WO2010110118A1

WO2010110118A1 - Method for producing liposomes

Info

Publication number: WO2010110118A1
Application number: PCT/JP2010/054409
Authority: WO
Inventors: 武寿磯田; 武志和田; 康之元杭
Original assignee: コニカミノルタホールディングス株式会社
Priority date: 2009-03-23
Filing date: 2010-03-16
Publication date: 2010-09-30
Also published as: JP5494054B2; JP2010248171A

Abstract

Disclosed is a method for producing liposomes, which is characterized by comprising: a primary emulsification step wherein a W1/O emulsion is prepared; a secondary emulsification step wherein a W1/O/W2 emulsion is prepared by emulsifying the W1/O emulsion and an aqueous solvent (W2) using a membrane that has a pore diameter of not less than 0.1 μm but not more than 5.0 μm; and a step wherein a suspension of liposomes is prepared by removing the organic solvent contained in the W1/O/W2 emulsion. By this method, liposomes including a large number of unilamellar (univesicular) liposomes which have a nano-sized particle diameter, while maintaining a relatively high encapsulation rate can be efficiently obtained. The method for producing liposomes enables the production of liposomes which include almost no multivesicular liposomes and are particularly suitable for water-soluble drugs.

Description

Method for producing liposome

The present invention relates to a method for producing liposomes used in the fields of pharmaceuticals, cosmetics, foods and the like.

Liposomes are closed vesicles composed of a single or multiple lipid bilayer membrane, and are known to be able to retain water-soluble and hydrophobic drugs in the inner aqueous phase and inside the lipid bilayer membrane, respectively. In addition, the lipid bilayer membrane of liposomes is similar to biological membranes, so it has high in-vivo safety, so various applications such as pharmaceuticals for DDS (Drug Delivery System) have been attracting attention, Research and development is ongoing.

So far, many methods for producing liposomes have been proposed, but there are few general-purpose technologies excellent in cost and industry that can produce liposomes that can be approved as pharmaceuticals. The water-soluble drug can be contained in the internal aqueous phase of the liposome. However, it is generally accepted that the encapsulation method (the final liposome suspension (liposome and external aqueous phase)) is included in the liposome production method. The ratio of the mass of the drug encapsulated in the liposome to the total mass of the drug contained is about 20%, and in many cases, the unencapsulated drug is lost. For example, the reverse phase evaporation method, such as a liposome production method having a higher encapsulation rate, is poor in reproducibility and is not industrially suitable. In recent years, attention has been paid to nucleic acid drugs and the like, but since they are unstable in a living body alone, a carrier that stabilizes them is desired. For this reason, a method capable of efficiently encapsulating a water-soluble drug and capable of producing liposomes having an average particle size of nano-size (preferably 200 nm or less suitable for intravenous injection) is desired.

In Japan, three types of liposome preparations, “VISUDYNE” (registered trademark), “AmBismome” (registered trademark), and “DOXIL” (registered trademark), are commercially available. However, bijudyne and ambisome encapsulate hydrophobic drugs that are easily encapsulated in liposomes. In addition, Doxil achieves a high encapsulation rate of 80% or more by a method called a remote loading method in which a drug is added to the external aqueous phase after liposome formation and the drug is encapsulated in the liposome using a difference in internal and external concentrations. . However, this method can be used only when it is a special low-molecular-weight water-soluble drug that is initially water-soluble but can form a salt in the inner aqueous phase and can pass through the lipid membrane of the liposome. Can not.

As a process for reducing the particle size of liposomes, sizing with an extruder has been conventionally performed, but although this process makes the particle size nano-sized, the amount of drug encapsulated in the liposome is greatly reduced. End up.

Non-Patent Document 1 proposes a microencapsulation method in which a W / O / W emulsion emulsified in two stages is dried in a liquid to form liposomes. Although this method can encapsulate drugs more efficiently than other methods, the size of the resulting liposome is large, and in Non-Patent Document 2, the encapsulation rate of drugs with high water solubility (low log P) is not so high It has been reported that it does not improve.

Patent Document 1 describes that, in a method for producing a liposome preparation including a two-stage emulsification process, the inclusion rate of drugs can be improved by using a lipid having a larger carbon number as the lipid component of the liposome. ing. However, since most of the liposomes obtained by this method are multivesicular liposomes (containing many non-concentric aqueous chambers) and the average particle size is micrometer size, this method allows uniform nano-sized liposomes. It is difficult to obtain monolayer liposomes (single cell liposomes).

