JP6000033B2 - Liposomes imparting heat resistance to encapsulated substance and method for producing the same - Google Patents

Description

  The present invention relates to a liposome encapsulating an easily heat-denaturing substance and imparting heat resistance, and a production method thereof.

  2. Description of the Related Art Conventionally, in the cosmetics field, it has been common to encapsulate active ingredients in liposomes as a means for delivering active ingredients having whitening, moisturizing and antioxidant effects to the skin. Liposomes are lipid bilayers or multilayers mainly composed of phospholipids, and have a structure similar to a cell membrane. Encapsulation of the active ingredient is often incorporated into the internal aqueous phase of liposomes for water-soluble drugs or incorporated into the lipid layer for fat-soluble drugs.

  Various methods are known for producing the liposome. For example, (1) prepared by dissolving lipid in an organic solvent, removing the solvent under reduced pressure to form a thin film, adding an aqueous solution such as a buffer solution to swell with shaking, and further peeling the thin film with a mechanical stirring means (2) A method in which a lipid is dissolved in an organic solvent such as ether or ethanol, poured into an aqueous solution such as a buffer solution, and then prepared by removing the solvent. Further, as a mechanical method, (3 ) Method by ultrasonic treatment (Patent Document 1), (4) Method by high-pressure homogenizer or high-speed rotary disperser (Patent Document 2), (5) Method by high-pressure filtration using a membrane filter made of polycarbonate, (6) Aqueous phase There is known a method in which an oil phase in which lipid is dissolved is prepared on the top of the lip, a droplet is introduced into the oil phase, and the droplet is dragged into the aqueous phase by centrifugal force. In addition, (7) a method in which lipid is dissolved in an organic solvent compatible with water such as ethanol and then replaced with water through a dialysis membrane (Patent Document 3).

  However, in the methods (1) and (2), the organic solvent remains, and in the method (3), the solution temperature rises due to ultrasonic irradiation, and degradation and denaturation of drugs and lipids are observed. It had drawbacks such as unevenness. The methods (4) and (5) also have drawbacks such as the risk of decomposition and oxidation of drugs and lipids because the treatment is performed at a temperature higher than the phase transition temperature of the lipid. Regarding (6), the liposome may be destroyed by mechanical stress due to the centrifuge. Furthermore, these methods have problems such as complicated operations and are not industrial, or require special devices. Although (7) is a simple method, there are still disadvantages such as the fact that the organic solvent remains or a high temperature is required to remove the organic solvent.

  On the other hand, many components that retain the original structure in nature are vulnerable to heat, and refrigeration or freezing storage is required during storage, or derivatization is necessary to improve heat resistance and stability. It was. For example, collagen, which is the most moisturizing ingredient blended as a cosmetic raw material, is a biologically derived raw material that originally has a triple helical structure when present in living organisms, but retains the triple helical structure. It is technically difficult to make the raw material as it is, and it has been made into a cosmetic raw material by acid treatment or heat treatment.

  Vitamin C has many functions as a cosmetic raw material such as whitening action, collagen synthesis promoting action, lipid peroxide inhibiting action, etc., but it is poorly oxidatively stable and easily colored, so it should be blended directly into a skin preparation. In the past, various derivatives have been developed (Patent Document 4).

JP 2005-41869 A Japanese Patent Laid-Open No. 11-139961 JP-A-01-224042 JP 2012-097030 A

  In recent years, for example, collagen has been developed as a raw material with a triple helix structure maintained, and a component having a triple helix structure is more effective than a component having no triple helix structure. In the cosmetics market, there is an increasing demand for applying ingredients that retain the original structure of such active ingredients as they are.

  The present invention provides a liposome containing an active ingredient composed of an easily heat-denaturing substance that is easily denatured particularly at low temperatures while suppressing the mixing of harmful components such as organic solvents, and a method for producing the liposome. With the goal.

