably 1:50 to 1:10. is there. Here, the amount of the water-immiscible organic solvent is the amount of the water-immiscible organic solvent that dissolves these amphiphilic polymers when using an amphiphilic polymer containing a polypeptide or an amphiphilic polymer containing polyethylene glycol. The total amount of the water-immiscible organic solvent that dissolves the hydrophobic polymer is used. When an amphiphilic polymer containing a polysaccharide is used, the amount of the water-immiscible organic solvent that dissolves the amphiphilic polymer containing the polysaccharide is used.
　水非混和性有機溶媒中の両親媒性ポリマーおよび疎水性ポリマーの濃度は、ポリペプチドを含む両親媒性ポリマーまたはポリエチレングリコールを含む両親媒性ポリマーを用いる場合、１～５００ｍｇ／ｍｌであればよく、好ましくは、１０～２５０ｍｇ／ｍｌであり、さらに好ましくは５０～１００ｍｇ／ｍｌである。この場合、両親媒性ポリマーに対する疎水性ポリマーの比は、１：１０００～１：１であればよく、好ましくは、１：１００～１：３、より好ましくは、１：５０～１：５である。 The concentration of the amphiphilic polymer and the hydrophobic polymer in the water-immiscible organic solvent may be 1 to 500 mg / ml when using an amphiphilic polymer containing a polypeptide or an amphiphilic polymer containing polyethylene glycol. It is preferably 10 to 250 mg / ml, more preferably 50 to 100 mg / ml. In this case, the ratio of the hydrophobic polymer to the amphiphilic polymer may be 1: 1000 to 1: 1, preferably 1: 100 to 1: 3, more preferably 1:50 to 1: 5. is there.
　また、水非混和性有機溶媒中の両親媒性ポリマーの濃度は、多糖を含む両親媒性ポリマーを用いる場合、１～５００ｍｇ／ｍｌであればよく、好ましくは、１０～２５０ｍｇ／ｍｌであり、さらに好ましくは５０～１００ｍｇ／ｍｌである。 In addition, the concentration of the amphiphilic polymer in the water-immiscible organic solvent may be 1 to 500 mg / ml, preferably 10 to 250 mg / ml when an amphiphilic polymer containing a polysaccharide is used. More preferably, it is 50 to 100 mg / ml.
（１次エマルション生成ステップＳ１０２）
　生成した１次溶液に超音波を照射し、逆相エマルション（Ｗ／Ｏエマルション）である１次エマルションを生成する。 (Primary emulsion generation step S102)
The produced primary solution is irradiated with ultrasonic waves to produce a primary emulsion which is a reverse phase emulsion (W / O emulsion).
　超音波の照射時間は、０．５～１５分間であればよく、好ましくは、１～１０分間であり、より好ましくは、３～５分間である。 The ultrasonic irradiation time may be 0.5 to 15 minutes, preferably 1 to 10 minutes, and more preferably 3 to 5 minutes.
　超音波の照射は、バス型ソニケータを用いても、プローブ型ソニケータを用いてもよく、バス型ソニケータおよびプローブ型ソニケータを用いてもよい。 The ultrasonic irradiation may be performed using a bus sonicator, a probe sonicator, or a bus sonicator and a probe sonicator.
　また、本実施形態において、生成した１次溶液に超音波を照射することで、逆相エマルションである１次エマルションを生成するが、これに限定されず、マグネチックスターラー等の攪拌装置、タービン型攪拌装置、ホモジナイザー等を用いて１次溶液を攪拌することで１次エマルションを生成してもよい。 Moreover, in this embodiment, the primary solution which is a reverse phase emulsion is produced | generated by irradiating the produced | generated primary solution with an ultrasonic wave, However, It is not limited to this, Stirring apparatuses, such as a magnetic stirrer, turbine type You may produce | generate a primary emulsion by stirring a primary solution using a stirring apparatus, a homogenizer, etc.
（２次溶液生成ステップＳ１０４）
　１次エマルションに、親水性ポリマーを溶解した水系溶媒を添加し、２次溶液を生成する。 (Secondary solution generation step S104)
An aqueous solvent in which a hydrophilic polymer is dissolved is added to the primary emulsion to form a secondary solution.
[２次溶液生成ステップＳ１０４における親水性ポリマー]
　親水性ポリマーは、上述した両親媒性ポリマーの疎水性セグメントまたは疎水性ポリマーと、化学結合し、または物理的に吸着するものであるとよい。また、親水性ポリマーは、溶媒中における１次エマルションの凝集を防止したり、凝集を遅延させたりするものであるとよい。なお、親水性ポリマーは、２次溶液生成ステップＳ１０４において、両親媒性ポリマーの疎水性セグメントまたは疎水性ポリマーと、化学結合し、または物理的に吸着するものであってもよく、後述する有機溶媒除去ステップＳ１０８における、溶媒除去時に両親媒性ポリマーの疎水性セグメントまたは疎水性ポリマーと、化学結合し、または物理的に吸着するものであってもよい。 [Hydrophilic polymer in secondary solution generation step S104]
The hydrophilic polymer may be chemically bonded or physically adsorbed with the hydrophobic segment or hydrophobic polymer of the above-described amphiphilic polymer. Further, the hydrophilic polymer may be one that prevents aggregation of the primary emulsion in the solvent or delays aggregation. The hydrophilic polymer may be chemically bonded or physically adsorbed with the hydrophobic segment or the hydrophobic polymer of the amphiphilic polymer in the secondary solution generation step S104. In the removal step S108, it may be chemically bonded or physically adsorbed with the hydrophobic segment or the hydrophobic polymer of the amphiphilic polymer when the solvent is removed.
　親水性ポリマーは、ポリビニルアルコール、ポリエチレングリコール、ポリペプチド、タンパク質または多糖類の群から選択される１または複数であればよく、好ましくは、ポリビニルアルコールである。 The hydrophilic polymer may be one or more selected from the group of polyvinyl alcohol, polyethylene glycol, polypeptide, protein, or polysaccharide, and is preferably polyvinyl alcohol.
　また、親水性ポリマーは、ポリビニルアルコール、ポリエチレングリコール、ポリペプチド、タンパク質または多糖類の群から選択される１または複数の類縁体であってもよい。 The hydrophilic polymer may be one or more analogs selected from the group of polyvinyl alcohol, polyethylene glycol, polypeptide, protein or polysaccharide.
[２次溶液生成ステップＳ１０４における水系溶媒]
　２次溶液生成ステップＳ１００における水系溶媒は、親水性ポリマーを溶解できればよく、例えば、水または無機塩類、糖類、有機塩類、アミノ酸等を含む水溶液である。 [Aqueous solvent in secondary solution generation step S104]
The aqueous solvent in the secondary solution generation step S100 only needs to dissolve the hydrophilic polymer, and is, for example, water or an aqueous solution containing inorganic salts, saccharides, organic salts, amino acids, and the like.
　水系溶媒中の親水性ポリマーの濃度は、０．１～５００ｍｇ／ｍｌであればよく、好ましくは、１０～２５０ｍｇ／ｍｌであり、さらに好ましくは５０～１００ｍｇ／ｍｌである。 The concentration of the hydrophilic polymer in the aqueous solvent may be 0.1 to 500 mg / ml, preferably 10 to 250 mg / ml, and more preferably 50 to 100 mg / ml.
　また、１次溶液生成ステップＳ１００における１次溶液に対する、２次溶液生成ステップＳ１００における親水性ポリマーを溶解した水系溶媒比は、１：５００～１：１０であればよく、好ましくは、１：３００～１：５０、さらに好ましくは、１：２００～１：１００である。 Further, the ratio of the aqueous solvent in which the hydrophilic polymer in the secondary solution generation step S100 is dissolved to the primary solution in the primary solution generation step S100 may be 1: 500 to 1:10, and preferably 1: 300. To 1:50, more preferably 1: 200 to 1: 100.
（２次エマルション生成ステップＳ１０６）
　生成した２次溶液に超音波を照射して２次エマルションを生成する。 (Secondary emulsion generation step S106)
The produced secondary solution is irradiated with ultrasonic waves to produce a secondary emulsion.
　超音波の照射時間は、０．５～２０分間であればよく、好ましくは、１～１５分間であり、さらに好ましくは、３～７分間である。 The ultrasonic irradiation time may be 0.5 to 20 minutes, preferably 1 to 15 minutes, and more preferably 3 to 7 minutes.
　超音波の照射は、バス型ソニケータを用いても、プローブ型ソニケータを用いてもよく、バス型ソニケータおよびプローブ型ソニケータを用いてもよい。 The ultrasonic irradiation may be performed using a bus sonicator, a probe sonicator, or a bus sonicator and a probe sonicator.
　また、本実施形態において、生成した２次溶液に超音波を照射することで、２次エマルションを生成するが、これに限定されず、マグネチックスターラー等の攪拌装置、タービン型攪拌装置、ホモジナイザー等を用いて２次溶液を攪拌することで２次エマルションを生成してもよい。 Moreover, in this embodiment, a secondary emulsion is produced | generated by irradiating the produced | generated secondary solution with an ultrasonic wave, However, It is not limited to this, Stirring apparatuses, such as a magnetic stirrer, a turbine type stirring apparatus, a homogenizer, etc. You may produce | generate a secondary emulsion by stirring a secondary solution using.
（有機溶媒除去ステップＳ１０８）
　２次エマルションから水非混和性有機溶媒を除去する。 (Organic solvent removal step S108)
Remove the water immiscible organic solvent from the secondary emulsion.
　水非混和性有機溶媒を除去する方法としては、液中乾燥、透析、遠心分離、凍結乾燥、濾過、再沈殿等を利用すればよく、好ましくは、液中乾燥、遠心分離、凍結乾燥を利用する。 As a method for removing the water-immiscible organic solvent, in-liquid drying, dialysis, centrifugation, freeze-drying, filtration, reprecipitation, etc. may be used, preferably in-liquid drying, centrifugation, or freeze-drying. To do.
　また、当該有機溶媒除去ステップＳ１０８を経た後に、２次エマルション（疎水性物質を内包する場合、エマルション）を、水系溶媒に導入し、さらに水非混和性有機溶媒を除去することにより、親水性物質もしくは疎水性物質のいずれか一方または両方を内包するナノスフェアの水系溶媒分散体としてもよい。 Further, after passing through the organic solvent removal step S108, the secondary emulsion (in the case of including a hydrophobic substance, an emulsion) is introduced into an aqueous solvent, and further the water-immiscible organic solvent is removed to thereby remove the hydrophilic substance. Or it is good also as the aqueous solvent dispersion of the nanosphere which encloses any one or both of hydrophobic substances.
　以上説明した１次溶液生成ステップＳ１００、１次エマルション生成ステップＳ１０２、２次溶液生成ステップＳ１０４、２次エマルション生成ステップＳ１０６および有機溶媒除去ステップＳ１０８により、親水性物質もしくは疎水性物質のいずれか一方または両方を内包するナノスフェアの水系溶媒分散体を得ることができる。なお、両親媒性ポリマー、疎水性ポリマーおよび親水性ポリマーに生分解性ポリマーを用いれば、薬物徐放型ＤＤＳ製剤等の医薬品や有効物質を内包する徐放型の皮膚外用組成物、有効成分を内包する化粧料に好適な、親水性物質もしくは疎水性物質のいずれか一方または両方を内包する生分解性ナノスフェアの水系溶媒分散体を得ることが可能となる。 By the primary solution generation step S100, the primary emulsion generation step S102, the secondary solution generation step S104, the secondary emulsion generation step S106, and the organic solvent removal step S108 described above, either a hydrophilic substance or a hydrophobic substance or An aqueous solvent dispersion of nanospheres enclosing both can be obtained. In addition, if a biodegradable polymer is used for the amphiphilic polymer, hydrophobic polymer and hydrophilic polymer, a sustained release type skin external composition and active ingredient containing a drug or an active substance such as a drug sustained release type DDS formulation can be obtained. It is possible to obtain an aqueous solvent dispersion of biodegradable nanospheres that enclose either or both of a hydrophilic substance and a hydrophobic substance, which is suitable for a cosmetic to be encapsulated.
　また、本実施形態により得られた親水性物質もしくは疎水性物質のいずれか一方または両方を内包するナノスフェアの水系溶媒分散体から、分散媒を除去することにより、親水性物質もしくは疎水性物質のいずれか一方または両方を内包するナノスフェアとすることも可能である。分散媒を除去する方法としては、特に限定されないが、液中乾燥、透析、遠心分離、凍結乾燥、濾過、再沈殿等を利用することができ、好ましくは、液中乾燥、遠心分離、凍結乾燥を利用する。 Further, by removing the dispersion medium from the aqueous dispersion of nanospheres enclosing one or both of the hydrophilic substance and the hydrophobic substance obtained by the present embodiment, either the hydrophilic substance or the hydrophobic substance is obtained. It is also possible to form nanospheres that include either or both. The method for removing the dispersion medium is not particularly limited, and submerged drying, dialysis, centrifugation, lyophilization, filtration, reprecipitation, and the like can be used. Preferably, submerged drying, centrifuging, and lyophilization are performed. Is used.
