JP2017190329A - Method for producing liposome and multi-lamellar liposome - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for producing liposomes using carbon dioxide and wave irradiation at a predetermined frequency in combination, and to provide a multi-lamellar liposome.SOLUTION: According to the present invention, there is provided a method for manufacturing a liposome. The method comprises placing a sphingophospholipid dispersed in an aqueous solvent in a high-pressure reactor, enclosing carbon dioxide having a temperature of 30 to 70°C and a pressure of 0.1 to 50 MPa in the high-pressure reactor, and irradiating a wave having a frequency of 10 to 500 kHz to react them. Without the use of any organic solvent, a multi-lamellar liposome having an average particle size of 5 to 200 nm can be obtained.SELECTED DRAWING: Figure 5

Description

  The present invention relates to a method for producing a liposome containing sphingophospholipid and a multilamellar liposome.

  A liposome is a closed endoplasmic reticulum composed of a lipid bilayer. In addition to being used as a cell model, it is applied to a drug delivery system (DDS) in which a pharmaceutical or the like is enclosed in a closed space. As a method for producing liposomes, for example, a chloroform solution containing phospholipid and cholesterol is charged into a flask, only the solvent is removed to form a lipid film on the inner wall of the flask, and a buffer solution such as phosphoric acid or lactic acid is added to the phospholipid. There is a method of heating above the transition temperature of the water and hydrating and dispersing with a stirrer (Non-patent Document 1). According to the method, multilamellar liposomes can be generated, and when the multilayer liposomes are irradiated with ultrasonic waves, a monolayer liposome of hundreds of nanometers is generated.

On the other hand, there is a method using supercritical carbon dioxide instead of the organic solvent. Supercritical carbon dioxide, liposome membrane components, pharmaceutical compounds, and formulation aids are mixed and reacted in a pressure vessel under conditions of 40 to 65 ° C. and 10 to 30 MPa. When the pressure vessel is depressurized to remove carbon dioxide, an aqueous dispersion of liposomes encapsulating the pharmaceutical compound can be obtained (Patent Document 1). Examples of the formulation aid include buffers such as trometamol, chelating agents, inorganic salts, pharmacologically active substances, osmotic pressure regulators, stabilizers, antioxidants, viscosity regulators, preservatives and the like. In the examples, dipalmitoyl phosphatidylcholine (DPPC) and hydrogenated soybean phosphatidylcholine (HSPC) are used as liposome raw materials, and liposomes are produced using trometamol and EDTANa ₂ -Ca in combination. In addition, the sizing operation is performed after liposome production, and the multilayer liposome whose average particle diameter is 0.26-0.30 micrometer is obtained.

  There is also a method for producing liposomes for encapsulating a water-soluble or hydrophilic substance or a mixture of substances (Patent Document 2). Water, phospholipids and auxiliaries are reacted with carbon dioxide and polar organic solvents above the critical pressure and temperature. In the examples, phospholipid [1-n-hexadecanoyl-2- (9-cis-octadecenoyl) -3-sn-phosphatidylcholine (POPC)] and cholesterol are used, and the POPC: cholesterol molar ratio is 3: 1 liposome is manufactured.

  Furthermore, a liposome encapsulating an encapsulated substance, wherein an aqueous phase containing the encapsulated substance is added to a homogeneous mixed fluid of carbon dioxide and phospholipid or glycolipid in a supercritical state or a temperature or pressure condition above the critical point. There is also a manufacturing method (Patent Document 3). A co-solvent such as ethanol is used in combination with a homogeneous mixture of phospholipid and supercritical carbon dioxide to increase the solubility of a poorly soluble encapsulating substance. According to this method, single-lamellar liposomes having a diameter of 50 nm to 800 nm can be produced. In the method using the supercritical carbon dioxide method, the liposome is not formed in the supercritical state, but the liposome is formed by mixing an aqueous solution with the phospholipid that is rapidly reduced in solubility and precipitated in the form of fine particles. Is said to be manufactured. In the examples, monolayer liposomes were produced using dipalmitoyl phosphatidylcholine (DPPC), hydrogenated soybean lecithin (HSPC), hydrogenated egg yolk lecithin, dioleyl phosphatidylcholine (DOPC), and distearoyl phosphatidylcholine (DSPC).

