JP6923155B2 - Liposomal manufacturing method and multilamellar liposome - Google Patents

Description

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

Liposomes are closed endoplasmic reticulum consisting of lipid bilayer membranes. In addition to being used as a cell model, it is also applied to a drug delivery system (DDS) in which a drug or the like is enclosed in a closed space. As a method for producing liposomes, for example, a chloroform solution containing phospholipids and cholesterol is placed in 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 phospholipids. There is a method of heating above the transition temperature of the flask and hydrating and dispersing it with a stirrer (Non-Patent Document 1). According to this method, multilayer liposomes can be produced, and when these multilayer liposomes are irradiated with ultrasonic waves, monolayer liposomes of a hundred and several tens of nanometers are produced.

On the other hand, there is also a method of using supercritical carbon dioxide instead of the organic solvent. In a pressure vessel, supercritical carbon dioxide is mixed and reacted with a liposome membrane component, a pharmaceutical compound, and a pharmaceutical additive under the conditions of 40 to 65 ° C. and 10 to 30 MPa. It is said that when the pressure vessel is depressurized to remove carbon dioxide, an aqueous dispersion of liposomes containing the pharmaceutical compound can be obtained (Patent Document 1). As the pharmaceutical additive, a buffer such as tromethamole, a chelating agent, an inorganic salt, a pharmacologically active substance, an osmoregulator, a stabilizer, an antioxidant, a viscosity modifier, a preservative and the like are described. In an embodiment, dipalmitoylphosphatidylcholine as a liposomal material (DPPC), using hydrogenated soybean phosphatidylcholine (HSPC), manufactures liposomes in combination with trometamol and EDTANa ₂ -Ca. After the liposome production, a granulation operation is performed to obtain a multilayer liposome having an average particle size of 0.26 to 0.30 μm.

There is also a method for producing liposomes for encapsulation of 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, phospholipids [1-n-hexadecanoyl-2- (9-cis-octadecenoyl) -3-sn-phosphatidylcholine (POPC)] and cholesterol were used, and the molar ratio of POPC: cholesterol was 3: 1 liposome is produced.

Further, a liposome containing an encapsulating substance, which comprises adding an aqueous phase containing an encapsulating substance to a homogeneous mixed fluid of carbon dioxide and phospholipids or glycolipids under a supercritical state or a temperature or pressure condition above the critical point. There is also a manufacturing method of (Patent Document 3). A homogeneous mixture of phospholipids and supercritical carbon dioxide is combined with an auxiliary solvent such as ethanol to increase the solubility of the sparingly soluble encapsulant. According to this method, monolayer lamellar liposomes having a diameter of 50 nm to 800 nm can be produced. In the method using the supercritical carbon dioxide method, liposomes are not formed in a supercritical state, but the solubility rapidly decreases due to reduced pressure, and liposomes are obtained by mixing an aqueous solution with phospholipids that precipitate in the form of fine particles. Is manufactured. In Examples, monolayer liposomes are produced using dipalmitoylphosphatidylcholine (DPPC), hydrogenated soybean lecithin (HSPC), hydrogenated egg yolk lecithin, dioleylphosphatidylcholine (DOPC), and distearoylphosphatidylcholine (DSPC).

On the other hand, a chloroform solution in which egg yolk-derived phosphatidylcholine is dissolved is charged in a test tube, only the solvent is removed to form a lipid film on the inner wall of the test tube, glass beads and a buffer solution are added to the test tube, and then the test tube is added. 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 to liposome inclusions and decomposition / denaturation of the raw material due to an increase in the solution temperature. When high-frequency ultrasonic irradiation of 500 kHz to 20 MHz is used in combination to make the liposome particle size extremely fine and uniform to the nano level under conditions with little cavitation damage, the particle size distribution is distributed in the size of several tens of nm to 150 nm. It is possible to prepare high-quality liposomes that are narrow and free of foreign substances.

Japanese Unexamined Patent Publication No. 2006-298844 Japanese Unexamined Patent Publication No. 06-315624 Japanese Patent No. 4296341 JP-A-2007-7625

Supervised by Kaoru Akiyoshi and Kaoru Tsujii; edited by Naoto Oku, Ryoichi Kuboi and Hirokazu Hotani, "New Developments in Liposomal Applications: Toward the Development of Artificial Cells", published by NTS, published in June 2005, page 33, 34 Tetsuro Yoshimura, "Development of New Liposomal Fabrication Technology and Application to Medical Care", [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 membrane of liposomes, and cholesterol-free liposomes have not been obtained.

Further, the method described in Patent Document 1 is a method for producing a multilayer liposome using supercritical carbon dioxide. It is necessary to use a formulation aid such as tromethamole or a chelating agent together with phospholipids. Therefore, it is desired to develop a method for producing a multilayer liposome without using a pharmaceutical aid.