　以上説明したように、１次エマルション生成ステップＳ１０２において１次エマルションを生成する際に、両親媒性ポリマーを添加する構成により、疎水性ポリマーに親水性物質もしくは疎水性物質のいずれか一方または両方を内包させることができる。また、疎水性ポリマーと、親水性物質もしくは疎水性物質のいずれか一方または両方とに両親媒性ポリマーを添加した１次溶液に上述した条件で超音波を照射して生成した１次エマルションに親水性ポリマーを添加した２次溶液に、上述した条件で超音波を照射する構成により、凝集を抑制しつつ、親水性物質もしくは疎水性物質のいずれか一方または両方を内包する高分子微粒子をナノメートルオーダーで製造することが可能となる。 As described above, when the primary emulsion is generated in the primary emulsion generation step S102, either one or both of the hydrophilic substance and the hydrophobic substance is added to the hydrophobic polymer by the configuration in which the amphiphilic polymer is added. Can be included. Further, the primary emulsion produced by irradiating the hydrophobic polymer and the primary solution obtained by adding the amphiphilic polymer to one or both of the hydrophilic substance and the hydrophobic substance under the above-described conditions is hydrophilic. The polymer solution containing the hydrophilic polymer and / or the hydrophobic substance is incorporated into the secondary solution to which the hydrophilic polymer is added under the above-described conditions while the aggregation is suppressed. It becomes possible to manufacture by order.
　また、１次エマルション生成ステップＳ１０２において１次エマルションを生成する際に、両親媒性ポリマーと、親水性物質もしくは疎水性物質のいずれか一方または両方と、を含む１次溶液に上述した条件で超音波を照射して生成した１次エマルションに親水性ポリマーを添加した２次溶液に、上述した条件で超音波を照射する構成により、凝集を抑制しつつ、親水性物質もしくは疎水性物質のいずれか一方または両方を内包する高分子微粒子をナノメートルオーダーで製造することが可能となる。 In addition, when the primary emulsion is generated in the primary emulsion generation step S102, the primary solution containing the amphiphilic polymer and either one or both of the hydrophilic substance and the hydrophobic substance is used under the above-described conditions. Either a hydrophilic substance or a hydrophobic substance is suppressed while agglomeration is suppressed by a structure in which ultrasonic waves are applied to the secondary solution in which a hydrophilic polymer is added to a primary emulsion formed by irradiating sound waves under the above-described conditions. It is possible to produce polymer fine particles enclosing one or both on the nanometer order.
　さらに、両親媒性ポリマー、疎水性ポリマーおよび親水性ポリマーに生分解性ポリマーを用いる構成により、タンパク質や核酸、生理活性を有する天然抽出物等の水溶性（親水性）高分子の薬物、および、脂質や、生理活性を有する天然抽出物等の疎水性の高分子の薬物を内包する医薬品や、親水性および疎水性の有効物質を内包する徐放型の化粧料を製造することができる。また、親水性物質もしくは疎水性物質のいずれか一方または両方を内包する高分子微粒子は、ナノメートルオーダーであるため、静脈または動脈注射用の医薬品、経皮吸収用の医薬品や化粧料等の皮膚外用組成物に利用することができ、薬物の全身投与、患部へのターゲッティングおよび皮膚への浸透が可能となる。 Furthermore, by using a biodegradable polymer as an amphiphilic polymer, a hydrophobic polymer, and a hydrophilic polymer, a water-soluble (hydrophilic) polymer drug such as a protein, a nucleic acid, or a natural extract having physiological activity, and Drugs encapsulating lipids and hydrophobic polymer drugs such as natural extracts having physiological activity and sustained-release cosmetics encapsulating hydrophilic and hydrophobic active substances can be produced. In addition, polymer fine particles encapsulating either or both of hydrophilic substances and hydrophobic substances are on the order of nanometers, so skin for pharmaceuticals for intravenous or arterial injection, pharmaceuticals for percutaneous absorption, cosmetics, etc. It can be used as an external composition, and enables systemic administration of drugs, targeting of affected areas, and penetration into the skin.
　皮膚外用組成物として、上記親水性物質もしくは疎水性物質のいずれか一方または両方を内包するナノスフェアの製造方法を用いて製造された親水性物質もしくは疎水性物質のいずれか一方または両方を内包するナノスフェアを含有する構成により、塗り薬等の皮膚外用組成物を皮膚表面の微細な凹凸にナノスフェアを略均一に塗布することができる。また、上述したようにナノスフェアを構成するポリマーに生分解性ポリマーを用いる構成により、ナノスフェアに内包させた親水性物質もしくは疎水性物質のいずれか一方または両方を皮膚表面で好適に徐放させることができ、従来と比較して、皮膚外用組成物の効果を維持しつつ、塗布する回数を減少させることが可能となる。 Nanospheres encapsulating either or both of a hydrophilic substance and a hydrophobic substance produced using the method for producing nanospheres encapsulating either or both of the hydrophilic substance and the hydrophobic substance as a composition for external use on the skin With the composition containing the nanospheres, the composition for external use such as a coating agent can be applied to the fine irregularities on the skin surface substantially uniformly. In addition, as described above, by using a biodegradable polymer as a polymer constituting the nanosphere, either one or both of the hydrophilic substance and the hydrophobic substance encapsulated in the nanosphere can be suitably and slowly released on the skin surface. In comparison with the prior art, it is possible to reduce the number of times of application while maintaining the effect of the external composition for skin.
　また、化粧料として、上記親水性物質もしくは疎水性物質のいずれか一方または両方を内包するナノスフェアの製造方法を用いて製造された親水性物質もしくは疎水性物質のいずれか一方または両方を内包するナノスフェアを含有する構成により、化粧料を皮膚表面の微細な凹凸にナノスフェアを略均一に塗布することができる。また、上述したようにナノスフェアを構成するポリマーに生分解性ポリマーを用いる構成により、ナノスフェアに内包させた親水性物質もしくは疎水性物質のいずれか一方または両方を皮膚表面で好適に徐放させることができ、従来と比較して化粧料の塗布回数を減らしても同程度の効果を得ることが可能となる。 Further, as a cosmetic, nanospheres encapsulating either one or both of the hydrophilic substance and the hydrophobic substance produced by using the method for producing nanospheres encapsulating one or both of the hydrophilic substance and the hydrophobic substance. With the constitution containing the nanosphere, the nanosphere can be applied substantially uniformly on the fine irregularities of the skin surface. In addition, as described above, by using a biodegradable polymer as a polymer constituting the nanosphere, either one or both of the hydrophilic substance and the hydrophobic substance encapsulated in the nanosphere can be suitably and slowly released on the skin surface. It is possible to obtain the same effect even if the number of times of applying the cosmetic is reduced as compared with the conventional case.
　例えば、親水性物質と疎水性物質の混合物であるステビア発酵エキスや、親水性物質もしくは疎水性物質のいずれか一方または両方の機能を有する両親媒性物質としてのスフィンゴミエリンを内包させたナノスフェアを含有させた皮膚外用組成物や、化粧料を製造することもできる。 For example, stevia fermented extract, which is a mixture of hydrophilic and hydrophobic substances, and nanospheres encapsulating sphingomyelin as an amphiphilic substance having the function of one or both of hydrophilic substances and hydrophobic substances It is also possible to produce a skin external composition and a cosmetic.
　ステビア発酵エキスおよびスフィンゴミエリンは、国際公開番号WO2008/126638A1や「ステビア発酵エキスの抗ヒスタミン作用に関する研究」薬理と治療 vol.36 no.8 2008に記載されているように、保湿効果が高く、荒れ肌を改善したり、かゆみを抑止したり、炎症を抑えたり、抗ヒスタミン作用を有したりする。したがって、ステビア発酵エキスまたはスフィンゴミエリンを内包させたナノスフェアを含有させた皮膚外用組成物や化粧料とする構成により、皮膚表面の微細な凹凸にステビア発酵エキスまたはスフィンゴミエリンを略均一に行き渡らせることができる。 Stevia fermented extract and sphingomyelin have high moisturizing effect and rough skin as described in International Publication No. WO2008 / 126638A1 and “Research on Antihistamine Action of Stevia Fermented Extract” Pharmacology and Treatment vol.36 no.8 2008 Improves it, suppresses itching, suppresses inflammation, and has an antihistamine effect. Therefore, it is possible to distribute the stevia fermented extract or sphingomyelin substantially evenly on the fine irregularities on the skin surface by using a composition for external skin or cosmetic containing nanospheres containing stevia fermented extract or sphingomyelin. it can.
　また本実施形態にかかるステビア発酵エキスもしくはスフィンゴミエリンのいずれか一方または両方を内包させたナノスフェアは、化粧料においては、頭髪用化粧品、整髪料、養毛料、頭皮料、毛髪着色料、洗髪料、ヘアリンス、皮膚用化粧品・化粧水、化粧液、クリーム、乳液、日焼け、日焼け止め、洗浄料、ひげそり、むだ毛そり、フェイシャルリンス、パック、化粧用油、ボディリンス、マッサージ料、仕上用化粧品・ファンデーション、化粧下地、おしろい、口紅、アイメークアップ、頬化粧料、ボディメークアップ、オーデコロン・香水、浴用化粧料、爪化粧料、ボディパウダー等、また、医薬品においては、散剤・細粒剤、顆粒剤、錠剤、カプセル剤、丸剤、桿剤、ペンシル剤、内容液剤、外用液剤、エキス剤、硬膏剤、坐剤、エアゾール、ガス剤、薬品吸着剤、眼科用剤、注射剤、絆創膏剤等幅広く使用可能である。 Further, nanospheres containing either or both of stevia fermented extract and sphingomyelin according to the present embodiment are cosmetics for hair, hair styling, hair nourishing, scalp, hair coloring, hair washing, Hair rinse, skin cosmetics / skin, cosmetic liquid, cream, milky lotion, tanning, sunscreen, cleaning agent, shaving, dead hair shave, facial rinse, pack, cosmetic oil, body rinse, massage, finishing cosmetics and foundation , Makeup base, funny, lipstick, eye makeup, cheek cosmetics, body makeup, eau de cologne / perfume, bath cosmetics, nail cosmetics, body powders, etc., and in pharmaceuticals, powders / fine granules, granules, Tablets, capsules, pills, glazes, pencils, liquid contents, external liquids, extracts, plasters, suppositories Aerosol, gas chemistry, chemical adsorbents, ophthalmic agents, injections, bandages and the like are widely available.
　以下に、ある実施例の詳細についてさらに説明する。 The details of a certain embodiment will be further described below.
（実施例１）
　本実施例では、両親媒性ポリマーとしてポリリジンとＬ－乳酸のジブロック共重合体（以下、単に、ＰＬｙｓ＋－ｂ－ＰＬＬＡと称する）を、疎水性ポリマーとして、ＰＬＧＡを、親水性ポリマーとしてポリビニルアルコール（以下、単に、ＰＶＡと称する）を、内包する親水性物質としてＢＳＡを用いた。 Example 1
In this example, a diblock copolymer of polylysine and L-lactic acid (hereinafter simply referred to as PLys + -b-PLLA) is used as an amphiphilic polymer, PLGA is used as a hydrophobic polymer, and polyvinyl is used as a hydrophilic polymer. BSA was used as a hydrophilic substance containing alcohol (hereinafter, simply referred to as PVA).
　図２は、実施例１にかかるＰＬｙｓ＋－ｂ－ＰＬＬＡの合成方法を説明するための説明図である。 FIG. 2 is an explanatory diagram for explaining the synthesis method of PLys + -b-PLLA according to the first example.
　図２に示すように、まず、２－ａｍｉｎｏｅｔｈａｎｏｌのアミノ基をt-ブトキシカルボニル（以下単に、Ｂｏｃと称する）基で保護した。次に、Ｂｏｃ―ａｍｉｎｏｅｔｈａｎｏｌの末端水酸基をカリウムナフタレンによりアルコキシド化し、これを開始剤としてラクチドの溶液系アニオン開環重合を行った。最後に、得られたポリマーの末端アミノ基の保護基（Ｂｏｃ基）を２５％臭化水素酢酸（２５％ＨＢｒ／ＡｃＯＨ）で除去した後、トリエチルアミン（ＴＥＡ）を用いて脱塩を行い、目的物であるＮＨ２－ＰＬＬＡを得た。 As shown in FIG. 2, first, the amino group of 2-aminoethanol was protected with a t-butoxycarbonyl (hereinafter simply referred to as Boc) group. Next, the terminal hydroxyl group of Boc-aminoethanol was alkoxided with potassium naphthalene, and solution based anionic ring-opening polymerization of lactide was performed using this as an initiator. Finally, after the protecting group (Boc group) of the terminal amino group of the obtained polymer was removed with 25% hydrobromic acetic acid (25% HBr / AcOH), desalting was performed using triethylamine (TEA). The product NH 2 -PLLA was obtained.