  On the other hand, a chloroform solution in which egg yolk-derived phosphatidylcholine is dissolved is placed in a test tube, and only the solvent is removed to form a lipid film on the inner wall of the test tube. After adding glass beads and a buffer solution to this test tube, There is also a method for producing liposomes that irradiates low frequency ultrasonic waves of 20 to 50 kHz and high frequency ultrasonic waves of 500 kHz to 20 MHz (Patent Document 4). Simply irradiating with ultrasonic waves causes problems such as damage of liposome inclusions and decomposition / denaturation of raw materials due to an increase in solution temperature. However, the raw materials are irradiated with low frequency ultrasonic waves of 20 to 50 kHz, and fine due to cavitation. Next, when combined with high-frequency ultrasonic irradiation of 500 kHz to 20 MHz that makes the liposome particle size extremely fine and uniform to the nano level under conditions with little cavitation damage, the particle size distribution in the size of several tens to 150 nm It is said that it is possible to prepare high-quality liposomes that are narrow and free from contamination.

JP 2006-298844 A Japanese Patent Laid-Open No. 06-315624 Japanese Patent No. 4296341 JP 2007-7625 A

Supervised by Kazunari Akiyoshi and Satoshi Sakurai; Naoto Oku, Ryoichi Kuboi, Junichi Takaya, “New Development of Liposome Application: Toward the Development of Artificial Cells”, NTS, June 2005, page 33, 34 Tetsuro Yoshimura, “Development of new liposome production technology and application to medicine” [online], [March 14, 2017], Internet <URL: http://www.nisri.jp/dor/report/2009 /yoshimura.pdf>

  However, the methods described in Non-Patent Document 1 and Patent Document 2 use cholesterol together with phospholipids. Cholesterol is used as a component of the lipid bilayer of liposomes, and no liposomes containing cholesterol have been obtained.

  The method described in Patent Document 1 is a method for producing multilamellar liposomes using supercritical carbon dioxide. It is necessary to use auxiliaries such as trometamol and chelating agents together with phospholipids. Therefore, it is desired to develop a method for producing multilamellar liposomes without using a formulation aid.

  The method described in Patent Document 3 uses supercritical carbon dioxide as in Patent Document 1, but in the examples, only unilamellar liposomes are produced. Compared to Patent Document 1, which uses supercritical carbon dioxide and uses the same phospholipid as a membrane component to produce multilayer liposomes, Patent Document 1 prepares multilayer liposomes using phospholipids together with cholesterol. On the other hand, Patent Document 3 does not use cholesterol and prepares only unilamellar liposomes. Therefore, development of a method for producing multilamellar liposomes without using cholesterol is desired.

  Patent Documents 1 to 4 all use glycerophospholipids as phospholipids and do not produce liposomes containing sphingophospholipids as membrane constituents. Liposomes are applied to DDS and the like, but glycerophospholipids and sphingophospholipids are also different in metabolism in vivo. Therefore, it is desired to develop a method for producing liposomes containing sphingophospholipid as a main constituent.

  Furthermore, the method described in Patent Document 4 irradiates ultrasonic waves in two steps after preparing a phospholipid membrane using an organic solvent in advance. Since an organic solvent is used, the production environment and product safety may be affected, and the process of multiple times of ultrasonic irradiation is complicated. Therefore, it is desired to develop a method for producing liposomes containing sphingophospholipids more easily without using an organic solvent.

  In view of the above situation, an object of the present invention is to provide a method for producing a sphingophospholipid liposome without using cholesterol or an auxiliary agent.

  A further object of the present invention is to provide multilamellar liposomes containing sphingophospholipid as a constituent component.