Patent Document 2 discloses a method for producing a W / O / W emulsion by allowing a W / O emulsion to permeate through a porous glass membrane, which is useful in an inner aqueous phase by using a predetermined ester compound as an aqueous emulsifier. A method of suppressing material leakage is described. However, this document does not specifically describe the use of the above method for liposome production.

Patent Document 3 describes a method for producing liposomes from a formed W / O / W emulsion, in which a solvent is removed from the emulsion and subjected to cross flow filtration. However, liposomes obtained by this method are multivesicular liposomes, and it is difficult to obtain uniform nano-sized monolayer liposomes (monovesicular liposomes).

JP-T-2001-505549 JP 2003-164754 A JP-T-2001-522870

The present invention can efficiently obtain liposomes (single-cell liposomes) having a nano-size particle size and containing a large amount of monolayers while maintaining a relatively high encapsulation rate, and there is almost no multivesicular liposome, It is an object of the present invention to provide a method for producing liposomes suitable for water-soluble drugs.

As a result of intensive studies, the present inventor performs membrane emulsification using a membrane having a pore diameter of 0.1 μm or more and 5.0 μm or less in a secondary emulsification step when preparing a W / O / W emulsion in two steps. As a result, the inventors have unexpectedly found that the above problems can be solved, and have completed the present invention.

That is, the method for producing a liposome of the present invention is characterized by having the following steps (1) to (3);
(1) Primary emulsification step: a step of preparing a W1 / O emulsion by emulsifying the organic solvent (O), the aqueous solvent (W1), and the mixed lipid component (F1);
(2) Secondary emulsification step: The W1 / O emulsion obtained in the above step (1) and the aqueous solvent (W2) are emulsified using a membrane having a pore diameter of 0.1 μm or more and 5.0 μm or less, Preparing a W1 / O / W2 emulsion;
(3) A step of preparing a liposome suspension by removing the organic solvent contained in the W1 / O / W2 emulsion obtained by the step (2);
However, you may perform the said primary emulsification process, after adding the substance further included in a liposome.

As the emulsification method of the step (2), the W1 / O emulsion is passed through a membrane having a pore diameter of 0.1 μm or more and 5.0 μm or less and dispersed as droplets in the aqueous solvent (W2), An emulsification method for preparing a W1 / O / W2 emulsion is preferred. In the step (2), a membrane treatment step of passing the W1 / O / W2 emulsion obtained by membrane emulsification of the W1 / O emulsion through a membrane having a pore diameter of 0.1 μm or more and 5.0 μm or less. It is also preferable to prepare the final W1 / O / W2 emulsion by further having it. As the membrane having a pore diameter of 0.1 μm or more and 5.0 μm or less in the step (2), an SPG membrane is preferably used. It is preferable that medical drugs are used as the substance to be encapsulated in the liposome, and the average particle size of the obtained liposome is 50 nm or more and 200 nm or less. In the step (2), in addition to the aqueous solvent (W2) and the W1 / O emulsion, it is preferable to use at least one of a water-soluble emulsifier or a mixed lipid component (F2) that does not break the liposome lipid membrane.

The “average particle diameter” in the present invention refers to the volume average particle diameter. The volume average particle size of the W1 / O emulsion or liposome was determined by diluting these solutions 10-fold with a chloroform / hexane mixed solvent (volume ratio: 4/6) as shown in the examples described later. It is a value calculated using an optical light scattering nanotrack particle size analyzer (UPA-EX150, Nikkiso Co., Ltd.).

According to the production method of the present invention, it becomes possible to efficiently produce liposomes having a nano-sized average particle size suitable for pharmaceuticals and maintaining a high encapsulation rate. Moreover, many of the obtained liposomes are monolayer (monovesicles), and the ratio of those remaining as multivesicular liposomes is remarkably reduced as compared with the prior art. In particular, by using an SPG membrane in the secondary emulsification step, it can be an industrially advantageous method with a low cost and a large throughput. In addition, when the primary emulsification step is performed after adding substances (especially water-soluble drugs) that encapsulate the liposomes, the above properties are imparted to the liposomes while being relatively higher than the conventional one. It is possible to achieve liposomes that can be applied to pharmaceutical products such as injection at an encapsulation rate. By using such a method for producing liposomes of the present invention, the productivity of liposomes suitable for various applications can be remarkably improved.

-Raw materials-
Mixed lipid component (F1) ・ (F2)
The mixed lipid component (F1) used in the primary emulsification step mainly constitutes the inner membrane of the lipid bilayer of the liposome, and in some cases also contributes to the outer membrane. The mixed lipid component (F2) mainly constitutes the outer membrane of the liposome. The mixed lipid components (F1) and (F2) may have the same composition or different compositions.