  The present inventors have formed a first lipid film at a water / oil interface in which an oil phase is disposed on an aqueous phase, and subsequently a liquid in which an aqueous solution containing a heat-modifying substance is encapsulated in the second lipid film. After forming the droplet, this is introduced into the oil phase, the droplet is transferred to the aqueous phase, the outer side of the second lipid membrane that becomes the inner lipid membrane is covered with the first lipid membrane, and the outer lipid membrane is formed, The present inventors have found that an active ingredient weak against heat can be made into a liposome while maintaining the original structure, and have completed the present invention. Specifically, the present invention provides the following.

(1) Liposomes containing an undenatured heat-denaturing substance.
(2) The liposome according to (1), wherein the heat-denaturing substance has a denaturation temperature of 10 to 50 ° C.
(3) A cosmetic, pharmaceutical product or food containing the liposome of (1) or (2).
(4) A method for producing a liposome encapsulating a non-denatured easily heat-denatured substance, the first step of forming a first lipid membrane at the water / oil interface where the oil phase is arranged in contact with the aqueous phase, A second step of forming droplets in which an aqueous solution containing a heat-denaturing substance is encapsulated in the second lipid membrane; introducing the droplets into the oil phase and transferring them to the water phase; A third step of covering the outer side of the lipid membrane with a first lipid membrane to form an outer lipid membrane, and a method for producing liposomes.
(5) The method for producing a liposome according to (4), which comprises a step of producing droplets at a temperature lower than the denaturation temperature of the heat-denaturing substance.
(6) The method for producing a liposome according to (4) or (5), wherein the first step and the second step are performed at a temperature lower than the denaturation temperature of the heat-denaturing substance.

  According to the present invention, it is possible to produce a liposome containing an active ingredient of an easily heat-denaturing substance that is easily denatured particularly at a low temperature while suppressing the mixing of harmful components such as organic solvents while retaining the original structure. In addition, heat resistance can be improved by encapsulating active ingredients that are sensitive to heat in liposomes, so that cosmetics, pharmaceuticals, foods, etc. containing active ingredients can be stored at room temperature, saving the trouble of storage management. Can do.

It is a figure which shows the liposome of this invention containing the collagen observed with the fluorescence microscope. It is a figure which shows the collagen observed with the fluorescence microscope.

  Hereinafter, embodiments of the present invention will be described.

  The liposome of the present invention needs to contain an easily heat-denaturing substance. An easily heat-denaturing substance is, for example, a substance whose structure is partially or entirely changed by heat at a relatively low temperature. The structural change includes not only the primary structure but also the secondary structure, tertiary structure, and quaternary structure as long as the effect obtained by application of the heat-denaturing substance is lost.

  As the denaturation temperature of these heat-denaturing substances, those having a wide temperature range can be used, and those having a temperature of 80 ° C. or lower can be used without any problem. The higher the denaturation temperature, the better in terms of stability. However, according to the present invention, an active ingredient having a relatively low denaturation temperature can be made into a liposome, and those having a temperature near room temperature, for example, 10 to 50 ° C. or 30 to 40 ° C. are used. can do.

  Desirable as an easily heat-denaturing substance of the present invention is an active ingredient having whitening, moisturizing, antioxidant effect and medical effect, specifically, for example, water-soluble collagen, placenta extract, elastin, silk protein, etc. Proteins, vitamins, nucleic acids such as DNA or RNA, enzymes and the like can be used.

  Collagen is one of the proteins constituting the dermis, ligaments, tendons, bones, cartilage and the like, and is the main component of the extracellular matrix (extracellular matrix) of multicellular animals. Conventionally, it has been mainly derived from cattle, pigs, etc., but collagens derived from fish, jellyfish, etc. have also increased recently. The denaturation temperature varies depending on which animal was taken, for example, 40 ° C for cattle, 40 ° C for pigs, 29 ° C for blue sharks, and 19 ° C for pink salmon.

  Placenta extract is a mammalian placenta extract and contains growth factors such as epidermal growth factor (EGF), and has been widely used for cosmetics with the expectation of a whitening effect. The normal delivery placenta of the mammal used is obtained from mammals such as pigs, cows, horses, humans or sheep, preferably the normal delivery placenta of humans or pigs. Porcine placenta is effective because its molecular structure is closest to that of humans.