  The inventors of the present invention reacted a mixture of sphingophospholipid and carbon dioxide in a critical state by irradiating a wave of a specific frequency, and the liposome suspension obtained after carbon dioxide removal contains multilamellar liposomes. As a result, the present invention has been completed. In addition, glycerophospholipid and sterols can be used in combination with sphingophospholipid.

  That is, the present invention accommodates sphingophospholipid dispersed in an aqueous solvent in a high-pressure reactor, encloses carbon dioxide at a temperature of 30 to 70 ° C. and a pressure of 0.1 to 50 MPa, and irradiates waves having a frequency of 10 to 500 kHz. The present invention provides a method for producing liposomes, characterized in that the reaction is carried out.

  The present invention also provides a method for producing the liposome, wherein glycerophospholipid and / or sterols are dispersed together with the sphingophospholipid in the aqueous solvent.

  The present invention also provides a method for producing the liposome, wherein the sphingophospholipid is sphingomyelin.

  The present invention also provides a method for producing the liposome, characterized in that the multi-lamellar liposome is produced by irradiating the carbon dioxide at a temperature of 40 to 60 ° C. and a pressure of 10 to 30 MPa and irradiating a wave with a frequency of 20 to 100 kHz. To do.

  The present invention also provides multilamellar liposomes composed of sphingophospholipids.

  Moreover, this invention provides the said multilamellar liposome containing glycerophospholipid and / or sterols with sphingomyelin.

  The present invention also provides the multilamellar liposome having a particle size of 5 to 200 nm.

  According to the present invention, liposomes containing sphingophospholipids can be produced without using organic solvents, formulation aids, and sterols.

  Also provided are multilamellar liposomes containing sphingophospholipids.

2 is an electron microscopic image of a liposome suspension prepared in Example 1. FIG. 2 is an electron microscopic image of a liposome suspension prepared in Example 2. FIG. 4 is an electron microscopic image of a liposome suspension prepared in Example 3. FIG. 6 is an electron microscopic image of a liposome suspension prepared in Example 5. FIG. 7 is an electron microscopic image of a liposome suspension prepared in Example 6. FIG. 2 is an electron microscopic image of a liposome suspension prepared in Example 7. FIG. 2 is an electron microscopic image of a liposome suspension prepared in Example 13. FIG. 2 is an electron microscopic image of a liposome suspension prepared in Example 14. FIG. 2 is an electron microscopic image of a liposome suspension prepared in Example 15. FIG. 10 (A) is an optical microscope image of the liposome suspension prepared in Example 16, and FIG. 10 (B) is an electron microscope image.

  In the first aspect of the present invention, a sphingophospholipid dispersed in an aqueous solvent is housed in a high-pressure reactor, carbon dioxide at a temperature of 30 to 70 ° C. and a pressure of 0.1 to 50 MPa is enclosed, and a wave with a frequency of 10 to 500 kHz is contained. It is a manufacturing method of a liposome characterized by reacting by irradiation. Hereinafter, the present invention will be described in detail.

  The production method of the present invention is characterized by containing sphingophospholipid as a constituent component of the liposome. As shown in the examples described later, it has been found that liposomes composed solely of sphingophospholipids can be produced without using sterols, auxiliary agents such as buffers and chelating agents, and organic solvents such as ethanol and chloroform. is there. The sphingophospholipid is a compound that contains ceramide in which a fatty acid is bonded to an acid amide to a sphingoid, and is further bonded to a phosphate and a base. Examples include sphingomyelin, ceramide phosphoryl ethanolamine, ceramide phosphoryl glycerol, ceramide phosphoryl glycerol phosphate, 1,2-dimyristoylamide-1,2-deoxyphosphatidyl choline.

  The production method of the present invention is characterized in that liposomes can be produced without using sterols, but sterols may be used in combination with the sphingophospholipid. Examples of sterols include cholesterol, dihydrocholesterol, cholesterol ester, phytosterol, sitosterol, stigmasterol, campesterol, cholestanol, and lanosterol. Further, it may be a sterol derivative such as 1-O-sterol glucoside, 1-O-sterol maltoside or 1-O-sterol galactoside.