The compounding composition of these mixed lipid components is not particularly limited, but in general, phospholipids (lecithin derived from animals and plants; phosphatidylcholine, phosphatidylserine, phosphatidylglycerol, phosphatidylinositol, phosphatidic acid or fatty acid esters thereof) Consists mainly of certain glycerophospholipids; sphingophospholipids; derivatives thereof, and sterols (cholesterol, phytosterols, ergosterol, derivatives thereof, etc.) that contribute to the stabilization of lipid membranes. , Aliphatic amines, long chain fatty acids (oleic acid, stearic acid, palmitic acid, etc.), and other compounds imparting various functionalities may be blended. In particular, F2 contains a lipid component necessary for imparting functionality as a DDS, such as PEGylated phospholipid, so that the liposome surface can be efficiently modified. The blending ratio of the mixed lipid component may be appropriately adjusted according to the application while taking into consideration properties such as the stability of the lipid membrane and the behavior of the liposome in vivo.

Aqueous solvent (W1) / (W2), organic solvent (O)
As the aqueous solvents (W1) and (W2) and the organic solvent (O), known general solvents can be used. Examples of the aqueous solvent include pure water and those to which salts, saccharides, and other compounds suitable for osmotic pressure adjustment and pH adjustment are added. Further, according to the use of liposome, W1 may contain a gelling agent such as gelatin, a thickening polysaccharide such as dextran, and a charged polymer such as chitin / chitosan and poly-L-lysine. . Examples of the organic solvent include those that are not miscible with water, such as hexane (n-hexane), chloroform, and methylene chloride, and these may be used alone or in combination. Moreover, it is preferable to use an organic solvent having a boiling point lower than that of water.

Substances to be encapsulated In the present invention, substances to be encapsulated in liposomes (collectively referred to as drugs) are not particularly limited, and various substances known in the fields of pharmaceuticals, cosmetics, foods, etc., depending on the use of liposomes Can be used.

Examples of water-soluble drugs for medical use include, for example, contrast agents (nonionic iodine compounds for X-ray contrast, complexes composed of gadolinium and chelating agents for MRI contrast), anticancer agents, and the like. (Adriamycin, viralubicin, vincristine, taxol, cisplatin, mitomycin, 5-fluorouracil, irinotecan, estrasite, epirubicin, carboplatin, intron, gemzar, methotrexate, cytarabine, etc.), antibacterial, antioxidant, anti-inflammatory, blood circulation promotion Agent, whitening agent, rough skin prevention agent, anti-aging agent, hair growth promoting agent, moisturizer, hormone agent, vitamins, nucleic acid (DNA or RNA sense strand or antisense strand, plasmid, vector, mRNA, siRNA, etc.) , Proteins (enzymes, antibodies, peptides, etc.) A substance having a pharmacological action, such as a vaccine preparation (toxoid such as tetanus as an antigen; diphtheria, Japanese encephalitis, polio, rubella, mumps, hepatitis or other virus as an antigen; DNA or RNA vaccine, etc.) Pharmaceutical aids such as dyes / fluorescent dyes, chelating agents, stabilizers, preservatives and the like can be mentioned.

-Method for producing liposome-
The method for producing a liposome of the present invention includes the following steps (1) to (3), and other steps can be appropriately combined as necessary.

(1) Primary emulsification step The primary emulsification step is a step of preparing a W1 / O emulsion by emulsifying the organic solvent (O), the aqueous solvent (W1), and the mixed lipid component (F1).

The method for preparing the W1 / O emulsion is not particularly limited, and can be performed using an apparatus such as an ultrasonic emulsifier, a stirring emulsifier, a membrane emulsifier, or a high-pressure homogenizer. The means is not particularly limited, but a method in which the average particle diameter can be controlled in a wide range and the obtained W1 / O emulsion is monodispersed is preferable.

The pH of the aqueous solvent (W1) is selected in the range of 3 to 10, and an appropriate buffer may be used to adjust the pH. For example, when oleic acid is used for the mixed lipid component, the pH is preferably 6 to 8.5.

In the primary emulsification step, the ratio of the mixed lipid component (F1) added to the organic solvent (O), the volume ratio of the organic solvent (O) and the aqueous solvent (W1), and other operating conditions are the conditions of the subsequent secondary emulsification step. In addition, it can be appropriately adjusted according to the emulsification method to be adopted, taking into consideration the aspect of the liposome finally prepared. Usually, the ratio of the mixed lipid component (F1) is 1 to 50% by mass with respect to the organic solvent (O), and the volume ratio of the organic solvent (O) and the aqueous solvent (W1) is 100: 1 to 1: 2. is there. Since the average particle size of the W1 / O emulsion needs to be at least ½ or less of the pore size of the membrane used in the secondary emulsification step, 50 to 1,000 nm within a range not exceeding this upper limit. Is more preferable, and 50 to 200 nm is more preferable.