　ＮＨ２－ＰＬＬＡのアミノ基を重合開始点として用い、Ｌｙｓ（Ｚ）－Ｎ－カルボキシ無水物（ＮＣＡ：N-Carboxy Anhydride）との脱炭酸重合を行った。蒸留したクロロホルムにＮＨ２－ＰＬＬＡを溶解した後、Ｌｙｓ（Ｚ）－ＮＣＡを加え、アルゴン気流下、４０℃で２４時間撹拌した。次に、得られたポリマーを２５％臭化水素酢酸で処理することで側鎖保護基であるベンジルオキシカルボニル基(Ｚ基)の脱保護を行い、目的物であるＰＬｙｓ＋－ｂ－ＰＬＬＡを得た。 Using the amino group of NH 2 -PLLA as a polymerization initiation point, decarboxylation polymerization with Lys (Z) -N-carboxyanhydride (NCA) was performed. After dissolving NH 2 -PLLA in distilled chloroform, Lys (Z) -NCA was added, and the mixture was stirred at 40 ° C. for 24 hours under an argon stream. Next, the resulting polymer is treated with 25% hydrobromic acetic acid to deprotect the benzyloxycarbonyl group (Z group) which is a side chain protecting group, and the target product, PLys + -b-PLLA, is removed. Obtained.
　本実施例にかかるＰＬｙｓ＋－ｂ－ＰＬＬＡは、リジンの重合度は１６であり、乳酸の重合度は３９であった。また、本実施例にかかるＰＬｙｓ＋－ｂ－ＰＬＬＡは両親媒性を有するため水中で自己会合してミセルを形成する。 The PLys + -b-PLLA according to this example had a polymerization degree of lysine of 16 and a polymerization degree of lactic acid of 39. In addition, PLys + -b-PLLA according to this example has amphipathic properties and thus self-associates in water to form micelles.
　図３は、実施例１にかかるＰＬｙｓ＋－ｂ－ＰＬＬＡのミセル状態における分解性を説明するための説明図である。ここでは、ＰＬｙｓ＋－ｂ－ＰＬＬＡのミセルを０．５ｍｇ／ｍｌとなるようにリン酸緩衝生理食塩水（ＰＢＳ：Phosphate Buffered Saline）（ｐＨ＝７．４、Ｉ＝０．１４）に分散させ、３７℃でインキュベートし、１日後、３日後、５日後、７日後のＰＬｙｓ＋－ｂ－ＰＬＬＡを、ＧＰＣ（Gel Permeation Chromatography）を用いた分子量分布測定を行い分子量の減少について検討した。 FIG. 3 is an explanatory diagram for explaining the decomposability in the micelle state of PLys + -b-PLLA according to the first example. Here, micelles of PLys + -b-PLLA are dispersed in phosphate buffered saline (PBS) (pH = 7.4, I = 0.14) so as to be 0.5 mg / ml. After incubation at 37 ° C., the molecular weight distribution measurement using GPC (Gel Permeation Chromatography) was performed on PLys + -b-PLLA after 1 day, 3 days, 5 days, and 7 days, and the decrease in molecular weight was examined.
　図３に示すように、１日後においても１％程度しか減少しておらず、３日後においては、１．５％程度、５日後においては、２．５％程度、７日後においては６％程度の減少であった。したがって、両親媒性ポリマーとしてＰＬｙｓ＋－ｂ－ＰＬＬＡを用いた親水性物質もしくは疎水性物質のいずれか一方または両方を内包する生分解性ナノスフェアを生体内に留置した場合に、分解に伴う炎症反応等を起こさない分解速度であり、かつ、生体内で必要とされる期間の経過後には生体内から消失している分解速度に調節することができる。 As shown in FIG. 3, it decreases only about 1% after 1 day, about 1.5% after 3 days, about 2.5% after 5 days, and about 6% after 7 days. Decrease. Therefore, when biodegradable nanospheres encapsulating either or both of hydrophilic substances and hydrophobic substances using PLys + -b-PLLA as an amphiphilic polymer are placed in vivo, the inflammatory reaction associated with the degradation It can be adjusted to a decomposition rate that does not cause the degradation and disappears from the living body after a period required in the living body.
　図４は、実施例１を説明するための説明図である。図４に示すように、試験管に、ＰＬｙｓ＋－ｂ－ＰＬＬＡ２０ｍｇおよび重量平均分子量が５０００～７５０００であるＰＬＧＡ１８０ｍｇを入れ、クロロホルム（ＣＨＣｌ３）２．５ｍｌに溶解させた。続いて、親水性物質としてＢＳＡ５ｍｇを溶解したＰＢＳ１００μｌを試験管に加え１次溶液を生成した。次に、１次溶液をスパチュラで激しく撹拌しながらプローブ型ソニケータで３分間超音波を照射し、１次エマルションを調製した。本実施例において、プローブ型ソニケータとして、株式会社トミー精工製　ＵＤ－２００を用い、レベル５～１０に設定して、超音波を照射した。以下、株式会社トミー精工製　ＵＤ－２００を用いて、レベル５～１０に設定し超音波を照射することを、単にプローブ型ソニケータで超音波を照射すると称する。 FIG. 4 is an explanatory diagram for explaining the first embodiment. As shown in FIG. 4, 20 mg of PLys + -b-PLLA and 180 mg of PLGA having a weight average molecular weight of 5000-75000 were placed in a test tube and dissolved in 2.5 ml of chloroform (CHCl 3 ). Subsequently, 100 μl of PBS in which 5 mg of BSA was dissolved as a hydrophilic substance was added to the test tube to produce a primary solution. Next, while the primary solution was vigorously stirred with a spatula, ultrasonic irradiation was performed with a probe sonicator for 3 minutes to prepare a primary emulsion. In this example, UD-200 manufactured by Tommy Seiko Co., Ltd. was used as a probe-type sonicator, and the ultrasonic wave was applied at a level of 5 to 10. Hereinafter, using UD-200 manufactured by Tommy Seiko Co., Ltd. and irradiating ultrasonic waves at levels 5 to 10 is simply referred to as irradiating ultrasonic waves with a probe sonicator.
　その後、１次エマルションを、０．１％ＰＶＡを含むＰＢＳ３００ｍｌに加えて２次溶液を調製した。次に、２次溶液にバス型ソニケータで３分間超音波を照射し、９０秒間スパチュラで激しく攪拌し、さらに、プローブ型ソニケータで９０秒間超音波を照射して２次エマルションを調製した。本実施例において、バス型ソニケータとして、株式会社エスエヌディ製　ＵＳ－２を用い、レベル５に設定して超音波を照射した。以下、株式会社エスエヌディ製　ＵＳ－２を用いて、レベル５に設定し超音波を照射することを、単にバス型ソニケータで超音波を照射すると称する。 Thereafter, the primary emulsion was added to 300 ml of PBS containing 0.1% PVA to prepare a secondary solution. Next, the secondary solution was irradiated with ultrasonic waves for 3 minutes with a bath sonicator, stirred vigorously with a spatula for 90 seconds, and further irradiated with ultrasonic waves for 90 seconds with a probe sonicator to prepare a secondary emulsion. In this example, US-2 manufactured by SND Co., Ltd. was used as a bath sonicator, and ultrasonic waves were applied with the level set to 5. Hereinafter, the irradiation with ultrasonic waves set to level 5 using US-2 manufactured by SND Corporation is simply referred to as irradiating ultrasonic waves with a bath-type sonicator.
　そして、２次エマルションを、０．１％ＰＶＡを含むＰＢＳ３００ｍｌにさらに加え、液中乾燥を行うことにより、クロロホルムを除去した懸濁液を得た。その後、懸濁液を２００００ｒｐｍで１０分間遠心分離し、ＢＳＡを内包する生分解性ナノスフェアを含む沈殿を得た。そして、かかる沈殿を超純水で洗浄した。この、遠心分離および洗浄からなる操作を数回繰り返した後、沈殿を凍結乾燥して水を除去することで、ＢＳＡを内包する生分解性ナノスフェアを得た。 Then, the secondary emulsion was further added to 300 ml of PBS containing 0.1% PVA and dried in liquid to obtain a suspension from which chloroform was removed. Thereafter, the suspension was centrifuged at 20000 rpm for 10 minutes to obtain a precipitate containing biodegradable nanospheres containing BSA. The precipitate was washed with ultrapure water. After repeating this operation consisting of centrifugation and washing several times, the precipitate was freeze-dried to remove water, thereby obtaining biodegradable nanospheres containing BSA.
（評価）
　ＢＳＡを内包する生分解性ナノスフェアの形状および粒径を、走査型電子顕微鏡（ＳＥＭ：Scanning Electron Microscope）を用いて検討した。走査型電子顕微鏡試料台にカーボン両面テープを貼り付け、その上に凍結乾燥後のＢＳＡを内包する生分解性ナノスフェアを載置し、２００Åの金蒸着を行うことにより、走査型電子顕微鏡の試料を作成した。ＢＳＡを内包する生分解性ナノスフェア粒径は無作為に選択したＢＳＡを内包する生分解性ナノスフェアの粒径を測定することにより求めた。 (Evaluation)
The shape and particle size of the biodegradable nanosphere encapsulating BSA were examined using a scanning electron microscope (SEM). A carbon double-sided tape is affixed to a scanning electron microscope sample stage, and biodegradable nanospheres containing BSA after freeze-drying are placed on the sample. Created. The particle size of the biodegradable nanosphere encapsulating BSA was determined by measuring the particle size of a biodegradable nanosphere encapsulating randomly selected BSA.
　図５は、ＢＳＡを内包する生分解性ナノスフェアを走査型電子顕微鏡で観察した結果を説明するための説明図である。図５に示すように、ＢＳＡを内包する生分解性ナノスフェアの粒径は、約１５０ｎｍ～約８００ｎｍであった。また、比較的凝集も抑えられており微粒子表面も、平滑なものが得られた。 FIG. 5 is an explanatory diagram for explaining the results of observation of biodegradable nanospheres containing BSA with a scanning electron microscope. As shown in FIG. 5, the particle size of the biodegradable nanosphere encapsulating BSA was about 150 nm to about 800 nm. Further, aggregation was relatively suppressed, and a fine particle surface was obtained.
　ナノスフェアの粒径が約１５０ｎｍ～約８００ｎｍと小さいため、親水性物質もしくは疎水性物質のいずれか一方または両方を内包した生分解性ナノスフェアを、静脈または動脈注射用の医薬品、経皮吸収用の医薬品や化粧料等の皮膚外用組成物に利用することができ、薬物の全身投与、患部へのターゲッティングおよび皮膚への浸透が可能となる。 Since the particle size of the nanospheres is as small as about 150 nm to about 800 nm, biodegradable nanospheres containing either or both of hydrophilic substances and hydrophobic substances are used as pharmaceuticals for intravenous or arterial injection, pharmaceuticals for transdermal absorption And can be used for compositions for external use of skin such as cosmetics and the like, and enables systemic administration of drugs, targeting to affected areas and penetration into skin.
　次に、生分解性ナノスフェアのＢＳＡ内包率およびＢＳＡの回収率を測定した。まず、サンプル管を６個用意し、そのうち５個のサンプル管には内包物なしの空の生分解性ナノスフェアを、残りの１個のサンプル管には内包率が未知である、ＢＳＡを内包した生分解性ナノスフェアを、それぞれ２０ｍｇ入れた。 Next, the BSA encapsulation rate and BSA recovery rate of the biodegradable nanospheres were measured. First, six sample tubes were prepared, of which five sample tubes contained empty biodegradable nanospheres without inclusions, and the remaining one sample tube contained BSA whose inclusion rate was unknown. 20 mg of each biodegradable nanosphere was added.
　空の生分解性ナノスフェアを入れた５個のサンプル管には、それぞれＢＳＡの濃度が０、０．３、０．５、０．７、０．９ｍｇ／ｍｌとなるように、ＢＳＡ含有１Ｎ水酸化ナトリウム水溶液を３ｍｌずつ入れ、ＢＳＡを内包した生分解性ナノスフェアを入れたサンプル管には、１Ｎ水酸化ナトリウム水溶液を３ｍｌ入れた。 Five sample tubes containing empty biodegradable nanospheres contain 1N water containing BSA so that the BSA concentrations are 0, 0.3, 0.5, 0.7, and 0.9 mg / ml, respectively. 3 ml each of an aqueous sodium oxide solution was placed, and 3 ml of a 1N aqueous sodium hydroxide solution was placed in a sample tube containing biodegradable nanospheres containing BSA.
　上記６個のサンプル管にキャップをして約２日間スターラーで攪拌した。２日後、溶液が透明になり、生分解性ナノスフェアが分解されたのを目視で確認した後、それぞれの溶液を、ディスポーサブルフィルターを用いて濾過した。ＢＳＡの濃度が０ｍｇ／ｍｌの空の生分解性ナノスフェアを分解した溶液をバックグラウンドとし、波長２９０ｎｍの吸光度を測定して、検量線を作成した。そして、ＢＳＡを内包した生分解性ナノスフェアを分解した溶液の吸光度を測定し、作成した検量線より、生分解性ナノスフェアに内包されたＢＳＡの定量（内包率の測定）を行った。 The above 6 sample tubes were capped and stirred with a stirrer for about 2 days. Two days later, the solution became transparent and it was visually confirmed that the biodegradable nanospheres were decomposed. Then, each solution was filtered using a disposable filter. A calibration curve was prepared by measuring absorbance at a wavelength of 290 nm using a solution obtained by decomposing empty biodegradable nanospheres having a BSA concentration of 0 mg / ml as a background. And the light absorbency of the solution which decomposed | disassembled the biodegradable nanosphere which included BSA was measured, and quantitative determination (measurement of the encapsulation rate) of BSA included in the biodegradable nanosphere was performed from the created calibration curve.