  Moreover, it may replace with sterols or may use glycerophospholipid together with sterols. Examples of the glycerophospholipid include phosphatidylcholine, dipalmitoylphosphatidylcholine, distearoylphosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidic acid, phosphatidylinositol, dimyristoylphosphatidylcholine, cardiopine, egg yolk lecithin, hydrogenated egg yolk lecithin, hydrogenated egg yolk lecithin, hydrogenated egg yolk lecithin, Examples include soy lecithin and hydrogenated soybean phosphatidylcholine.

  The ratio of the sphingophospholipid in the liposome constituent component is 50 to 100% by mass, more preferably 60 to 100% by mass. Glycerophospholipids and sterols can be used in combination, and the combined amount in the case of using these in combination does not exceed 50% by mass of the liposome constituents, and each is less than 50% by mass, more preferably 0 to 30% by mass. %, Particularly preferably 0 to 20% by mass.

  In the present invention, sphingophospholipid dispersed in an aqueous solvent is used. This is characterized in that liposomes can be produced without using organic solvents or auxiliaries when mixed with carbon dioxide of a predetermined condition and irradiated with wave irradiation of a specific frequency. By using the sphingophospholipid dispersed in an aqueous solvent, a liposome in which the aqueous solvent is encapsulated in the liposome is produced. Accordingly, water such as purified water, distilled water, or pure water is suitable as the aqueous solvent. It is not necessary to add other components to the aqueous solvent.

  On the other hand, liposomes encapsulating any compound A can also be produced. In this case, a solution obtained by dissolving or dispersing component A in water or the like can be used as the aqueous solvent. At this time, an organic solvent such as a buffer solution, a chelate, and alcohol can be included as a solubilizing agent for component A.

  Liposome components such as sphingophospholipids, glycerophospholipids, and sterols are solid at room temperature. In order to disperse this in water, for example, a container charged with liposome constituents and an aqueous solvent may be stored in a bath part of a bath-type Sony caterer and subjected to Sony application to refine the liposome constituents. Liposome constituents can be dispersed in water without using an organic solvent. In addition, the addition amount of a liposome structural component is 0.001-10 mass parts with respect to 100 mass parts of water, Preferably it is 0.01-1 mass part.

  An aqueous solvent in which liposome components are dispersed is stored in a high-pressure reactor. Next, in the same reactor, the temperature is 30 to 70 ° C., preferably 30 to 60 ° C., more preferably 40 to 60 ° C., particularly preferably 50 to 60 ° C., pressure 0.1 to 50 MPa, preferably 0.1 to 30 MPa, More preferably, carbon dioxide is supplied so as to be 1 to 30 MPa.

  Next, the mixture is irradiated with a wave having a frequency of 10 to 500 kHz, more preferably 10 to 300 kHz, and particularly preferably 20 to 100 kHz. When carbon dioxide is removed under reduced pressure after wave irradiation, liposomes are formed in the resulting suspension. As described in Non-Patent Document 2, when ultrasonic treatment is applied to MLV (multi lameller vesicle; multilamellar liposome), SUV (small unilameller vesicle; small unilamellar liposome) having a particle size of hundreds of nanometers is obtained. Become. However, in the present invention, when sphingophospholipid is mixed with carbon dioxide in a supercritical state and further irradiated with a predetermined wave, the mechanism is unknown, but multilamellar liposomes composed of sphingophospholipid can be produced. found. The reaction time, that is, the wave irradiation time is not particularly limited as long as it is 10 minutes or longer. For example, it is 10 minutes to 5 hours, preferably 30 minutes to 2 hours. The method of the present invention is characterized in that the liposome is composed only of the sphingophospholipid, and the produced liposome is not limited to the multilamellar liposome, and may include a unilamellar liposome.