The method described in Patent Document 3 also uses supercritical carbon dioxide as in Patent Document 1, but in the examples, only monolayer liposomes are produced. Compared with Patent Document 1 in which supercritical carbon dioxide is used and the same phospholipid is used as a membrane component to produce a multilayer liposome, Patent Document 1 prepares a multilayer liposome in combination with phospholipid and cholesterol. On the other hand, in Patent Document 3, only monolayer liposomes are prepared without using cholesterol. Therefore, it is desired to develop a method for producing multilamellar liposomes without using cholesterol.

All of the above Patent Documents 1 to 4 use glycerophospholipid as a phospholipid, and do not produce liposomes containing sphingolipid as a membrane constituent. Liposomes are applied to DDS and the like, but glycerophospholipids and sphingolipids also differ in metabolism in vivo. Therefore, it is desired to develop a method for producing liposomes containing sphingolipid as a main component.

Further, the method described in Patent Document 4 is to prepare a phospholipid membrane in advance using an organic solvent and then irradiate ultrasonic waves in two steps. Since an organic solvent is used, it may affect the manufacturing environment and product safety, and the process of multiple ultrasonic irradiations is also complicated. Therefore, it is desired to develop a method for producing a liposome containing a sphingolipid more easily without using an organic solvent.

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

A further object of the present invention is to provide a multilamellar liposome containing a sphingolipid as a constituent.

The present inventors reacted a mixture of sphingolipid and carbon dioxide in a critical state by irradiating it with a wave of a specific frequency, and the liposome suspension obtained after carbon dioxide removal contained multilamella liposomes. We found that and completed the present invention. In addition, glycerophospholipids and sterols can be used in combination with sphingolipids.

That is, in the present invention, a sphingolipid dispersed in an aqueous solvent is stored in a high-pressure reactor, carbon dioxide having a temperature of 30 to 70 ° C. and a pressure of 0.1 to 50 MPa is sealed, and a wave having a frequency of 10 to 500 kHz is irradiated. The present invention provides a method for producing a liposome, which comprises reacting with the same.

The present invention also provides a method for producing the liposome, which comprises dispersing glycerophospholipids and / or sterols together with the sphingolipid in the aqueous solvent.

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

The present invention also provides a method for producing a liposome, which comprises producing a multilamella liposome by irradiating the carbon dioxide with a wave of a frequency of 20 to 100 kHz at a temperature of 40 to 60 ° C. and a pressure of 10 to 30 MPa. Is what you do.
The present invention also provides a method for producing the liposome, wherein the multilamellar liposome is composed of a sphingolipid and has a particle size of 30 to 150 nm.
The present invention also provides a method for producing the liposome, wherein the multilamellar liposome contains sphingomyelin and glycerophospholipids and / or sterols.

The present invention also provides multilamellar liposomes composed only of sphingolipids and having a particle size of 30 to 150 nm.

According to the present invention, liposomes containing sphingolipids can be produced without using an organic solvent, a pharmaceutical additive, or sterols.

Also provided are multilamellar liposomes containing sphingolipids.

It is an electron microscope image of the liposome suspension prepared in Example 1. It is an electron microscope image of the liposome suspension prepared in Example 2. It is an electron microscope image of the liposome suspension prepared in Example 3. It is an electron microscope image of the liposome suspension prepared in Example 5. It is an electron microscope image of the liposome suspension prepared in Example 6. It is an electron microscope image of the liposome suspension prepared in Example 7. It is an electron microscope image of the liposome suspension prepared in Example 13. It is an electron microscope image of the liposome suspension prepared in Example 14. It is an electron microscope image of the 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 of the liposome suspension.

The first of the present invention is that a sphingolipid dispersed in an aqueous solvent is stored in a high-pressure reactor, carbon dioxide having a temperature of 30 to 70 ° C. and a pressure of 0.1 to 50 MPa is sealed, and a wave having a frequency of 10 to 500 kHz. It is a method for producing a liposome, which comprises irradiating and reacting with. Hereinafter, the present invention will be described in detail.

The production method of the present invention is characterized by containing sphingolipid as a constituent component of liposome. As shown in the examples described later, it was found that liposomes consisting only of sphingolipids can be produced without using sterols, auxiliaries such as buffers and chelating agents, and organic solvents such as ethanol and chloroform. be. The sphingolipid is a compound containing ceramide in which a fatty acid is acid-amide bonded to sphingoid, and further, phosphoric acid and a base are bonded. For example, sphingomyelin, ceramide phosphatidylethanolamine, ceramide phosphatidylglycerol, ceramide phosphatidylglycerol phosphate, 1,2-dimyristylamide-1,2-deoxyphosphatidylcholine and the like.