　図６は、実施例における内包率、回収率、収率および内包効率を算出する式を説明するための説明図である。ここで内包率は、得られた単位重量あたりのナノスフェアに対する内包された物質（本実施例では、親水性物質）の重量の比率（％）であり、回収率は、用いられた内包される物質（本実施例では、親水性物質）に対する回収された内包される物質（本実施例では、親水性物質）の比率（％）であり、収率は、ナノスフェアの構成成分として用いられた内包される物質（本実施例では、親水性物質）、両親媒性ポリマー、疎水性ポリマーおよび親水性ポリマーの重量に対する回収されたナノスフェアの重量の比率（％）であり、内包効率は、用いられた内包される物質（本実施例では、親水性物質）と用いられたポリマーの合計との割合に対する、内包される親水性物質もしくは疎水性物質のいずれか一方または両方と回収したナノスフェア中のポリマーとの割合、の比を百分率（％）で示す。 FIG. 6 is an explanatory diagram for explaining formulas for calculating the inclusion rate, recovery rate, yield, and inclusion efficiency in the examples. Here, the encapsulation rate is the ratio (%) of the weight of the encapsulated substance (in this embodiment, hydrophilic substance) to the nanospheres per unit weight obtained, and the recovery rate is the encapsulated substance used. It is the ratio (%) of the collected encapsulated substance (in this example, hydrophilic substance) to the (in this example, hydrophilic substance), and the yield is the encapsulated used as a component of the nanosphere. The ratio of the recovered nanosphere weight to the weight of the substance (hydrophilic substance in this example), amphiphilic polymer, hydrophobic polymer and hydrophilic polymer, and the encapsulation efficiency is the encapsulated efficiency used. Nanospheres recovered with either or both of the included hydrophilic substance and / or hydrophobic substance with respect to the ratio of the substance to be used (hydrophilic substance in this example) and the total of the polymers used Shows the proportion of the polymer, the ratio of a percentage (%).
　図６に示す、内包率を算出する式を用いて、実施例１の生分解性ナノスフェアのＢＳＡの内包率を算出すると、２．９％となった。 Using the equation for calculating the encapsulation rate shown in FIG. 6, the BSA encapsulation rate of the biodegradable nanosphere of Example 1 was 2.9%.
　また、懸濁液を２００００ｒｐｍで１０分間遠心分離した結果、得られた上澄におけるＢＳＡの濃度から、図６に示す、回収率を算出する式を用いて、実施例１におけるＢＳＡの回収率を求めた。その結果、実施例１におけるＢＳＡの回収率は、９１．０％となった。 Further, as a result of centrifuging the suspension at 20000 rpm for 10 minutes, the BSA recovery rate in Example 1 was calculated using the formula for calculating the recovery rate shown in FIG. 6 from the BSA concentration in the obtained supernatant. Asked. As a result, the recovery rate of BSA in Example 1 was 91.0%.
　ＢＳＡの回収率が９０％以上と高いことから、医薬品等の高価な物質を内包させる場合であっても、好適に医薬品等の高価な物質を回収することができる。したがって、回収した物質を再利用することができ、薬価の不要な上昇を抑えることが可能となる。 Since the recovery rate of BSA is as high as 90% or more, even when expensive substances such as pharmaceuticals are included, it is possible to suitably recover expensive substances such as pharmaceuticals. Therefore, the collected substance can be reused, and an unnecessary increase in drug price can be suppressed.
（実施例２）
　本実施例では、両親媒性ポリマーとして数平均分子量３０００のポリエチレングリコールとＬ－乳酸のジブロック共重合体（以下、単に、ＰＥＧ３Ｋ－ｂ－ＰＬＬＡと称する）を、疎水性ポリマーとして、ＰＬＧＡを、親水性ポリマーとしてＰＶＡを、内包する物質は、親水性物質および疎水性物質の機能を備える両親媒性物質としてスフィンゴミエリンを用いた。 (Example 2)
In this example, a diblock copolymer of polyethylene glycol having a number average molecular weight of 3000 and L-lactic acid (hereinafter simply referred to as PEG3K-b-PLLA) as an amphiphilic polymer, PLGA as a hydrophobic polymer, Sphingomyelin was used as an amphiphilic substance having functions of a hydrophilic substance and a hydrophobic substance as a substance encapsulating PVA as a hydrophilic polymer.
　図７は、実施例２にかかるＰＥＧ３Ｋ－ｂ－ＰＬＬＡの合成方法を説明するための説明図である。 FIG. 7 is an explanatory diagram for explaining a method of synthesizing PEG3K-b-PLLA according to Example 2.
　図７に示すように、まず、Ｌ－ラクチド、ＭｅＯ－ＰＥＧ３Ｋ、２－エチルヘキサン酸スズをそれぞれ秤量し、ナス型フラスコにいれ、攪拌子もいれて減圧乾燥を行った。乾燥後、ナス型フラスコを１５０℃のｏｉｌ　ｂａｔｈに浸漬して内容物を溶融させ、ナス型フラスコを１３０℃のｏｉｌ　ｂａｔｈに１２時間浸漬することで開環重合反応を行った。反応後、反応物を少量のクロロホルムに溶解させ、大量のエーテルにより再沈殿を行った。これを遠心分離にかけ、沈殿物を減圧乾燥することでＰＥＧ３Ｋ－ｂ－ＰＬＬＡを得た。 As shown in FIG. 7, first, L-lactide, MeO-PEG3K, and tin 2-ethylhexanoate were weighed, placed in an eggplant-shaped flask, and stirred under a vacuum to dry under reduced pressure. After drying, the eggplant-shaped flask was immersed in an oil bath at 150 ° C. to melt the contents, and the eggplant-shaped flask was immersed in an oil bath at 130 ° C. for 12 hours to perform a ring-opening polymerization reaction. After the reaction, the reaction product was dissolved in a small amount of chloroform and reprecipitated with a large amount of ether. This was centrifuged and the precipitate was dried under reduced pressure to obtain PEG3K-b-PLLA.
　図８は、実施例２を説明するための説明図である。図８に示すように、試験管に、ＰＥＧ３Ｋ－ｂ－ＰＬＬＡ２０ｍｇおよび重量平均分子量が５０００～７５０００であるＰＬＧＡ１８０ｍｇを入れ、クロロホルム２．５ｍｌに溶解させた。続いて、スフィンゴミエリン５ｍｇを試験管に加え１次溶液を生成した。次に、１次溶液をスパチュラで激しく撹拌しながらプローブ型ソニケータで３分間超音波を照射し、１次エマルションを調製した。 FIG. 8 is an explanatory diagram for explaining the second embodiment. As shown in FIG. 8, 20 mg of PEG3K-b-PLLA and 180 mg of PLGA having a weight average molecular weight of 5000-75000 were placed in a test tube and dissolved in 2.5 ml of chloroform. Subsequently, 5 mg of sphingomyelin was added to the test tube to produce a primary solution. Next, while the primary solution was vigorously stirred with a spatula, ultrasonic irradiation was performed with a probe sonicator for 3 minutes to prepare a primary emulsion.
　その後、１次エマルションを、０．１％ＰＶＡを含むＰＢＳ３００ｍｌに加えて２次溶液を調製した。次に、２次溶液にバス型ソニケータで３分間超音波を照射し、９０秒間スパチュラで激しく攪拌し、さらに、プローブ型ソニケータで９０秒間超音波を照射して２次エマルションを調製した。 Thereafter, the primary emulsion was added to 300 ml of PBS containing 0.1% PVA to prepare a secondary solution. Next, the secondary solution was irradiated with ultrasonic waves for 3 minutes with a bath sonicator, stirred vigorously with a spatula for 90 seconds, and further irradiated with ultrasonic waves for 90 seconds with a probe sonicator to prepare a secondary emulsion.
　そして、２次エマルションを、０．１％ＰＶＡを含むＰＢＳ３００ｍｌにさらに加え、液中乾燥を行うことにより、クロロホルムを除去した懸濁液を得た。その後、懸濁液を２００００ｒｐｍで１０分間遠心分離し、両親媒性物質としてのスフィンゴミエリンを内包する生分解性ナノスフェアを含む沈殿を得た。そして、かかる沈殿を超純水で洗浄した。この、遠心分離および洗浄からなる操作を数回繰り返した後、沈殿を凍結乾燥して水を除去することで、スフィンゴミエリンを内包する生分解性ナノスフェアを得た。 Then, the secondary emulsion was further added to 300 ml of PBS containing 0.1% PVA and dried in liquid to obtain a suspension from which chloroform was removed. Thereafter, the suspension was centrifuged at 20000 rpm for 10 minutes to obtain a precipitate containing biodegradable nanospheres encapsulating sphingomyelin as an amphiphilic substance. The precipitate was washed with ultrapure water. After repeating this operation consisting of centrifugation and washing several times, the precipitate was freeze-dried to remove water, thereby obtaining biodegradable nanospheres containing sphingomyelin.
（評価）
　スフィンゴミエリンを内包する生分解性ナノスフェアの形状および粒径を、走査型電子顕微鏡を用いて検討した。走査型電子顕微鏡の試料の作成方法は、上述した実施例１の作成方法と同一であるため説明を省略する。 (Evaluation)
The shape and particle size of the biodegradable nanosphere encapsulating sphingomyelin were examined using a scanning electron microscope. Since the method for preparing the sample of the scanning electron microscope is the same as the method for preparing the first embodiment, the description thereof will be omitted.
　図９は、スフィンゴミエリンを内包する生分解性ナノスフェアを、走査型電子顕微鏡で観察した結果を説明するための説明図である。図９に示すように、スフィンゴミエリンを内包する生分解性ナノスフェアの粒径は、比較的凝集も抑えられており微粒子表面も、平滑なものが得られた。 FIG. 9 is an explanatory diagram for explaining the results of observing biodegradable nanospheres containing sphingomyelin with a scanning electron microscope. As shown in FIG. 9, the particle size of the biodegradable nanosphere encapsulating sphingomyelin was relatively suppressed from aggregation and a fine particle surface was obtained.
　また、懸濁液を２００００ｒｐｍで１０分間遠心分離した結果、得られた上澄におけるスフィンゴミエリンの濃度から、図６に示す、収率を算出する式を用いて、実施例２におけるスフィンゴミエリンの収率を求めた。その結果、実施例２におけるスフィンゴミエリンの収率は、５３．７％となった。 Further, as a result of centrifuging the suspension at 20000 rpm for 10 minutes, the concentration of sphingomyelin in Example 2 was calculated from the concentration of sphingomyelin in the obtained supernatant using the formula for calculating the yield shown in FIG. The rate was determined. As a result, the yield of sphingomyelin in Example 2 was 53.7%.
　実施例２において、親水性物質だけでなく、親水性物質および疎水性物質の機能を備える両親媒性物質のスフィンゴミエリンを内包したナノスフェアを製造することができた。したがって、親水性物質だけでなく、親水性物質および疎水性物質を内包するナノスフェアを製造することが可能となった。また、スフィンゴミエリンを内包する高分子微粒子は、ナノメートルオーダーであるため、静脈または動脈注射用の医薬品、経皮吸収用の医薬品や化粧料等の皮膚外用組成物に利用することができ、薬物の全身投与、患部へのターゲッティングおよび皮膚への浸透が可能となる。 In Example 2, nanospheres including not only hydrophilic substances but also amphiphile sphingomyelin having functions of hydrophilic substances and hydrophobic substances could be produced. Therefore, it has become possible to produce nanospheres that contain not only hydrophilic substances but also hydrophilic substances and hydrophobic substances. In addition, since the fine polymer particles encapsulating sphingomyelin are on the order of nanometers, they can be used for pharmaceutical preparations for intravenous or arterial injection, pharmaceutical preparations for percutaneous absorption, cosmetic preparations, etc. Can be administered systemically, targeted to the affected area, and penetrated into the skin.
　また、スフィンゴミエリンのセラミドに基づく保湿効果により、スフィンゴミエリンを内包するナノスフェアを含有する皮膚外用組成物または化粧料を製造すれば、皮膚外用組成物または化粧料を皮膚表面の微細な凹凸にナノスフェアを略均一に塗布することができるため、皮膚外用組成物または化粧料が有する保湿効果を満遍なく皮膚に与えることが可能となる。また、上述したようにナノスフェアを構成するポリマーに生分解性ポリマーを用いる構成により、スフィンゴミエリンを皮膚表面で好適に徐放させることができ、従来と比較して皮膚外用組成物または化粧料の塗布回数を減らしても同程度の保湿効果を得ることが可能となる。 In addition, if a skin external composition or cosmetic containing nanospheres containing sphingomyelin is produced by the moisturizing effect based on ceramide of sphingomyelin, the skin external composition or cosmetic can be applied to fine irregularities on the skin surface. Since it can be applied substantially uniformly, the moisturizing effect of the external composition for skin or cosmetic can be evenly applied to the skin. Further, as described above, by using a biodegradable polymer as the polymer constituting the nanosphere, sphingomyelin can be suitably and slowly released on the skin surface, and the application of a composition for external skin or cosmetic compared to the conventional case Even if the number of times is reduced, a similar moisturizing effect can be obtained.
（実施例３）
　本実施例では、両親媒性ポリマーとしてＤｅｘ－ｇ－ＰＬＬＡを、親水性ポリマーとしてＰＶＡを、内包する物質は、親水性物質および疎水性物質の混合物としてステビア発酵エキス（stevia ferment extract）を用い、疎水性ポリマーは使用しなかった。 (Example 3)