  Elastin is a protein present in tissues having elasticity and elasticity such as ligaments and arteries. In addition, there is a slight amount in the dermis connective tissue together with collagen as a component involved in skin elasticity, and the reduction or degeneration of elastin in the skin due to aging or ultraviolet rays contributes to skin wrinkles and tarmi . For this reason, elastin is currently applied to many products, mainly in the cosmetics and health food fields. On the other hand, since elastin usually exists in vivo as an insoluble protein having a three-dimensional network structure, water-soluble elastin can be obtained by hydrolysis with acid or alkali or treatment with an enzyme. That is widely known.

  Attempts have been made to use silk protein obtained by extraction means as a cosmetic ingredient in addition to the use of silk protein obtained by extraction means from rabbits and wild silkworms produced by silk insects. It has been clarified that silk proteins have various functions such as cell growth, antioxidant properties, antibacterial properties, alcohol digestibility, and anticoagulant properties. Silk proteins are classified into globular proteins (albumin, casein, globulin, gelatin, nucleus / sugar / pigment / phosphoprotein) and hard proteins (fibroin, sericin, keratin, collagen, elastin) according to the molecular shape of the components. However, it is not yet clarified at all about the site or structure of the silk protein, but for example, the peptide chain at a specific site of silk fibroin has a cell growth promoting function. In addition, sericin is used as a cosmetic having a moist feeling (moisturizing property) and also having an antioxidant action (whitening effect).

  As vitamins, water-soluble vitamins such as niacin, vitamin B2, vitamin B6, vitamin C and biotin can be used. Vitamin C is one of the typical cosmetic ingredients, and has a function of inhibiting melamine synthesis of pigment cells on the skin surface and a role of assisting synthesis of collagen closely related to wrinkles.

  As the enzyme, various enzymes used in cosmetics can be used without particular limitation. For example, an active oxygen removing enzyme (superoxide dismutase: SOD), peroxidase, thioredoxin reductase and the like can be used.

  In the method for producing liposomes of the present invention, an oil phase is arranged on an aqueous phase, and a lipid monomolecular film (first lipid membrane) that becomes an outer lipid membrane of the liposome is formed at the oil-water interface, An aqueous solution droplet containing an easily heat-denaturing substance, which is covered with an inner lipid membrane (second lipid membrane) of liposomes, is introduced into the phase, and the droplet is transferred into the aqueous phase, so that the outer side of the inner lipid membrane And forming an outer lipid membrane.

  Here, in order to pass the droplet covered with the inner lipid membrane through the oil-water interface on which the lipid monomolecular film as the outer lipid membrane is formed and transfer it into the aqueous phase, an aqueous solution that forms droplets and an aqueous phase is formed. It is preferable to use the specific gravity difference of each solution, that is, gravity. Gravity of the droplets into the aqueous phase is carried out under very mild conditions, so there is no exotherm during the formation of liposomes, the heat-denaturing substance is not denatured, and mechanical stress, etc. Is not added, so that the formed liposome is not destroyed. In addition, in the transfer of droplets by gravity, for example, by performing an operation in a transparent container, the formation process of liposomes can be visually confirmed.

  Below, the manufacturing method of the liposome of this invention is explained in full detail.

  A lipid monomolecular film (first lipid film) formed at the oil-water interface between the water phase and the oil phase can be easily formed by adding lipid components such as various phospholipids to the oil phase. . The lipid components added in the oil phase are aligned at the oil / water interface so that the hydrophobic group part of each lipid molecule is on the oil phase side and the hydrophilic group part is on the water phase side, and all lipid molecules are in a certain direction. A facing lipid monolayer is formed at the oil-water interface.