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 sphingolipid. Examples of sterols include cholesterol, dihydrocholesterol, cholesterol ester, phytosterol, cytosterol, stigmasterol, campesterol, cholestanol, lanosterol and the like. It may also be a sterol derivative such as 1-O-sterol glucoside, 1-O-sterol maltoside or 1-O-sterol galactoside.

In addition, glycerophospholipids may be used in combination with sterols or instead of sterols. Examples of glycerophospholipid lipids include phosphatidylcholine, dipalmitoylphosphatidylcholine, distearoylphosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidic acid, phosphatidylglycerol, phosphatidylinositol, dimyristylphosphatidylcholine, cardiopine, egg yolk lecithin, and hydrogenated egg lecithin. There are supplemented soybean lecithin, hydrogenated soybean phosphatidylcholine and the like.

The proportion of sphingolipid in the liposome component is 50 to 100% by mass, more preferably 60 to 100% by mass. Glycerophospholipids and sterols can be used in combination, and the total amount of 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. %, Especially preferably 0 to 20% by mass.

In the present invention, a sphingolipid dispersed in an aqueous solvent is used. When carbon dioxide under predetermined conditions is mixed with this and irradiated with wave irradiation of a specific frequency, liposomes can be produced without using an organic solvent or an auxiliary agent. By using a sphingolipid dispersed in an aqueous solvent, liposomes in which the aqueous solvent is encapsulated are produced. Therefore, as the aqueous solvent, purified water, distilled water, pure water or the like is suitable. It is not necessary to add other components to the aqueous solvent.

On the other hand, liposomes in which any compound A is encapsulated can also be produced. In this case, a solvent in which the component A is dissolved or dispersed in water or the like can be used as the aqueous solvent. At this time, a buffer solution, a chelate, an organic solvent such as alcohol, or the like can be included as a solubilizing agent for the component A.

Liposomal constituents such as sphingolipids, glycerophospholipids, and sterols are solid at room temperature. In order to disperse this in water, for example, a container in which the liposome constituent component and the aqueous solvent are charged may be stored in the bath portion of the bath-type Sony Kata and Sonyation may be performed to make the liposome constituent component finer. Liposomal constituents can be dispersed in water without the use of organic solvents. The amount of the liposome constituent component added is 0.001 to 10 parts by mass, preferably 0.01 to 1 part by mass with respect to 100 parts by mass of water.

The aqueous solvent in which the liposome constituents are dispersed is stored in a high-pressure reactor. Next, the reactor was charged with a temperature of 30 to 70 ° C., preferably 30 to 60 ° C., more preferably 40 to 60 ° C., particularly preferably 50 to 60 ° C., a pressure of 0.1 to 50 MPa, preferably 0.1 to 30 MPa. Carbon dioxide is supplied so as to be more preferably 1 to 30 MPa.

The mixture is then irradiated with waves at a frequency of 10-500 kHz, more preferably 10-300 kHz, particularly preferably 20-100 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 MLV (multi lameller vesicles) are subjected to sonication, SUVs (small unilameller vesicles) having a particle size of a hundred and several tens of nanometers are obtained. Become. However, in the present invention, when sphingolipid 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 sphingolipid 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 more. For example, 10 minutes to 5 hours, preferably 30 minutes to 2 hours. The method of the present invention is characterized in that liposomes are composed only of sphingolipids, and the liposomes produced are not limited to multilamella liposomes, and may include unilamella liposomes.

In the present invention, the layer structure and the average particle size 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, preferably 20 to 50 kHz. When the reaction is carried out at a pressure of 15 MPa in the above temperature range, multilamellar liposomes having a substantially uniform particle size can be produced in a temperature range of 40 to 60 ° C. Further, when the reaction is carried out at a temperature higher than 40 ° C., the separability of the multilamellar liposome can be improved. In addition, more multi-layered multilamellar liposomes can be produced in the temperature range of 40 to 60 ° C. by ultrasonic irradiation at 28 to 45 kHz.

The liposome is composed of a lipid bilayer membrane. In the present specification, the unilamella liposome means a liposome composed of one layer of lipid bilayer membrane, and the multilamella liposome means a liposome composed of two or more lipid bilayer membranes. do.

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

The liposome produced in the present invention contains sphingolipid as an essential component. In principle, the size of liposomes depends on the spontaneous curvature based on the morphology of phospholipid molecules containing hydrated water, etc., and is affected by the preparation environment leading to the formation. Then, it becomes a huge monolayer liposome (GUV: Giant Unilamellar Vesicle), becomes a multilamella liposome under a vortex such as vortex treatment, and becomes a unilamella liposome having an average particle size of 200 nm or less by sonication (Non-Patent Document 2). ). However, according to the present invention, as shown in Examples described later, multilamellar liposomes composed only of sphingolipids can be produced. The average particle size when converted from the average molecular weight measured by the particle size distribution measuring device was more than 1,000 nm, but the particle size of the multilamellar liposome was 5 to 200 nm according to the calculation based on the electron microscope image. rice field. However, the liposomes that can be produced in the present invention are not limited to multilamellar liposomes. Further, regardless of the layer structure, liposomes having a large average particle size such as GUV can be used as a research material for biological membranes.