In this example, Dex-g-PLLA is used as an amphiphilic polymer, PVA is used as a hydrophilic polymer, and a stevia ferment extract is used as a mixture of a hydrophilic substance and a hydrophobic substance as an encapsulating substance, Hydrophobic polymer was not used.
　図１０は、実施例３にかかるＤｅｘ－ｇ－ＰＬＬＡの合成方法を説明するための説明図である。 FIG. 10 is an explanatory diagram for explaining the Dex-g-PLLA synthesis method according to the third embodiment.
　図１０（ａ）に示すように、まず、トリメチルシリル（ＴＭＳ）化デキストラン（ＴＭＳＤｅｘ）を合成した。合成前日にデキストランの減圧乾燥を行った。アルゴンで満たして常圧に戻し、デキストランを取り出した。デキストランをホルムアミドに溶解させ、８０℃に加熱し、ヘキサメチルジシラザンを攪拌しながら注いだ。 As shown in FIG. 10 (a), first, trimethylsilyl (TMS) dextran (TMSDex) was synthesized. The day before synthesis, dextran was dried under reduced pressure. It was filled with argon and returned to normal pressure, and dextran was taken out. Dextran was dissolved in formamide, heated to 80 ° C., and hexamethyldisilazane was poured with stirring.
　２時間後分液漏斗を用い抽出し、濃縮して、さらに減圧乾燥した。生成物をクロロホルムに溶解させ、メタノールを貧溶媒とした再沈殿を行い精製した。沈殿物は超遠心分離を行い回収し、減圧乾燥した。 After 2 hours, the mixture was extracted using a separatory funnel, concentrated, and dried under reduced pressure. The product was dissolved in chloroform and purified by reprecipitation using methanol as a poor solvent. The precipitate was collected by ultracentrifugation and dried under reduced pressure.
　図１０（ｂ）に示すように、まず、合成前日にＬ－ラクチドと図１０（ａ）で得られたＴＭＳＤｅｘの減圧乾燥をそれぞれ行なった。Ｌ－ラクチドはクライゼンフラスコに秤量し、常温で直引減圧乾燥した。水酸基をカリウムナフタレンによりアルコキシド化し、これを開始剤としてラクチドの溶液系アニオン開環重合を行った。 As shown in FIG. 10 (b), L-lactide and TMSDex obtained in FIG. 10 (a) were first dried under reduced pressure on the day before the synthesis. L-lactide was weighed into a Claisen flask and directly drawn under reduced pressure at room temperature. The hydroxyl group was alkoxided with potassium naphthalene, and this was used as an initiator for solution-based anionic ring-opening polymerization of lactide.
　次に、ＴＭＳ基の脱保護を行った。ＴＭＳＤｅｘ－ｇ－ＰＬＬＡを、ＤＭＳＯ（Dimethylsulfoxide）を加えてスターラーで攪拌し溶解した。ＴＭＳＤｅｘ－ｇ－ＰＬＬＡが完全に溶解したことを確認してからメタノールを加えた。そして、酢酸を加え４時間攪拌した。４時間後、メタノールを貧溶媒とした再沈殿を行ない、超遠心分離を行い、沈殿を回収し、デシケータ内で減圧乾燥した。さらなる精製は糖含有率により、良溶媒にＤＭＳＯまたはクロロホルム、貧溶媒にメタノールを用いた再沈殿により行なった。これを数回繰り返し、Ｄｅｘ－ｇ－ＰＬＬＡを得た。 Next, the TMS group was deprotected. TMSDex-g-PLLA was dissolved by adding DMSO (Dimethylsulfoxide) and stirring with a stirrer. After confirming that TMSDex-g-PLLA was completely dissolved, methanol was added. Then, acetic acid was added and stirred for 4 hours. After 4 hours, reprecipitation using methanol as a poor solvent was performed, ultracentrifugation was performed, and the precipitate was collected and dried under reduced pressure in a desiccator. Further purification was performed by reprecipitation using DMSO or chloroform as a good solvent and methanol as a poor solvent, depending on the sugar content. This was repeated several times to obtain Dex-g-PLLA.
　図１１は、実施例３にかかるＤｅｘ－ｇ－ＰＬＬＡの分解性を説明するための説明図であり、図１２は、分解性実験に用いたＤｅｘ－ｇ－ＰＬＬＡを説明するための説明図である。ここでは、クロロホルムを用いたキャスト法で作成したＤｅｘ－ｇ－ＰＬＬＡフィルムおよびＰＬＬＡフィルムの質量を電子天秤で量り、ＰＢＳに浸漬して３７℃でインキュベートし、１日後、２日後、４日後、７日後、１４日後、２８日後に取り出し、水分を減圧乾燥により除去した後の質量を測定した。分解前のフィルムの重さと分解後のフィルムの重さから、図１１（ａ）に示す式を用いてフィルムの重量減少率を求めた。また、取り出したサンプルの一部をＤＭＦで溶解し、ＧＰＣを用いた分子量分布測定を行い数平均分子量の減少について検討した。分解前のＤｅｘ－ｇ－ＰＬＬＡの数平均分子量と分解後のＤｅｘ－ｇ－ＰＬＬＡの数平均分子量から、図１１（ｃ）に示す式を用いてＤｅｘ－ｇ－ＰＬＬＡの分子量減少率を求めた。 FIG. 11 is an explanatory diagram for explaining the degradability of Dex-g-PLLA according to Example 3, and FIG. 12 is an explanatory diagram for explaining Dex-g-PLLA used in the decomposability experiment. is there. Here, the mass of the Dex-g-PLLA film and PLLA film prepared by the casting method using chloroform was weighed with an electronic balance, immersed in PBS, incubated at 37 ° C., 1 day, 2 days, 4 days, 7 days After 14 days and after 28 days, the mass was measured after removing moisture by drying under reduced pressure. From the weight of the film before decomposition and the weight of the film after decomposition, the weight reduction rate of the film was determined using the formula shown in FIG. Further, a part of the sample taken out was dissolved in DMF, and molecular weight distribution measurement using GPC was performed to examine the reduction of the number average molecular weight. From the number average molecular weight of Dex-g-PLLA before decomposition and the number average molecular weight of Dex-g-PLLA after decomposition, the molecular weight reduction rate of Dex-g-PLLA was determined using the formula shown in FIG. .
　図１１（ｂ）に示すように、ＰＬＬＡと比較して、Ｄｅｘ－ｇ－ＰＬＬＡは、フィルムの重量減少率が高い。また、Ｄｅｘ－ｇ－ＰＬＬＡにおけるＬ－ラクチドの重合度が低い方が、フィルムの重量減少率が高くなった。 As shown in FIG. 11B, Dex-g-PLLA has a higher weight reduction rate than the PLLA. In addition, the lower the degree of polymerization of L-lactide in Dex-g-PLLA, the higher the weight reduction rate of the film.
　また、図１１（ｄ）に示すように、ＰＬＬＡと比較して、Ｄｅｘ－ｇ－ＰＬＬＡは、分子量減少率が低い。また、Ｄｅｘ－ｇ－ＰＬＬＡにおける糖含有率が高い方が、分子量減少率が高い。したがって、両親媒性ポリマーとして、Ｄｅｘ－ｇ－ＰＬＬＡにおけるＬ－ラクチドの重合度または糖含有率を調節することで、生分解性ナノスフェアを生体内に留置した場合に、分解に伴う炎症反応等を起こさない分解速度であり、かつ、生体内で必要とされる期間の経過後には生体内から消失している生分解速度に調節することができる。 In addition, as shown in FIG. 11 (d), Dex-g-PLLA has a lower molecular weight reduction rate than PLLA. Further, the higher the sugar content in Dex-g-PLLA, the higher the molecular weight reduction rate. Therefore, by adjusting the polymerization degree or sugar content of L-lactide in Dex-g-PLLA as an amphiphilic polymer, when biodegradable nanospheres are placed in vivo, the inflammatory reaction associated with the degradation can be reduced. It can be adjusted to a biodegradation rate that does not occur and disappears from the living body after a lapse of a period required in the living body.
　図１３は、実施例３を説明するための説明図である。図１３に示すように、試験管に、Ｄｅｘ－ｇ－ＰＬＬＡであるＧ４５－１２－２０またはＧ８０－９－１６を、２００ｍｇを入れ、クロロホルム（ＣＨＣｌ３）２．４ｍｌに溶解させた。続いて、ステビア発酵エキス５ｍｇを試験管に加え１次溶液を生成した。次に、１次溶液をスパチュラで激しく撹拌しながらバス型ソニケータで５分間超音波を照射し、１次エマルションを調製した。 FIG. 13 is an explanatory diagram for explaining the third embodiment. As shown in FIG. 13, 200 mg of G45-12-20 or G80-9-16, which is Dex-g-PLLA, was put in a test tube and dissolved in 2.4 ml of chloroform (CHCl 3 ). Subsequently, 5 mg of stevia fermented extract was added to the test tube to produce a primary solution. Next, a primary emulsion was prepared by irradiating ultrasonic waves with a bath sonicator for 5 minutes while stirring the primary solution vigorously with a spatula.
　その後、１次エマルションを、０．１％ＰＶＡを含むＰＢＳ３００ｍｌに加えて２次溶液を調製した。次に、２次溶液にプローブ型ソニケータで３分間超音波を照射して２次エマルションを調製した。 Thereafter, the primary emulsion was added to 300 ml of PBS containing 0.1% PVA to prepare a secondary solution. Next, a secondary emulsion was prepared by irradiating the secondary solution with ultrasonic waves for 3 minutes using a probe sonicator.
　そして、２次エマルションを、０．１％ＰＶＡを含むＰＢＳ３００ｍｌにさらに加え、液中乾燥を行うことにより、クロロホルムを除去した懸濁液を得た。その後、懸濁液を２００００ｒｐｍで１０分間遠心分離し、ステビア発酵エキスを内包する生分解性ナノスフェアを含む沈殿を得た。そして、かかる沈殿を超純水で洗浄した。この、遠心分離および洗浄からなる操作を数回繰り返した後、沈殿を凍結乾燥して水を除去することで、ステビア発酵エキスを内包する生分解性ナノスフェアを得た。 Then, the secondary emulsion was further added to 300 ml of PBS containing 0.1% PVA and dried in liquid to obtain a suspension from which chloroform was removed. Thereafter, the suspension was centrifuged at 20000 rpm for 10 minutes to obtain a precipitate containing biodegradable nanospheres containing stevia fermentation extract. The precipitate was washed with ultrapure water. After repeating this operation consisting of centrifugation and washing several times, the precipitate was freeze-dried and water was removed to obtain biodegradable nanospheres containing stevia fermentation extract.
（評価）
　ステビア発酵エキスを内包する生分解性ナノスフェアの形状および粒径を、走査型電子顕微鏡を用いて検討した。走査型電子顕微鏡の試料の作成方法は、上述した実施例１の作成方法と同一であるため説明を省略する。 (Evaluation)
The shape and particle size of the biodegradable nanospheres containing the stevia fermented extract were examined using a scanning electron microscope. Since the method for preparing the sample of the scanning electron microscope is the same as the method for preparing the first embodiment, the description thereof will be omitted.
　図１４は、ステビア発酵エキスを内包する生分解性ナノスフェアを、走査型電子顕微鏡で観察した結果を説明するための説明図である。図１４に示すように、ステビア発酵エキスを内包する生分解性ナノスフェアの粒径は、比較的凝集も抑えられており微粒子表面も、平滑なものが得られた。 FIG. 14 is an explanatory diagram for explaining the result of observing the biodegradable nanosphere containing the stevia fermented extract with a scanning electron microscope. As shown in FIG. 14, the particle size of the biodegradable nanosphere encapsulating the stevia fermented extract was relatively suppressed from aggregation and a fine particle surface was obtained.
　また、懸濁液を２００００ｒｐｍで１０分間遠心分離した結果、得られた上澄におけるステビア発酵エキスの濃度から、図６に示す、収率を算出する式を用いて、実施例３におけるステビア発酵エキスの収率を求めた。その結果、実施例３におけるステビア発酵エキスの収率は、Ｇ４５－１２－２０で６５．８％、Ｇ８０－９－１６で６３．７％となった。 Further, as a result of centrifuging the suspension at 20000 rpm for 10 minutes, the stevia fermented extract in Example 3 was calculated from the concentration of stevia fermented extract in the obtained supernatant using the formula for calculating the yield shown in FIG. The yield of was determined. As a result, the yield of stevia fermented extract in Example 3 was 65.8% for G45-12-20 and 63.7% for G80-9-16.
　実施例３において、親水性物質だけでなく、親水性物質および疎水性物質の混合物であるステビア発酵エキスを内包したナノスフェアを製造することができた。したがって、親水性物質だけでなく、親水性物質および疎水性物質の混合物を内包するナノスフェアを製造することが可能となった。また、ステビア発酵エキスを内包する高分子微粒子は、ナノメートルオーダーであるため、静脈または動脈注射用の医薬品、経皮吸収用の医薬品や化粧料等の皮膚外用組成物に利用することができ、薬物の全身投与、患部へのターゲッティングおよび皮膚への浸透が可能となる。 In Example 3, it was possible to produce nanospheres containing not only hydrophilic substances but also stevia fermented extract, which is a mixture of hydrophilic substances and hydrophobic substances. Therefore, it has become possible to produce nanospheres that include not only hydrophilic substances but also mixtures of hydrophilic substances and hydrophobic substances. In addition, the fine polymer particles encapsulating the stevia fermented extract are on the order of nanometers, and therefore can be used for pharmaceutical preparations for intravenous or arterial injection, pharmaceutical preparations for transdermal absorption, cosmetic compositions such as cosmetics, Systemic administration of drugs, targeting to affected areas and penetration into the skin are possible.