In the present invention, in order to encapsulate the water-soluble drug in the liposome, (i) the water-soluble drug is preliminarily dissolved in the aqueous solvent (W1) in the primary emulsification step, and is encapsulated at the end of the secondary emulsification step. (Ii) after obtaining (empty) liposomes that do not contain water-soluble drugs, and then redispersing the aqueous solvent in which the liposomes are dispersed or the lyophilized product of the liposomes. Any method can be used in which a water-soluble drug is added to an aqueous solvent, and the liposome is incorporated into the liposome by stirring. In the production method of the present invention, even when the method (i) is used, the encapsulation rate is relatively high, and water-soluble drugs can be efficiently encapsulated in liposomes. Note that water-insoluble drugs are also added to the aqueous solvent (W1) or the organic solvent (O) in advance at the time of the primary emulsification step as in (i) above, or empty as in (ii) above. It is possible to encapsulate the liposome by adding it after obtaining the liposome.

(2) Secondary emulsification step In the secondary emulsification step, the W1 / O emulsion obtained by the above step (1) has a pore diameter of 0.1 µm to 5.0 µm, preferably 0.1 µm to 3.0 µm. This is a step of preparing a W1 / O / W2 emulsion dispersed as droplets in an aqueous solvent (W2) by a membrane emulsification method using a membrane having the same.

As the porous film having the above pore diameter, an SPG (Shirasu Porous Glass) film is suitable, and can be purchased from, for example, SPG Techno Co., Ltd.

In this secondary emulsification step, for example, the W1 / O emulsion is passed through a membrane having a pore diameter of 0.1 μm or more and 5.0 μm or less, and dispersed as droplets in the aqueous solvent (W2), whereby W1 / O An O / W2 emulsion can be prepared. Further, after obtaining a W1 / O / W2 emulsion by membrane emulsification by the above method or other methods, the obtained W1 / O / W2 emulsion is further passed through a membrane having a pore diameter of 0.1 μm or more and 5.0 μm or less. The final W1 / O / W2 emulsion can also be prepared by performing the membrane treatment. This film treatment may be performed not only once but a plurality of times. The membrane used for membrane emulsification and the membrane used for membrane treatment may be the same or different, and the pore diameters of the membrane may be the same or different. By performing the membrane treatment, the monodispersity of the average particle diameter of the W1 / O / W2 emulsion can be improved (contributing to narrowing the spread of the particle size distribution).

In particular, by making the pore size of the membrane used for membrane treatment smaller than the pore size of the membrane used for membrane emulsification, compared to the case of preparing a W1 / O / W2 emulsion by one membrane emulsification without membrane treatment. It is more preferable because the load on the membrane can be reduced. The pressure required to allow the emulsion to pass through the membrane can be reduced, which can contribute to extending the life of the membrane and shorten the processing time required for the secondary emulsification process, improving the productivity of liposomes and lower costs. It is also advantageous to make it easier.

In the secondary emulsification step, in addition to the aqueous solvent (W2) and W1 / O emulsion, a water-soluble emulsifier that does not break the liposomal lipid membrane composed of the mixed lipid component (F1), the mixed lipid component (F2), or both It may be used. In particular, it is preferable to use a water-soluble emulsifier that does not destroy the liposomal lipid membrane because it can reduce the number of multivesicular liposomes. Many emulsifiers are known in the field of surface chemistry, and typically, proteins, polysaccharides, nonionic surfactants, and the like are used in the emulsification / dispersion process as water-soluble emulsifiers. Among these known water-soluble emulsifiers, water-soluble emulsifiers that do not destroy the liposome lipid membrane can be appropriately selected and used. Examples of such water-soluble emulsifiers that do not break the liposome lipid membrane include sodium caseinate as a protein, dextran as a polysaccharide, and polyalkylene glycol derivatives as nonionic surfactants. In particular, when phosphatidylcholine is blended with the mixed lipid component (F1), a protein emulsifier such as sodium caseinate is preferable as a water-soluble emulsifier that does not destroy such a liposomal lipid membrane.

The mixing mode (addition order, etc.) of the aqueous solvent (W2), W1 / O emulsion, water-soluble emulsifier and / or mixed lipid component (F2) is not particularly limited, and an appropriate mode may be selected. For example, when F2 is mainly composed of a water-soluble lipid, such F2 and / or a water-soluble emulsifier can be added to W2 in advance, and a W1 / O emulsion can be added thereto for emulsification. On the other hand, when F2 is mainly composed of a fat-soluble lipid, (after preparation of the W1 / O emulsion) such F2 is added to the oil phase of the W1 / O emulsion in advance, and if necessary, a water-soluble emulsifier is added. The emulsification treatment can be performed by adding to the added W2.