  In the present invention, the layer structure and the average particle diameter can be adjusted by adjusting the temperature, pressure, frequency, and reaction time within the above ranges. For example, as shown in Examples described later, multilamellar liposomes can be mainly produced by irradiation at a temperature of 40 to 60 ° C., a pressure of 10 to 30 MPa, a frequency of 20 to 100 kHz, and preferably 20 to 50 kHz. When the reaction is performed at a pressure of 15 MPa in the above temperature range, multilamellar liposomes having a substantially uniform particle diameter can be produced in the temperature range of 40 to 60 ° C. Moreover, when it reacts at temperature higher than 40 degreeC, the separability of a multilamellar liposome can be improved. Moreover, more multilayer multilamellar liposomes can be produced in the temperature range of 40 to 60 ° C. by 28 to 45 kHz ultrasonic irradiation.

  Liposomes are composed of lipid bilayers. In the present specification, the unilamellar liposome means a liposome composed of one layer of lipid bilayer, and the multilamellar liposome means a liposome composed of two or more lipid bilayers. To do.

  Liposomes produced by the production method of the present invention form a multilamellar structure without using membrane components such as sterols. Conventionally, for the preparation of multilamellar liposomes, it is necessary to use a membrane component such as sterols together (see Patent Document 1), and phospholipid-only multilamellar liposomes and methods for producing the same are not known. However, in the present invention, sterols and other combinations are not excluded, and sterols and glycerophospholipids can also be used in combination as liposome constituents.

  The liposome produced in the present invention contains sphingophospholipid as an essential component. The size of liposomes depends in principle on the spontaneous curvature based on the form of phospholipid molecules containing hydration water, etc., and is influenced by the preparation environment leading to the formation. For example, the static hydration method under mild conditions Then, it becomes a giant unilamellar liposome (GUV: Giant Unilamellar Vesicle), becomes a multi-lamellar liposome under a vortex like vortex treatment, and becomes an unilamellar liposome having an average particle size of 200 nm or less by sonication (Non-patent Document 2). ). However, according to the present invention, multilamellar liposomes composed solely of sphingophospholipids can be produced as shown in the examples described later. The average particle size when converted from the average molecular weight measured with the particle size distribution analyzer was more than 1,000 nm, but according to the calculation based on the electron microscope image, the particle size of the multilamellar liposome was 5 to 200 nm. It was. However, liposomes that can be produced in the present invention are not limited to multilamellar liposomes. Regardless of the layer structure, liposomes having a large average particle size such as GUV can be used as research materials for biological membranes.

  The second of the present invention is a multilamellar liposome composed of sphingophospholipid. This liposome can be produced by the first method of the present invention. However, the manufacturing method is not limited to this.

  The particle size of the multilamellar liposome of the present invention is 5 to 200 nm suitable as a liposome preparation for medical use. In order to deliver liposomes to a target site in a living body, there is a method of utilizing vascular permeability. Since the permeability of blood vessel walls where angiogenesis is active, such as cancer tissues and inflamed sites, is enhanced, particles of several tens to 200 nm that are difficult to pass through in normal tissues can permeate. The particle size of the multilamellar liposome of the present invention is preferably 30 to 150 nm, more preferably 50 to 120 nm. Multilamellar liposomes of this particle size can reach cancerous tissues and inflammatory sites.

  EXAMPLES Next, although an Example is given and this invention is demonstrated concretely, these Examples do not restrict | limit this invention at all.

Example 1
Sphingomyelin was added to purified water so as to have a concentration of 0.1 wt%, and the mixture was stirred for 10 minutes with an ultrasonic washer (STN USM Co., Ltd.). 3 ml of the mixture was charged into a high-pressure reactor, and carbon dioxide was sealed at a temperature of 40 ° C. so that the pressure was 10 MPa. Furthermore, the ultrasonic wave of 28kHz was irradiated and it was made to react for 60 minutes. After completion of the reaction, carbon dioxide was removed under reduced pressure, and a liposome suspension was recovered.

  The liposome suspension obtained in a reaction time of 60 minutes was diluted 10-fold with distilled water, and dispersed with a 400-mesh Cu grid with a carbon support membrane at room temperature or 80 ° C., and 2% phosphotungstic acid, room temperature or 80 ° C. The electron staining by the negative staining method was performed. An electron microscope image is shown in FIG. Multilamellar liposomes composed of multiple layers were included.