　また、ステビア発酵エキスの回収率が６０％以上と高いことから、医薬品や、皮膚外用組成物、化粧料、に利用される有効物質等の高価な物質を内包させる場合であっても、好適に有効成分等の高価な物質を回収することができる。したがって、回収した物質を再利用することができ、薬価の不要な上昇を抑えることが可能となる。 In addition, since the recovery rate of stevia fermented extract is as high as 60% or more, it is suitable even when encapsulating expensive substances such as effective substances used in pharmaceuticals, compositions for external use of skin, and cosmetics. Expensive substances such as active ingredients can be recovered. Therefore, the collected substance can be reused, and an unnecessary increase in drug price can be suppressed.
　また、ステビア発酵エキスは、保湿効果が高く、荒れ肌を改善したり、かゆみを抑止したり、炎症を抑えたり、抗ヒスタミン作用を有したりする。したがって、ステビア発酵エキスを内包するナノスフェアを含有する皮膚外用組成物または化粧料を製造すれば、皮膚表面の微細な凹凸にステビア発酵エキスを略均一に行き渡らせることができ、ステビア発酵エキスの保湿効果、荒れ肌改善効果、かゆみ抑止効果、炎症抑止効果および抗ヒスタミン効果を皮膚表面に満遍なく塗布することが可能となる。 Stevia fermented extract has a high moisturizing effect, improves rough skin, suppresses itching, suppresses inflammation, and has an antihistaminic action. Therefore, if a composition for external skin or cosmetics containing nanospheres containing stevia fermented extract is manufactured, stevia fermented extract can be distributed almost evenly on the fine irregularities of the skin surface, and the moisturizing effect of stevia fermented extract It is possible to uniformly apply rough skin improving effect, itching inhibiting effect, inflammation inhibiting effect and antihistamine effect to the skin surface.
（実施例４）
　本実施例では、両親媒性ポリマーとしてＤｅｘ－ｇ－ＰＬＬＡを、疎水性ポリマーとしてＥｔＯ－ＰＬＬＡを、親水性ポリマーとしてＰＶＡを、内包する親水性物質としてＢＳＡを用いた。 Example 4
In this example, Dex-g-PLLA was used as the amphiphilic polymer, EtO-PLLA was used as the hydrophobic polymer, PVA was used as the hydrophilic polymer, and BSA was used as the hydrophilic substance to be included.
　図１５は、実施例４を説明するための説明図である。図１５に示すように、試験管に、ＥｔＯ－ＰＬＬＡを１７０ｍｇ、Ｄｅｘ－ｇ－ＰＬＬＡであるＧ７１－１３－１３（図１３（ｂ）参照）を、３０ｍｇを入れ、塩化メチレン（ＣＨ２Ｃｌ２）２．４ｍｌに溶解させた。続いて、ＢＳＡ５ｍｇを溶解させたＰＢＳ１００μｌを試験管に加え１次溶液を生成した。次に、１次溶液をスパチュラで激しく撹拌しながら、バス型ソニケータで５分間、プローブ型ソニケータで１．５分間超音波を照射し、１次エマルションを調製した。 FIG. 15 is an explanatory diagram for explaining the fourth embodiment. As shown in FIG. 15, 170 mg of EtO-PLLA and G71-13-13 which is Dex-g-PLLA (see FIG. 13B) were placed in a test tube, and methylene chloride (CH 2 Cl 2 ) It was dissolved in 2.4 ml. Subsequently, 100 μl of PBS in which 5 mg of BSA was dissolved was added to the test tube to form a primary solution. Next, while the primary solution was vigorously stirred with a spatula, ultrasonic waves were irradiated for 5 minutes with a bath sonicator and 1.5 minutes with a probe sonicator to prepare a primary emulsion.
　その後、１次エマルション８００μｌを、０．１％ＰＶＡを含むＰＢＳ５ｍｌに加えて２次溶液を調製した。次に、２次溶液にプローブ型ソニケータで１．５分間超音波を照射して２次エマルションを調製した。 Thereafter, 800 μl of the primary emulsion was added to 5 ml of PBS containing 0.1% PVA to prepare a secondary solution. Next, a secondary emulsion was prepared by irradiating the secondary solution with ultrasonic waves with a probe-type sonicator for 1.5 minutes.
　そして、２次エマルションを、０．１％ＰＶＡを含むＰＢＳ２００ｍｌに加え、１時間攪拌し、約４０℃で２時間液中乾燥することにより塩化メチレンを除去した懸濁液を得た。その後、懸濁液を２００００ｒｐｍで１０分間遠心分離し、ＢＳＡを内包する生分解性ナノスフェアを含む沈殿を得た。かかる沈殿を超純水で洗浄した。この、遠心分離および洗浄からなる操作を数回繰り返した後、沈殿を凍結乾燥して水を除去することで、ＢＳＡを内包する生分解性ナノスフェアを得た。 The secondary emulsion was added to 200 ml of PBS containing 0.1% PVA, stirred for 1 hour, and dried in liquid at about 40 ° C. for 2 hours to obtain a suspension from which methylene chloride was removed. Thereafter, the suspension was centrifuged at 20000 rpm for 10 minutes to obtain a precipitate containing biodegradable nanospheres containing BSA. The precipitate was washed with ultrapure water. After repeating this operation consisting of centrifugation and washing several times, the precipitate was freeze-dried to remove water, thereby obtaining biodegradable nanospheres containing BSA.
（評価）
　ＢＳＡを内包する生分解性ナノスフェアの形状および粒径を、走査型電子顕微鏡を用いて検討した。走査型電子顕微鏡の試料の作成方法は、上述した実施例１の作成方法と同一であるため説明を省略する。 (Evaluation)
The shape and particle size of the biodegradable nanosphere encapsulating BSA were examined using a scanning electron microscope. Since the method for preparing the sample of the scanning electron microscope is the same as the method for preparing the first embodiment, the description thereof will be omitted.
　図１６は、ＢＳＡを内包する生分解性ナノスフェアを、走査型電子顕微鏡で観察した結果を説明するための説明図である。図１６に示すように、ＢＳＡを内包する生分解性ナノスフェアの粒径は、約４００ｎｍ～約６００ｎｍであった。また、比較的凝集も抑えられており微粒子表面も、平滑なものが得られた。 FIG. 16 is an explanatory diagram for explaining the results of observation of biodegradable nanospheres containing BSA with a scanning electron microscope. As shown in FIG. 16, the particle size of the biodegradable nanosphere encapsulating BSA was about 400 nm to about 600 nm. Further, aggregation was relatively suppressed, and a fine particle surface was obtained.
　実施例４におけるナノスフェアの粒径が約４００ｎｍ～約６００ｎｍと小さいため、親水性物質を内包した生分解性ナノスフェアを、静脈または動脈注射用の医薬品、経皮吸収用の医薬品や化粧料等の皮膚外用組成物に利用することができ、薬物の全身投与、患部へのターゲッティングおよび皮膚への浸透が可能となる。 Since the particle size of the nanosphere in Example 4 is as small as about 400 nm to about 600 nm, the biodegradable nanosphere encapsulating a hydrophilic substance is used as a skin for pharmaceuticals for intravenous or arterial injection, pharmaceuticals for transdermal absorption, cosmetics, etc. It can be used as an external composition, and enables systemic administration of drugs, targeting of affected areas, and penetration into the skin.
　次に、生分解性ナノスフェアのＢＳＡ内包率およびＢＳＡの回収率を測定した。まず、サンプル管を６個用意し、そのうち５個のサンプル管には内包物なしの空の生分解性ナノスフェアを、残りの１個のサンプル管には内包率が未知である、ＢＳＡを内包した生分解性ナノスフェアを、それぞれ２０ｍｇ入れた。 Next, the BSA encapsulation rate and BSA recovery rate of the biodegradable nanospheres were measured. First, six sample tubes were prepared, of which five sample tubes contained empty biodegradable nanospheres without inclusions, and the remaining one sample tube contained BSA whose inclusion rate was unknown. 20 mg of each biodegradable nanosphere was added.
　空の生分解性ナノスフェアを入れた５個のサンプル管には、それぞれＢＳＡの濃度が０、０．１、０．１５、０．２３、０．３ｍｇ／ｍｌとなるように、ＢＳＡ含有１Ｎ水酸化ナトリウム水溶液を３ｍｌずつ入れ、ＢＳＡを内包した生分解性ナノスフェアを入れたサンプル管には、１Ｎ水酸化ナトリウム水溶液を３ｍｌ入れた。 Five sample tubes containing empty biodegradable nanospheres contain 1N water containing BSA so that the BSA concentrations are 0, 0.1, 0.15, 0.23, and 0.3 mg / ml, respectively. 3 ml each of an aqueous sodium oxide solution was placed, and 3 ml of a 1N aqueous sodium hydroxide solution was placed in a sample tube containing biodegradable nanospheres containing BSA.
　上記６個のサンプル管にキャップをして約２日間スターラーで攪拌した。２日後、溶液が透明になり、生分解性ナノスフェアが分解されたのを目視で確認した後、それぞれの溶液を、ディスポーサブルフィルターを用いて濾過した。ＢＳＡの濃度が０ｍｇ／ｍｌの空の生分解性ナノスフェアを分解した溶液をバックグラウンドとし、波長２９１ｎｍの吸光度を測定して、検量線を作成した。そして、ＢＳＡを内包した生分解性ナノスフェアを分解した溶液の吸光度を測定し、作成した検量線より、生分解性ナノスフェアに内包されたＢＳＡの定量（内包率の測定）を行った。 The above 6 sample tubes were capped and stirred with a stirrer for about 2 days. Two days later, the solution became transparent and it was visually confirmed that the biodegradable nanospheres were decomposed. Then, each solution was filtered using a disposable filter. A calibration curve was prepared by measuring the absorbance at a wavelength of 291 nm using a solution obtained by decomposing empty biodegradable nanospheres having a BSA concentration of 0 mg / ml as a background. And the light absorbency of the solution which decomposed | disassembled the biodegradable nanosphere which included BSA was measured, and quantitative determination (measurement of the encapsulation rate) of BSA included in the biodegradable nanosphere was performed from the created calibration curve.
　図６に示す、内包効率を算出する式を用いて、実施例４の生分解性ナノスフェアのＢＳＡの内包効率を算出すると、５２．９％となった。 Using the formula for calculating the encapsulation efficiency shown in FIG. 6, the BSA encapsulation efficiency of the biodegradable nanosphere of Example 4 was calculated to be 52.9%.
　また、懸濁液を２００００ｒｐｍで１０分間遠心分離した結果、得られた上澄におけるＢＳＡの濃度から、図６に示す、回収率を算出する式を用いて、実施例４におけるＢＳＡの回収率を求めた。その結果、実施例４におけるＢＳＡの回収率は、８２．７％となった。 Further, as a result of centrifuging the suspension at 20000 rpm for 10 minutes, the BSA recovery rate in Example 4 was calculated using the formula for calculating the recovery rate shown in FIG. 6 from the BSA concentration in the obtained supernatant. Asked. As a result, the recovery rate of BSA in Example 4 was 82.7%.
　ＢＳＡの回収率が８０％以上と高いことから、医薬品や、皮膚外用組成物、化粧料、に利用される有効成分等の高価な物質を内包させる場合であっても、好適に有効成分等の高価な物質を回収することができる。したがって、回収した物質を再利用することができ、薬価の不要な上昇を抑えることが可能となる。 Since the recovery rate of BSA is as high as 80% or more, even when expensive substances such as active ingredients used for pharmaceuticals, compositions for external use of skin, and cosmetics are encapsulated, the active ingredients etc. Expensive material can be recovered. Therefore, the collected substance can be reused, and an unnecessary increase in drug price can be suppressed.
　以上、添付図面を参照しながら本発明の好適な実施形態について説明したが、本発明はかかる実施形態に限定されないことは言うまでもない。当業者であれば、特許請求の範囲に記載された範疇内において、各種の変更例または修正例に想到し得ることは明らかであり、それらについても当然に本発明の技術的範囲に属するものと了解される。 The preferred embodiments of the present invention have been described above with reference to the accompanying drawings, but the present invention is not limited to such embodiments. It will be apparent to those skilled in the art that various changes and modifications can be made within the scope of the claims, and these are naturally within the technical scope of the present invention. Understood.
　なお、本明細書のナノスフェアの製造方法における各工程は、必ずしもフローチャートとして記載された順序に沿って時系列に処理する必要はなく、並列的あるいはサブルーチンによる処理を含んでもよい。 In addition, each process in the manufacturing method of the nanosphere of this specification does not necessarily need to process in time series along the order described as a flowchart, and may include the process by parallel or a subroutine.
　本発明は、例えば、タンパク質、核酸、薬物、造影剤等の親水性物質、もしくは、脂質、薬物等の疎水性物質のいずれか一方または両方を内包できるナノスフェアの製造方法、ナノスフェア、これを含有する皮膚外用組成物および化粧料に利用することができる。 The present invention includes, for example, a method for producing a nanosphere capable of enclosing one or both of a hydrophilic substance such as a protein, a nucleic acid, a drug and a contrast agent, or a hydrophobic substance such as a lipid and a drug. It can be used for an external composition for skin and cosmetics.