The second of the present invention is a multilamellar liposome composed of sphingolipids. The 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 multilamella liposome of the present invention is 5 to 200 nm, which is suitable as a liposome preparation for medical use. There is a method of utilizing vascular permeability to deliver liposomes to a target site in vivo. Since the permeability is enhanced in the blood vessel wall where angiogenesis is active such as cancer tissue and inflamed site, particles having a diameter of several to 200 nm, which are difficult to pass through in normal tissue, 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 inflamed sites.

Next, the present invention will be specifically described with reference to examples, but these examples do not limit the present invention in any way.

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

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

(Examples 2 to 15)
The liposome suspension was recovered according to Example 1 except that the temperature, pressure, and ultrasonic conditions were changed to Table 1. In addition, the average molecular weight was measured using a particle size distribution measuring device (Shimadzu SALD-2200) and converted to the average particle size of liposomes. The results are shown in Table 1. "-" In the table means unmeasured. In addition, electron microscopic images of liposomes contained in the liposome suspension are shown in FIGS. 2 to 9.

(Example 16)
A liposome suspension was obtained in the same manner as in Example 1 except that the mixture was sealed so that the temperature was 50 ° C. and the pressure was 20 MPa, and the mixture was stirred at 1,500 rpm for 60 minutes instead of ultrasonic irradiation. The liposomes constituting the liposome suspension were multilamella liposomes. The average particle size of the liposomes obtained from the average molecular weight measured using the particle size distribution measuring device was converted to 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) It was found that when sphingolipids dispersed in an aqueous solvent were stored in a high-pressure reactor and reacted at a temperature of 40 to 60 ° C. and a pressure of 10 to 30 MPa, multilamellar liposomes were produced under any of the conditions.
(2) As shown in the electron microscope images of FIGS. 2, 5 and 8 showing the results of Examples 2, 6 and 14, when the reaction is carried out at a pressure of 15 MPa in a temperature range of 40 to 60 ° C. , It was observed that the particle size of the multilamellar liposomes tended to be uniform.
(3) As shown in the electron microscope images of FIGS. 1, 4, and 7 showing the results of Examples 1, 5, and 13, when the reaction was carried out at a pressure of 10 MPa, the temperature was 50 rather than 40 ° C. The separability of the multilamellar liposome was superior at ° C. and 60 ° C.
(4) According to the results of Examples 7, 8 and 11, it depended on the irradiation time of ultrasonic waves, and according to the results of Examples 3, 7 and 15, it depended on the reaction temperature. When the temperature is 50 ° C. and the pressure is 20 MPa and the ultrasonic wave is 28 kHz, the average particle size is the shortest when the irradiation time is 60 minutes, and when the pressure is 20 MPa and the ultrasonic wave is 28 kHz, the average particle size is the average when the temperature is 50 ° C. The particle size was the shortest.
(5) As shown in the comparison between FIGS. 6 and 10 (B) showing the results of Example 7 and Example 16, when the temperature is 50 ° C. and the pressure is 20 MPa, it depends on the ultrasonic treatment or the stirring treatment. It was observed that the layer thickness was different and the layer was more multi-layered when irradiated with ultrasonic waves of 28 kHz.

Claims (7)

A sphingolipid dispersed in an aqueous solvent is stored in a high-pressure reactor, carbon dioxide having a temperature of 30 to 70 ° C. and a pressure of 0.1 to 50 MPa is sealed, and a wave with a frequency of 10 to 500 kHz is irradiated to react. A method for producing a liposome. The method for producing a liposome according to claim 1, wherein the glycerophospholipid and / or sterols are dispersed together with the sphingolipid in the aqueous solvent. The method for producing a liposome according to claim 1 or 2, wherein the sphingolipid is sphingomyelin. The liposome according to any one of claims 1 to 3, wherein the carbon dioxide has a temperature of 40 to 60 ° C. and a pressure of 10 to 30 MPa, and is irradiated with a wave having a frequency of 20 to 100 kHz to produce a multilamella liposome. Manufacturing method. The method for producing a liposome according to claim 4, wherein the multilamellar liposome is composed of a sphingolipid and has a particle size of 30 to 150 nm. The multilamellar liposomes comprise a glycerophospholipids and / or sterol with sphingomyelin, method for producing a liposome according to claim 5, wherein. Multilamellar liposomes composed only of sphingolipids and having a particle size of 30 to 150 nm.

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