(Examples 2 to 15)
A liposome suspension was recovered according to Example 1 except that the temperature, pressure, and ultrasonic conditions were changed to Table 1. Moreover, the average molecular weight was measured using the particle size distribution measuring apparatus (Shimadzu SALD-2200), and it converted into the average particle diameter of a liposome. The results are shown in Table 1. In the table, “-” means unmeasured. Moreover, the electron microscope image of the liposome contained in a liposome suspension is shown to FIGS.

(Example 16)
A liposome suspension was obtained by operating in the same manner as in Example 1 except that sealing was performed so that the temperature was 50 ° C. and the pressure was 20 MPa, and stirring was performed at 1,500 rpm for 60 minutes instead of ultrasonic irradiation. The liposome constituting the liposome suspension was a multilamellar liposome. The average particle size of the liposome obtained from the average molecular weight measured using a particle size distribution analyzer was 3,421 ± 392 nm. An optical microscope image of the liposome suspension is shown in FIG. 10 (A), and an electron microscope image is shown in FIG. 10 (B).

(result)
(1) When sphingophospholipid dispersed in an aqueous solvent is stored in a high-pressure reactor and reacted at a temperature of 40 to 60 ° C. and a pressure of 10 to 30 MPa, it has been found that multilamellar liposomes are produced under any conditions.
(2) As shown in the electron microscope images of FIGS. 2, 5, and 8 showing the results of Example 2, Example 6 and Example 14, when the reaction is performed at a pressure of 15 MPa in the temperature range of 40 to 60 ° C. The tendency that the particle size of the multilamellar liposome becomes uniform was observed.
(3) As shown in the electron microscope images of FIGS. 1, 4 and 7 showing the results of Example 1, Example 5 and Example 13, when the reaction was performed at a pressure of 10 MPa, the temperature was 50 ° C. higher than 40 ° C. The separability of multilamellar liposomes was excellent at ℃ and 60 ℃.
(4) According to the results of Example 7, Example 8, and Example 11, it depended on the irradiation time of ultrasonic waves, and according to the results of Example 3, Example 7, and Example 15, it depended on the reaction temperature. In the case of a temperature of 50 ° C., a pressure of 20 MPa, and an ultrasonic wave of 28 kHz, the average particle diameter becomes the shortest when the irradiation time is 60 minutes, and in the case of a pressure of 20 MPa and an ultrasonic wave of 28 kHz, the average is obtained at a temperature of 50 ° C. The particle diameter was the shortest.
(5) As shown in the comparison between FIG. 6 and FIG. 10 (B) showing the results of Example 7 and Example 16, when the temperature is 50 ° C. and the pressure is 20 MPa, depending on the ultrasonic treatment or the stirring treatment It was observed that the layer thickness was different and the layer irradiated with 28 kHz ultrasonic waves was more multilayered.

Claims (7)

  A sphingophospholipid dispersed in an aqueous solvent is stored in a high-pressure reactor, carbon dioxide at a temperature of 30 to 70 ° C. and a pressure of 0.1 to 50 MPa is enclosed, and a reaction is performed by irradiating a wave with a frequency of 10 to 500 kHz. A method for producing liposomes, characterized in that   The method for producing a liposome according to claim 1, wherein glycerophospholipid and / or sterols are dispersed together with the sphingophospholipid in the aqueous solvent.   The method for producing a liposome according to claim 1 or 2, wherein the sphingophospholipid is sphingomyelin.   The liposome according to any one of claims 1 to 3, wherein a multilamellar liposome is produced by irradiating a wave with a frequency of 20 to 100 kHz by setting the carbon dioxide to a temperature of 40 to 60 ° C and a pressure of 10 to 30 MPa. Manufacturing method.   Multilamellar liposomes composed of sphingophospholipids.   The multilamellar liposome according to claim 5, comprising glycerophospholipid and / or sterols together with sphingomyelin.   The multilamellar liposome according to claim 5 or 6, wherein the particle size is 5 to 200 nm.