Claims (17)

  1. 　Ｗ／Ｏ／Ｗエマルション法を用いた、ナノスフェアの製造方法であって、
    　親水性物質を溶解した水系溶媒と、両親媒性ポリマーを溶解した水非混和性有機溶媒と、疎水性ポリマーを溶解した水非混和性有機溶媒とを混合し、１次溶液を生成する１次溶液生成ステップと、
    　前記１次溶液に超音波を照射して１次エマルションを生成する１次エマルション生成ステップと、
    　前記１次エマルションに、親水性ポリマーを溶解した水系溶媒を添加し、２次溶液を生成する２次溶液生成ステップと、
    　前記２次溶液に超音波を照射して２次エマルションを生成する２次エマルション生成ステップと、
    を含み、
    　前記ナノスフェアは、親水性物質を内包するナノスフェアの製造方法。     A method for producing nanospheres using a W / O / W emulsion method,
    A primary solution is produced by mixing a water-based solvent in which a hydrophilic substance is dissolved, a water-immiscible organic solvent in which an amphiphilic polymer is dissolved, and a water-immiscible organic solvent in which a hydrophobic polymer is dissolved. A solution generation step;
    A primary emulsion generating step of irradiating the primary solution with ultrasonic waves to generate a primary emulsion;
    A secondary solution generating step of adding an aqueous solvent in which a hydrophilic polymer is dissolved to the primary emulsion to generate a secondary solution;
    A secondary emulsion generating step of generating a secondary emulsion by irradiating the secondary solution with ultrasonic waves;
    Including
    The said nanosphere is a manufacturing method of the nanosphere which includes a hydrophilic substance.
  2. 　Ｗ／Ｏ／Ｗエマルション法を用いた、ナノスフェアの製造方法であって、
    　親水性物質を溶解した水系溶媒と、両親媒性ポリマーを溶解した水非混和性有機溶媒とを混合し、１次溶液を生成する１次溶液生成ステップと、
    　前記１次溶液に超音波を照射して１次エマルションを生成する１次エマルション生成ステップと、
    　前記１次エマルションに、親水性ポリマーを溶解した水系溶媒を添加し、２次溶液を生成する２次溶液生成ステップと、
    　前記２次溶液に超音波を照射して２次エマルションを生成する２次エマルション生成ステップと、
    を含み、
    　前記ナノスフェアは、親水性物質を内包するナノスフェアの製造方法。     A method for producing nanospheres using a W / O / W emulsion method,
    A primary solution generating step of mixing a water-based solvent in which a hydrophilic substance is dissolved and a water-immiscible organic solvent in which an amphiphilic polymer is dissolved to generate a primary solution;
    A primary emulsion generating step of irradiating the primary solution with ultrasonic waves to generate a primary emulsion;
    A secondary solution generating step of adding an aqueous solvent in which a hydrophilic polymer is dissolved to the primary emulsion to generate a secondary solution;
    A secondary emulsion generating step of generating a secondary emulsion by irradiating the secondary solution with ultrasonic waves;
    Including
    The said nanosphere is a manufacturing method of the nanosphere which includes a hydrophilic substance.
  3. 　Ｏ／Ｗエマルション法を用いた、ナノスフェアの製造方法であって、
    　疎水性物質を溶解した水非混和性有機溶媒と、両親媒性ポリマーを溶解した水非混和性有機溶媒と、疎水性ポリマーを溶解した水非混和性有機溶媒とを混合し、１次溶液を生成する１次溶液生成ステップと、
    　前記１次溶液に、親水性ポリマーを溶解した水系溶媒を添加し、２次溶液を生成する２次溶液生成ステップと、
    　前記２次溶液に超音波を照射してエマルションを生成するエマルション生成ステップと、
    を含み、
    　前記ナノスフェアは、疎水性物質を内包するナノスフェアの製造方法。     A method for producing nanospheres using an O / W emulsion method,
    A water-immiscible organic solvent in which a hydrophobic substance is dissolved, a water-immiscible organic solvent in which an amphiphilic polymer is dissolved, and a water-immiscible organic solvent in which a hydrophobic polymer is dissolved are mixed to form a primary solution. A primary solution generation step to generate;
    A secondary solution generating step of adding an aqueous solvent in which a hydrophilic polymer is dissolved to the primary solution to generate a secondary solution;
    An emulsion generating step of generating an emulsion by irradiating the secondary solution with ultrasonic waves;
    Including
    The said nanosphere is a manufacturing method of the nanosphere which includes a hydrophobic substance.
  4. 　Ｏ／Ｗエマルション法を用いた、ナノスフェアの製造方法であって、
    　疎水性物質を溶解した水非混和性有機溶媒と、両親媒性ポリマーを溶解した水非混和性有機溶媒とを混合し、１次溶液を生成する１次溶液生成ステップと、
    　前記１次溶液に、親水性ポリマーを溶解した水系溶媒を添加し、２次溶液を生成する２次溶液生成ステップと、
    　前記２次溶液に超音波を照射してエマルションを生成するエマルション生成ステップと、
    を含み、
    　前記ナノスフェアは、疎水性物質を内包するナノスフェアの製造方法。     A method for producing nanospheres using an O / W emulsion method,
    A primary solution generation step of mixing a water-immiscible organic solvent in which a hydrophobic substance is dissolved and a water-immiscible organic solvent in which an amphiphilic polymer is dissolved to generate a primary solution;
    A secondary solution generating step of adding an aqueous solvent in which a hydrophilic polymer is dissolved to the primary solution to generate a secondary solution;
    An emulsion generating step of generating an emulsion by irradiating the secondary solution with ultrasonic waves;
    Including
    The said nanosphere is a manufacturing method of the nanosphere which includes a hydrophobic substance.
  5. 　Ｗ／Ｏ／Ｗエマルション法を用いた、ナノスフェアの製造方法であって、
    　親水性物質を溶解した水系溶媒と、疎水性物質を溶解した水非混和性有機溶媒と、両親媒性ポリマーを溶解した水非混和性有機溶媒と、疎水性ポリマーを溶解した水非混和性有機溶媒とを混合し、１次溶液を生成する１次溶液生成ステップと、
    　前記１次溶液に超音波を照射して１次エマルションを生成する１次エマルション生成ステップと、
    　前記１次エマルションに、親水性ポリマーを溶解した水系溶媒を添加し、２次溶液を生成する２次溶液生成ステップと、
    　前記２次溶液に超音波を照射して２次エマルションを生成する２次エマルション生成ステップと、
    を含み、
    　前記ナノスフェアは、親水性物質および疎水性物質を内包するナノスフェアの製造方法。     A method for producing nanospheres using a W / O / W emulsion method,
    A water-based solvent in which a hydrophilic substance is dissolved, a water-immiscible organic solvent in which a hydrophobic substance is dissolved, a water-immiscible organic solvent in which an amphiphilic polymer is dissolved, and a water-immiscible organic in which a hydrophobic polymer is dissolved A primary solution generation step of mixing a solvent to generate a primary solution;
    A primary emulsion generating step of irradiating the primary solution with ultrasonic waves to generate a primary emulsion;
    A secondary solution generating step of adding an aqueous solvent in which a hydrophilic polymer is dissolved to the primary emulsion to generate a secondary solution;
    A secondary emulsion generating step of generating a secondary emulsion by irradiating the secondary solution with ultrasonic waves;
    Including
    The said nanosphere is a manufacturing method of the nanosphere which includes a hydrophilic substance and a hydrophobic substance.
  6. 　Ｗ／Ｏ／Ｗエマルション法を用いた、ナノスフェアの製造方法であって、
    　親水性物質を溶解した水系溶媒と、疎水性物質を溶解した水非混和性有機溶媒と、両親媒性ポリマーを溶解した水非混和性有機溶媒とを混合し、１次溶液を生成する１次溶液生成ステップと、
    　前記１次溶液に超音波を照射して１次エマルションを生成する１次エマルション生成ステップと、
    　前記１次エマルションに、親水性ポリマーを溶解した水系溶媒を添加し、２次溶液を生成する２次溶液生成ステップと、
    　前記２次溶液に超音波を照射して２次エマルションを生成する２次エマルション生成ステップと、
    を含み、
    　前記ナノスフェアは、親水性物質および疎水性物質を内包するナノスフェアの製造方法。     A method for producing nanospheres using a W / O / W emulsion method,
    A primary solution is produced by mixing a water-based solvent in which a hydrophilic substance is dissolved, a water-immiscible organic solvent in which a hydrophobic substance is dissolved, and a water-immiscible organic solvent in which an amphiphilic polymer is dissolved. A solution generation step;
    A primary emulsion generating step of irradiating the primary solution with ultrasonic waves to generate a primary emulsion;
    A secondary solution generating step of adding an aqueous solvent in which a hydrophilic polymer is dissolved to the primary emulsion to generate a secondary solution;
    A secondary emulsion generating step of generating a secondary emulsion by irradiating the secondary solution with ultrasonic waves;
    Including
    The said nanosphere is a manufacturing method of the nanosphere which includes a hydrophilic substance and a hydrophobic substance.
  7. 　前記両親媒性ポリマーは、親水性セグメントと疎水性セグメントとで構成される共重合体であり、
    　前記親水性セグメントは、電荷を有するアミノ酸を３個以上有するポリペプチド、数平均分子量５００～１０００００であるポリエチレングリコール、および、多糖、の群から選択される１または複数を含み、
    　前記疎水性セグメントは、生分解性ポリエステルを含む請求項１から６のいずれか１項に記載の方法。     The amphiphilic polymer is a copolymer composed of a hydrophilic segment and a hydrophobic segment,
    The hydrophilic segment includes one or more selected from the group consisting of a polypeptide having three or more charged amino acids, a polyethylene glycol having a number average molecular weight of 500 to 100,000, and a polysaccharide,
    The method according to claim 1, wherein the hydrophobic segment comprises a biodegradable polyester.
  8. 　前記アミノ酸は、正電荷を有するアミノ酸であるリジン、アルギニンおよびヒスチジンの群および負電荷を有するアミノ酸であるアスパラギン酸およびグルタミン酸の群から選択される１または複数のアミノ酸である請求項７に記載の方法。 The method according to claim 7, wherein the amino acid is one or more amino acids selected from the group of lysine, arginine and histidine which are positively charged amino acids and the group of aspartic acid and glutamic acid which are negatively charged amino acids. .
  9. 　前記多糖は、ヒアルロン酸、アミロース、プルラン、コンドロイチン、コンドロイチン硫酸、デキストラン、デキストラン硫酸、デルマタン硫酸、ケラタン硫酸、ヘパラン硫酸、キチン、キトサン、および、βグルカン、の群から選択される１または複数である請求項７に記載の方法。 The polysaccharide is one or more selected from the group of hyaluronic acid, amylose, pullulan, chondroitin, chondroitin sulfate, dextran, dextran sulfate, dermatan sulfate, keratan sulfate, heparan sulfate, chitin, chitosan, and β-glucan. The method of claim 7.
  10. 　前記両親媒性ポリマーにおける前記多糖の含有量が１～５０質量％である請求項７または５に記載の方法。 The method according to claim 7 or 5, wherein the content of the polysaccharide in the amphiphilic polymer is 1 to 50% by mass.
  11. 　前記生分解性ポリエステルの構成単位は、乳酸、グリコール酸、および、アミノ酸、の群から選択される１または複数である請求項７に記載の方法。 The method according to claim 7, wherein the structural unit of the biodegradable polyester is one or more selected from the group consisting of lactic acid, glycolic acid, and amino acid.
  12. 　請求項１から１１のいずれか１項に記載のナノスフェアの製造方法を用いて製造されたナノスフェア。 A nanosphere manufactured using the method of manufacturing a nanosphere according to any one of claims 1 to 11.
  13. 　前記親水性物質もしくは疎水性物質のいずれか一方または両方として、ステビア発酵エキスまたはスフィンゴミエリンを内包する請求項１２に記載のナノスフェア。 13. The nanosphere according to claim 12, which contains stevia fermented extract or sphingomyelin as one or both of the hydrophilic substance and the hydrophobic substance.
  14. 　請求項１から１１いずれか１項に記載のナノスフェアの製造方法を用いて製造されたナノスフェアを含有する皮膚外用組成物。 An external composition for skin containing nanospheres produced using the method for producing nanospheres according to any one of claims 1 to 11.
  15. 　前記親水性物質もしくは疎水性物質のいずれか一方または両方として、ステビア発酵エキスまたはスフィンゴミエリンを含有する請求項１４に記載の皮膚外用組成物。 The external composition for skin according to claim 14, containing stevia fermented extract or sphingomyelin as one or both of the hydrophilic substance and the hydrophobic substance.
  16. 　請求項１から１１いずれか１項に記載のナノスフェアの製造方法を用いて製造されたナノスフェアを含有する化粧料。 Cosmetics containing nanospheres produced using the method for producing nanospheres according to any one of claims 1 to 11.
  17. 　前記親水性物質もしくは疎水性物質のいずれか一方または両方として、ステビア発酵エキスまたはスフィンゴミエリンを含有する請求項１６に記載の化粧料。 The cosmetic according to claim 16, comprising stevia fermented extract or sphingomyelin as one or both of the hydrophilic substance and the hydrophobic substance.
PCT/JP2010/060630 2009-07-02 2010-06-23 Method for producing nanospheres, nanospheres, external composition for skin containing same, and cosmetic preparation WO2011001869A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2009-157496 2009-07-02
JP2009157496A JP5031001B2 (en) 2009-07-02 2009-07-02 Method for producing nanosphere, nanosphere, external composition for skin containing the same, and cosmetic