In the secondary emulsification step, the total proportion of the water-soluble emulsifier and the mixed lipid component (F2) added to the aqueous solvent (W2) or the mixed lipid component (F2) added to the organic solvent (O) of the W1 / O emulsion, The volume ratio between the W1 / O emulsion and the aqueous solvent (W2) and other operating conditions can be appropriately adjusted in consideration of the application of the liposome to be finally prepared. Usually, the total ratio of the components added to the aqueous solvent (W2) to the organic solvent (O) is 0.01 to 10% by mass with respect to them, and the volume ratio of the W1 / O emulsion to the aqueous solvent (W2) is 1: 100 to 2: 1.

(3) Solvent removal step The solvent removal step is a suspension of liposomes having lipid bilayers by removing the organic solvent (O) contained in the W1 / O / W2 emulsion obtained by the secondary emulsification step. This is a step of preparing a liquid.

The method for removing the solvent from the W1 / O / W2 emulsion is not limited to this, although the solvent of the W1 / O / W2 emulsion can be distilled off by heating or decompression according to a conventional method. Although it is affected by the type of solvent used, a condition range in which the solvent does not bump suddenly is set, and the temperature condition is preferably in the range of 0 to 60 ° C., more preferably 0 to 25 ° C. The decompression condition is preferably set within the range of the saturated vapor pressure of the solvent to atmospheric pressure, and more preferably within the range of + 1% to 10% of the saturated vapor pressure of the solvent. When different solvents are used in combination, conditions that match the solvent species having a higher saturated vapor pressure are preferred. These removal conditions may be combined within a range in which the solvent does not bump. For example, when a heat-sensitive chemical is used, it is preferable that the solvent is distilled off at a lower temperature and under reduced pressure. The solvent removal may not require stirring of the W1 / O / W2 emulsion, but the solvent removal proceeds more uniformly with stirring. Moreover, the time required for solvent removal can be shortened by widening the gas-liquid interface.

The liposome finally obtained by the production method of the present invention contains almost no multivesicular liposome, and the average particle size can be controlled between 50 and 1,000 nm. In particular, liposomes having an average particle size of 50 to 200 nm have almost no risk of occluding capillaries and can pass through gaps formed in blood vessels in the vicinity of cancer tissues. Convenient above.

(Measurement method of calcein inclusion rate)
The fluorescence intensity (F _total ) of the entire liposome aqueous solution (1 ml) was measured with a spectrophotometer (U-3310, JASCO Corporation). Next, 30 μL of 0.01 M CoCl ₂ Tris-HCl buffer was added and the fluorescence of calcein leaked into the aqueous solvent (W2) was quenched by Co ²⁺ to measure the fluorescence intensity (F _in ) in the vesicle. Furthermore, vesicles were prepared under the same conditions as the sample without adding calcein, and the fluorescence (F ₁ ) emitted by the lipids themselves was measured. The inclusion rate was calculated from the following formula;
Inclusion rate E (%) = (F _in −F _l ) / (F _total −F _l ) × 100.

(Measurement method of inclusion rate of cytarabine)
The solid (liposome) was separated (decanted) with an ultracentrifuge, and the supernatant and solid were each quantified by HPLC. Assayed using a Varian Polaris C18-A (3 μm, 2 × 40 mm) as the HPLC column. The inclusion rate of cytarabine was calculated from the compound not included and the absolute value of the compound included.

(Measurement method of volume average particle diameter)
The aqueous liposome solution was diluted 10 times with a chloroform / hexane mixed solvent (volume ratio: 4/6), and the volume average particle size was determined using a dynamic light scattering nanotrack particle size analyzer (UPA-EX150, Nikkiso Co., Ltd.). Calculated.

(Method for observing multivesicular liposomes)
Using a slide glass with a depression, a preparation of 25 μL of an aqueous liposome solution was prepared and observed under bright field conditions with an upright microscope (Axio Imager. A1, manufactured by Carl Zeiss) to measure the number of multivesicular liposomes.