  Subsequently, W / O droplets covered with an inner lipid membrane are introduced into the oil phase. These W / O droplets are formed by forming an inner lipid membrane (second lipid membrane) around droplets of an aqueous solution containing an easily heat-denaturing substance. The inner lipid membrane has a hydrophilic group portion on the inner side. And each lipid molecule is formed as a monomolecular film with the hydrophobic group portion facing outward. W / O droplets may be introduced separately into the oil phase, or may be formed in the oil phase.

  When W / O droplets are introduced into the oil phase, the W / O droplets sink due to gravity and reach the oil / water interface. This is because the aqueous solution constituting the droplets is heavier than the oil constituting the oil phase. However, since both the W / O droplet and the aqueous phase are composed of an aqueous solution, for example, when the aqueous solution constituting the droplet of the W / O droplet and the aqueous solution constituting the aqueous phase are the same, The W / O droplet cannot descend any further and cannot be transferred into the aqueous phase.

  Therefore, in the present invention, a specific gravity difference is given to the aqueous solution constituting the W / O droplet and the aqueous solution constituting the aqueous phase, and the W / O droplet moves into the aqueous phase due to gravity. It is better to do so. When the W / O droplets pass through the oil-water interface, a lipid monomolecular film (first lipid film) is wound around the periphery, and the lipid monomolecular film becomes an outer lipid film to form a lipid bimolecular film. Provided liposomes are formed. The specific gravity difference can be imparted by dissolving the easily heat-denaturing substance in the aqueous solution constituting the droplets of the W / O droplets, but other water-soluble substances may be further added.

  Lipid membrane components constituting the lipid membrane of the liposome include at least phospholipids, glycolipids, sterols, glycols, cationic lipids, lipids having a polyethylene glycol (PEG) group (for example, PEG-phospholipid), and the like. . Of these, phospholipids and / or glycolipids are preferably used.

  Examples of phospholipids that can be used include phosphatidylcholine, phosphatidylserine, phosphatidic acid, phosphatidylglycerin, dioleoylphosphatidylcholine, distearoylphosphatidylcholine, dipalmitoylphosphatidylcholine, palmitoyloleoylphosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, Phosphate, lysophosphatidylcholine (lysolecithin), egg yolk lecithin, soybean lecithin or hydrogenated phospholipids thereof can be used without any particular limitation.

  The phospholipids can be used alone or in combination of two or more. Moreover, you may make it provide the asymmetric lipid bimolecular membrane from which the composition of an inner-layer lipid membrane and an outer-layer lipid membrane differs.

  Examples of the glycolipid include glycerolipids such as digalactosyl diglyceride and galactosyl diglyceride sulfate, galactosylceramide, galactosylceramide sulfate, lactosylceramide, sphingoglycolipids such as ganglioside G7, ganglioside G6, and ganglioside G4.

  As a constituent component of the liposome membrane, other substances can be added as necessary in addition to the lipid. For example, sterols acting as a lipid membrane stabilizer such as cholesterol, dihydrocholesterol, cholesterol ester, phytosterol, sitosterol, stigmasterol, campesterol, cholestanol, lanosterol and the like can be mentioned. It is also known that sterol derivatives such as 1-O-sterol glucoside, 1-O-sterol maltoside or 1-O-sterol galactoside are effective in stabilizing liposomes. Among these, cholesterol is particularly preferable.

  As the amount of sterols used, the molar ratio of phospholipid (PEG-phospholipid not included) / sterols is 100/60 to 100/90, preferably 100/70 to 100/85. This molar ratio is based on the amount of phospholipid excluding PEG-phospholipid. When the molar ratio is less than 100/60, stabilization by sterols that improve the dispersibility of the mixed lipid is not sufficiently exhibited.

  In addition to the sterols, glycols may be added as a constituent of the liposome membrane. When preparing a liposome, if glycols are added together with phospholipid or the like, the retention efficiency of the water-soluble compound in the liposome is increased. Examples of glycols include ethylene glycol, diethylene glycol, triethylene glycol, propylene glycol, dipropylene glycol, trimethylene glycol, and 1,4-butanediol. As a usage-amount of glycols, the ratio of 0.01-20 mass% with respect to lipid total mass, Preferably the ratio of 0.5-10 mass% is desirable.