Publications (1)

Publication Number Publication Date
WO2011001869A1 true WO2011001869A1 (en) 2011-01-06

Family

ID=43410944

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2010/060630 WO2011001869A1 (en) 2009-07-02 2010-06-23 Method for producing nanospheres, nanospheres, external composition for skin containing same, and cosmetic preparation

Country Status (2)

Country Link
JP (1) JP5031001B2 (en)
WO (1) WO2011001869A1 (en)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109046194A (en) * 2018-08-21 2018-12-21 武汉佰玛生物科技有限公司 A kind of method that limonene emulsification prepares chitosan microball
WO2022185082A1 (en) * 2021-03-05 2022-09-09 Uea Enterprises Limited Functionalised biodegradable polyester polymers
CN115645545A (en) * 2022-09-13 2023-01-31 浙江大学 Preparation method and application of red cell membrane-wrapped hair nano-particles modified by cRGD polypeptide

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2014051464A (en) * 2012-09-07 2014-03-20 Panasonic Corp Bio-active molecule encapsulating particle, bio-active pharmaceutical drug, method for permeation of bio-active molecule encapsulating particle into living body, and dissipating apparatus of bio-active molecule encapsulating particle and air cleaning device
EP4299058A3 (en) * 2014-06-24 2024-03-27 The Trustees of Princeton University Process for encapsulating soluble biologics, therapeutics, and imaging agents
JP7097566B2 (en) * 2018-02-27 2022-07-08 国立大学法人 鹿児島大学 Manufacturing method of colored resin fine particles
KR102182794B1 (en) * 2019-09-04 2020-11-25 (주)코스메디션 Method for preparing resveratrol and ferulic acid homogenizing composition using ultrasonic emulsification and high pressure gas injection

Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2001226294A (en) * 2000-02-09 2001-08-21 Nano Career Kk Method for producing polymer micelle containing included drug and polymer micelle composition
JP2003026812A (en) * 2001-07-13 2003-01-29 Nano Career Kk Method for producing medicament-containing polymeric micelle
JP2004285026A (en) * 2003-03-25 2004-10-14 Kao Corp Oil-in-water type multi-phase emulsified composition
WO2006057429A1 (en) * 2004-11-24 2006-06-01 Nanocarrier Co., Ltd. Method of changing morphology of block copolymer
JP2006321763A (en) * 2005-05-20 2006-11-30 Hosokawa Funtai Gijutsu Kenkyusho:Kk Biocompatibilie nanoparticle and method for production of the same
JP2007099631A (en) * 2005-09-30 2007-04-19 Hosokawa Funtai Gijutsu Kenkyusho:Kk Method for producing anionic medicine-encapsulated nanoparticle and pharmaceutical formulation using the same
WO2007145276A1 (en) * 2006-06-14 2007-12-21 Shalom Co., Ltd. Cosmetic and method of producing the same
WO2008126638A1 (en) * 2007-04-06 2008-10-23 Shalom Co., Ltd. Anti-histamine substance and method for production thereof
JP2009107941A (en) * 2007-10-26 2009-05-21 Hosokawa Funtai Gijutsu Kenkyusho:Kk Hair-restoring agent for scalp

Patent Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2001226294A (en) * 2000-02-09 2001-08-21 Nano Career Kk Method for producing polymer micelle containing included drug and polymer micelle composition
JP2003026812A (en) * 2001-07-13 2003-01-29 Nano Career Kk Method for producing medicament-containing polymeric micelle
JP2004285026A (en) * 2003-03-25 2004-10-14 Kao Corp Oil-in-water type multi-phase emulsified composition
WO2006057429A1 (en) * 2004-11-24 2006-06-01 Nanocarrier Co., Ltd. Method of changing morphology of block copolymer
JP2006321763A (en) * 2005-05-20 2006-11-30 Hosokawa Funtai Gijutsu Kenkyusho:Kk Biocompatibilie nanoparticle and method for production of the same
JP2007099631A (en) * 2005-09-30 2007-04-19 Hosokawa Funtai Gijutsu Kenkyusho:Kk Method for producing anionic medicine-encapsulated nanoparticle and pharmaceutical formulation using the same
WO2007145276A1 (en) * 2006-06-14 2007-12-21 Shalom Co., Ltd. Cosmetic and method of producing the same
WO2008126638A1 (en) * 2007-04-06 2008-10-23 Shalom Co., Ltd. Anti-histamine substance and method for production thereof
JP2009107941A (en) * 2007-10-26 2009-05-21 Hosokawa Funtai Gijutsu Kenkyusho:Kk Hair-restoring agent for scalp

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
NOUVEL C ET AL.: "Biodegradable nanoparticles made from polyactide-grafted dextran copolymers", JOURNAL OF COLLOID AND INTERFACE SCIENCE, vol. 330, no. 2, 15 February 2009 (2009-02-15), pages 337 - 343, XP025799226, DOI: doi:10.1016/j.jcis.2008.10.069 *
OUCHI T ET AL.: "Encapsulation and/or Release Behavior of Bovine Serum Albumin within and from Polylactide-Grafted Dextran Microspheres", MACROMOL BIOSCI, vol. 4, no. 4, 19 April 2004 (2004-04-19), pages 458 - 463, XP002697192, DOI: doi:10.1002/mabi.200300106 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109046194A (en) * 2018-08-21 2018-12-21 武汉佰玛生物科技有限公司 A kind of method that limonene emulsification prepares chitosan microball
WO2022185082A1 (en) * 2021-03-05 2022-09-09 Uea Enterprises Limited Functionalised biodegradable polyester polymers
CN115645545A (en) * 2022-09-13 2023-01-31 浙江大学 Preparation method and application of red cell membrane-wrapped hair nano-particles modified by cRGD polypeptide
CN115645545B (en) * 2022-09-13 2024-01-30 浙江大学 Preparation method and application of hair nanoparticle coated with cRGD polypeptide modified erythrocyte membrane

Also Published As

Publication number Publication date
JP2011011144A (en) 2011-01-20
JP5031001B2 (en) 2012-09-19

Similar Documents

Publication Publication Date Title
Afshar et al. Preparation and characterization of sodium alginate/polyvinyl alcohol hydrogel containing drug-loaded chitosan nanoparticles as a drug delivery system
JP5031001B2 (en) Method for producing nanosphere, nanosphere, external composition for skin containing the same, and cosmetic
Basu et al. Poly (lactic acid) based hydrogels
US11517538B2 (en) Shear-thinning self-healing networks
EP2642984B1 (en) Method for encapsulating particles
KR101399341B1 (en) amphiphilic triblock copolymer for accelerating percutaneous absorption, method for preparation the amphiphilic triblock copolymer, and cosmetic composition containing the amphiphilic triblock copolymer
Cavallaro et al. PHEA–PLA biocompatible nanoparticles by technique of solvent evaporation from multiple emulsions
Bos et al. Tissue reactions of in situ formed dextran hydrogels crosslinked by stereocomplex formation after subcutaneous implantation in rats
WO2019139380A1 (en) Vitamin c-containing polycaprolactone microsphere filler and preparation method therefor
Kulkarni et al. Synthesis of polymeric nanomaterials for biomedical applications
Mahajan et al. Development of grafted xyloglucan micelles for pulmonary delivery of curcumin: in vitro and in vivo studies
Zhang et al. In vivo inducing collagen regeneration of biodegradable polymer microspheres
Rong et al. Applications of polymeric nanocapsules in field of drug delivery systems
CN111298195A (en) Composite micron material, dermal filler, and preparation method and application thereof
CN104262638A (en) Hyaluronic acid-cystamine-polylactic acid-glycollic acid graft polymer and preparation method thereof
JP2013530235A (en) Compositions and methods for improving delivery of nucleic acids
Bertram et al. Functionalized poly (lactic-co-glycolic acid) enhances drug delivery and provides chemical moieties for surface engineering while preserving biocompatibility
Soni et al. Biodegradable block copolymers and their applications for drug delivery
Kang et al. In vivo release of bovine serum albumin from an injectable small intestinal submucosa gel
Papaneophytou et al. Polyhydroxyalkanoates applications in drug carriers
Lee et al. A biodegradable and biocompatible drug-delivery system based on polyoxalate microparticles
Kakade et al. Transfection activity of layer-by-layer plasmid DNA/poly (ethylenimine) films deposited on PLGA microparticles
Shrestha et al. Versatile use of nanosponge in the pharmaceutical arena: a mini-review
Snežana et al. Polymeric matrix systems for drug delivery
Jiang et al. Multiple Natural Polymers in Drug and Gene Delivery Systems

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 10794038

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 10794038

Country of ref document: EP

Kind code of ref document: A1