Example 1
(Production of W1 / O emulsion by primary emulsification process)
15 ml of hexane containing 0.3 g of egg yolk lecithin “COATSOME NC-50” (manufactured by NOF Corporation) having a phosphatidylcholine content of 95%, 0.152 g of cholesterol (Chol) and 0.108 g of oleic acid (OA) (O) and 5 ml of Tris-HCl buffer solution (pH 8, 50 mmol / L) containing calcein (0.4 mM) was used as the aqueous dispersion phase (W1) for the inner aqueous phase. The mixed solution was put into a 50 ml beaker, and an ultrasonic dispersion device “UH-600S” (manufactured by SMT Co., Ltd.) equipped with a 20 mm diameter probe was irradiated with ultrasonic waves for 15 minutes at 25 ° C. went. The average particle size of the obtained W1 / O emulsion at room temperature was 247 nm.

(Production of W1 / O / W2 emulsion by secondary emulsification process)
Using the W1 / O emulsion obtained by the primary emulsification step as a dispersed phase, a W1 / O / W2 emulsion was produced by the SPG emulsification method. A cylindrical SPG membrane having a diameter of 10 mm, a length of 20 mm, and a pore diameter of 2.0 μm was used for an SPG membrane emulsifying device (trade name “external pressure type micro kit” manufactured by SPG Techno Co., Ltd.). ) 3% sodium caseinate tris-hydrochloric acid buffer solution (pH 8, 50 mmol / L) was filled, and the W1 / O emulsion was supplied from the apparatus inlet side to produce a W1 / O / W2 emulsion. . The pressure required for membrane emulsification was about 25 kPa.

(Production of liposomes by removal of organic solvent phase)
The W1 / O / W2 emulsion obtained by the secondary emulsification step was transferred to an open glass container without a lid and stirred at room temperature for about 24 hours to volatilize hexane. A suspension of fine liposome particles was obtained, and it was confirmed that calcein was contained in the particles. The obtained liposomes had an average particle size at room temperature of 233 nm and a calcein encapsulation rate of 71%, and multivesicular liposomes were not confirmed.

Example 2
The same operation as in Example 1 was performed except that an SPG membrane having a pore diameter of 5.0 μm was used in the secondary emulsification step and the pressure required for membrane emulsification was about 10 kPa. A suspension of fine liposome particles was obtained, and it was confirmed that calcein was contained in the particles. The obtained liposome had an average particle size at room temperature of 244 nm, a calcein encapsulation rate of 70%, and three multivesicular liposomes.

Example 3
(Production of W1 / O emulsion by primary emulsification process)
15 ml of a mixed solvent (hexane: dichloromethane = 8: 2) containing the same lipid composition as in Example 1 was used as the organic solvent phase (O), and Tris-HCl buffer (pH 7.4, 50 mmol) containing calcein (0.4 mM) was used. / L) 5 ml was used as an aqueous dispersion phase (W1) for the inner aqueous phase. These mixed liquids were put into a 50 ml beaker, and the same emulsification treatment as in Example 1 was performed. The average particle size of the obtained W1 / O emulsion at room temperature was 207 nm.

(Production of W1 / O / W2 emulsion by secondary emulsification process)
Using the W1 / O emulsion obtained by the primary emulsification step as a dispersed phase, a W1 / O / W2 emulsion was produced by the SPG emulsification method. A SPG membrane having a pore diameter of 1.0 μm was used in the same membrane emulsification apparatus as in Example 1, and Tris-hydrochloric acid buffer (pH 7.4) containing 3% sodium caseinate as an external aqueous phase solution (W2) on the outlet side of the apparatus. 50 mmol / L), and the W1 / O emulsion was supplied from the apparatus inlet side to produce a W1 / O / W2 emulsion. The pressure required for membrane emulsification was about 10 kPa.

(Production of liposomes by removal of organic solvent phase)
The W1 / O / W2 emulsion obtained by the secondary emulsification step was transferred to a closed container, stirred for about 4 hours under a reduced pressure room temperature of 500 mbar, and then stirred for about 18 hours under a reduced pressure room temperature of 180 mbar. Was volatilized. A suspension of fine liposome particles was obtained, and it was confirmed that calcein was contained in the particles. The obtained liposomes had an average particle size at room temperature of 203 nm and a calcein encapsulation rate of 49%, and multivesicular liposomes were not confirmed.

Example 4
In the primary emulsification step, emulsification was performed under a pressure condition of 100 MPa using a high-pressure homogenizer (Nanomizer, manufactured by Yoshida Kikai Kogyo Co., Ltd.). The same operation as in Example 3 was performed. The average particle size of the obtained W1 / O emulsion at room temperature was 127 nm, the average particle size of the obtained liposome at room temperature was 123 nm, the calcein encapsulation rate was 53%, and one multivesicular liposome Met. The pressure required for membrane emulsification was about 20 kPa.