  The liposome of the present invention can be widely applied to a drug delivery system (DDS) in which an active ingredient having a target medicinal effect reaches a target organ of interest. Depending on the purpose, the drug containing the liposome of the present invention can be introduced into the human body orally or through blood vessels. In addition, as an external preparation for skin, it is not only for cosmetics such as skin lotion, emulsion, cream, pack agent, peeling agent for the purpose of whitening, improving skin quality, improving freckles, rejuvenating skin, and tightening skin, but also for hair. It can be suitably used for any of quasi-drugs such as cleaning agents, bath preparations, pharmaceuticals such as drugs, and foods such as health foods useful for maintaining and promoting health.

  EXAMPLES Hereinafter, the present invention will be specifically described by way of examples, but these do not limit the scope of the present invention.

(Reagent used)
Collagen: A raw material having a triple helical structure with a denaturation temperature of about 37 ° C. was used.
Phospholipid: Lecithin

(Preparation of evaluation collagen)
A fluorescent reagent (NBD Chloride) was bound to the amino group of triple helical collagen according to a conventional method to prepare a 0.3 wt% aqueous solution.

(Preparation of W / O droplet)
Phospholipid (lecithin) was dissolved in a mineral oil solvent to a concentration of 7.5 mg / mL. In a microtube container, 1000 mg of a phospholipid solution and 50 mg of an aqueous collagen solution combined with a fluorescent reagent were mixed. Furthermore, this mixed liquid was stirred with a vortex mixer.

(Preparation of liposome)
In a sample bottle, 4000 mg of purified water serving as an aqueous phase was placed, and 1000 mg of an oil phase containing phospholipid having a concentration of 7.5 mg / mL was placed thereon. This phospholipid becomes an outer lipid membrane. Further, W / O droplets prepared in the oil phase were added. The W / O droplets spontaneously migrated from the oil phase to the aqueous phase by gravity through the oil-water interface, and thus a liposome having a lipid bimolecular film formed thereon.

  The aqueous phase containing the collagen-encapsulated liposome prepared above was taken out. The aqueous phase was placed on a hot plate, heated to 45 ° C. while observing with a fluorescence microscope, and photographed (FIG. 1). On the other hand, as a comparative example, the same observation was performed on collagen modified with a fluorescent reagent not encapsulated in liposomes (FIG. 2).

  In the case of liposomes encapsulating collagen, fluorescence derived from triple helical collagen was confirmed only in the liposomes even at 45 ° C. It is considered that the triple helix structure is maintained even at 45 ° C. On the other hand, in the case of collagen alone, it was confirmed that the fluorescence disappeared. It is considered that collagen molecule movement due to thermal energy is suppressed by encapsulating collagen molecules in liposomes.

Claims (4)

A method for producing a liposome encapsulating a non-denatured heat-denaturing substance,
A first step of forming a first lipid membrane at the water / oil interface where the oil phase is arranged in contact with the water phase;
A second step in which an aqueous solution containing an easily heat-denaturing substance forms droplets encapsulated in a second lipid membrane;
The droplets are introduced into the oil phase, and the droplets are transferred to the water phase due to the difference in specific gravity between the aqueous solution containing the heat-denaturing substance and the water phase, and the second lipid membrane serving as the inner lipid membrane A third step of covering the outside with the first lipid membrane to form an outer lipid membrane, wherein the specific gravity difference is imparted by the heat-denaturing substance;
A method for producing a liposome, comprising: The method for producing a liposome according to claim 1 , further comprising a step of creating the droplet at a temperature lower than the denaturation temperature of the heat-denaturing substance. The method for producing a liposome according to claim 1 or 2 , wherein the first step and the second step are performed at a temperature lower than a denaturation temperature of the heat-denaturing substance. The method for producing a liposome according to any one of claims 1 to 3 , wherein the non-denatured heat-denaturing substance is collagen having a triple helical structure.