Example 5
Except that 0.3 g of egg yolk lecithin “PL-100M” (manufactured by Kewpie) having a phosphatidylcholine content of 80%, 0.152 g of cholesterol (Chol) and 0.108 g of oleic acid (OA) were used as lipids. The same operation as 3 was performed. The average particle size of the obtained W1 / O emulsion at room temperature was 232 nm, the average particle size of the obtained liposome at room temperature was 237 nm, the calcein encapsulation rate was 62%, and multivesicular liposomes were confirmed. There wasn't. The pressure required for membrane emulsification was about 10 kPa.

Example 6
The same operation as in Example 3 was performed except that a mixed solvent (hexane: ethyl acetate = 2: 1) was used for the organic solvent phase (O) and the reduced pressure condition for removing the organic solvent was 180 mbar and 22 hours. The average particle size of the obtained W1 / O emulsion at room temperature was 189 nm, the average particle size of the obtained liposome at room temperature was 197 nm, the calcein encapsulation rate was 42%, and there were 6 multivesicular liposomes Met. The pressure required for membrane emulsification was about 10 kPa.

Example 7
The same operation as in Example 3 was performed except that an SPG membrane having a pore diameter of 5.0 μm was used in the secondary emulsification step. The average particle size of the obtained W1 / O emulsion at room temperature was 203 nm, the average particle size of the obtained liposome at room temperature was 210 nm, the calcein encapsulation rate was 46%, and one multivesicular liposome Met. The pressure required for membrane emulsification was about 2 kPa.

Example 8
The same operation as in Example 4 was performed except that an SPG membrane having a pore diameter of 0.2 μm was used in the secondary emulsification step. The obtained liposomes had an average particle size of 120 nm at room temperature, a calcein encapsulation rate of 40%, and two multivesicular liposomes. The pressure required for membrane emulsification was about 25 kPa.

Example 9
(Production of W1 / O emulsion by primary emulsification process)
Calcein was prepared by using 15 ml of a mixed solvent (hexane: dichloromethane = 8: 2) containing 0.25 g of dipalmitoylphosphatidylcholine (COATSOME MC-6060, manufactured by NOF Corporation) and 0.152 g of cholesterol (Chol) as the organic solvent phase (O). 5 ml of Tris-HCl buffer solution (pH 7.4, 50 mmol / L) containing (0.4 mM) was used as the aqueous dispersion phase (W1) for the inner aqueous phase. These mixed liquids were put into a 50 ml beaker, and the same emulsification treatment as in Example 1 was performed. The average particle size of the obtained W1 / O emulsion at room temperature was 219 nm.

(Production of W1 / O / W2 emulsion by secondary emulsification process)
SPG emulsification with a dispersion phase obtained by further adding 0.05 g of dipalmitoylphosphatidylglycerol (COATSOME MG-6060LA, manufactured by NOF Corporation) to the organic solvent (O) of the W1 / O emulsion obtained by the primary emulsification step. W1 / O / W2 emulsion was produced by the method. A SPG membrane having a pore diameter of 1.0 μm was used in the same membrane emulsification apparatus as in Example 1, and Tris-hydrochloric acid buffer (pH 7.4) containing 3% sodium caseinate as an external aqueous phase solution (W2) on the outlet side of the apparatus. 50 mmol / L), and the W1 / O emulsion was supplied from the apparatus inlet side to produce a W1 / O / W2 emulsion. The pressure required for membrane emulsification was about 10 kPa.

(Production of liposomes by removal of organic solvent phase)
The W1 / O / W2 emulsion obtained by the secondary emulsification step was transferred to a closed container, stirred for about 4 hours under a reduced pressure room temperature of 500 mbar, and then stirred for about 18 hours under a reduced pressure room temperature of 180 mbar. Was volatilized. A suspension of fine liposome particles was obtained, and it was confirmed that calcein was contained in the particles. The obtained liposomes had an average particle size at room temperature of 231 nm and a calcein encapsulation rate of 70%, and multivesicular liposomes were not confirmed.

Comparative Example 1
The same operation as in Example 1 was performed except that an SPG membrane having a pore size of 7.0 μm was used in the secondary emulsification step, and the pressure required for membrane emulsification was about 7 kPa. A suspension of fine liposome particles was obtained, and it was confirmed that calcein was contained in the particles. The average particle diameter of the obtained liposomes at room temperature was 255 nm and the calcein encapsulation rate was 70%, but the number of multivesicular liposomes was slightly high at 58.

Comparative Example 2
The same operation as in Example 1 was performed except that an SPG membrane having a pore size of 10.0 μm was used in the secondary emulsification step, and the pressure required for membrane emulsification was about 5 kPa. A suspension of fine liposome particles was obtained, and it was confirmed that calcein was contained in the particles. The obtained liposome had an average particle size at room temperature of 277 nm and a calcein encapsulation rate of 68%, but the number of multivesicular liposomes was as high as 170.

Example 10
(Manufacture of W1 / O emulsion by primary emulsification process with mixed solvent)
0.25 g of dipalmitoyl phosphatidylcholine (DPPC) “COATSOME MC-6060” (NOF Corporation), 0.152 g of cholesterol, and dipalmitoyl phosphatidylglycerol (DPPG) “COATSOME MG-6060LA” (NOF Corporation) 0.25 g 15 ml of a mixed solvent containing hexane (dichloromethane = 8/2) is used as the organic solvent phase (O), and 5 ml of Tris-HCl buffer solution (pH 7.4, 50 mM) containing calcein (0.4 mM) is used for the inner aqueous phase. An aqueous dispersion phase (W1) was obtained. These mixed liquids were put into a 50 ml beaker and subjected to an emulsification treatment by irradiating ultrasonic waves at 25 ° C. for 15 minutes with an ultrasonic dispersion apparatus (UH-600S, SMT Co., Ltd.) equipped with a φ20 mm probe. When measured according to the above method, the W1 / O emulsion obtained in this primary emulsification step had a volume average particle size of about 212 nm.

(Production of W1 / O / W2 emulsion by secondary emulsification process)
Using the W1 / O emulsion obtained by the primary emulsification step as a dispersed phase, a W1 / O / W2 emulsion was produced by the SPG emulsification method. First, an SPG membrane having a pore diameter of 5.0 μm as in Example 2 was used in the membrane emulsifying device, and Tris-HCl buffer (pH 7) containing 3% sodium caseinate as an external aqueous phase solution (W2) on the outlet side of the device. 4, 50 mmol / L) was satisfied, and the W1 / O emulsion was supplied from the apparatus inlet side to produce a W1 / O / W2 emulsion. The pressure required for membrane emulsification was about 10 kPa.

Subsequently, using the obtained W1 / O / W2 emulsion as a dispersed phase, an SPG membrane having a pore diameter of 0.5 μm similar to that in Example 4 was used for the membrane emulsification device, and the outer aqueous phase solution (W2) was used on the device outlet side. A tris-hydrochloric acid buffer solution (pH 7.4, 50 mmol / L) containing 3% sodium casein is filled, and the above W1 / O / W2 emulsion is supplied from the inlet side of the apparatus to perform membrane treatment. A W1 / O / W2 emulsion was prepared. The pressure required for this membrane treatment was about 5 Pa.

(Production of liposomes by removal of organic solvent phase)
The final W1 / O / W2 emulsion obtained by the secondary emulsification step is transferred to a closed container and stirred for about 8 hours under a reduced pressure condition of 20 ° C. and 500 mbar, and then under a reduced pressure condition of 20 ° C. and 180 mbar. The mixture was stirred for about 8 hours, and the solvent was volatilized stepwise. It was confirmed that calcein was contained in the particles of the obtained liposome suspension. The average particle diameter of the obtained liposome was 190 nm, and the calcein encapsulation rate was 62%. Multivesicular liposomes were not confirmed.

Example 11
(Production of W1 / O emulsion by primary emulsification process)
A mixed solvent (hexane: dichloromethane) containing 6.0 g of egg yolk lecithin “COATSOME NC-50” (manufactured by NOF CORPORATION), 3.04 g of cholesterol (Chol) and 2.16 g of oleic acid (OA) having a phosphatidylcholine content of 95% = 8: 2) 300 ml of organic solvent phase (O) and 100 ml of Tris-HCl buffer solution (pH 7.4, 50 mmol / L) containing calcein (0.4 mM) as an aqueous dispersion phase (W1) for the inner aqueous phase It was. The same primary emulsification process as in Example 4 was performed. The average particle size of the obtained W1 / O emulsion at room temperature was 135 nm.

(Production of W1 / O / W2 emulsion by secondary emulsification process)
Using the W1 / O emulsion obtained by the primary emulsification step as a dispersed phase, a W1 / O / W2 emulsion was produced by the SPG emulsification method. Same as Example 4 except that a cylindrical SPG membrane having a diameter of 10 mm, a length of 125 mm, and a pore diameter of 1.0 μm is used in an SPG membrane emulsifying device (trade name “High Speed Mini Kit KH-125” manufactured by SPG Techno Co., Ltd.). Thus, a W1 / O / W2 emulsion was produced. The pressure required for membrane emulsification was about 10 kPa.

(Production of liposomes by removal of organic solvent phase)
The W1 / O / W2 emulsion obtained by the secondary emulsification step was transferred to a closed container, stirred for about 4 hours under a reduced pressure room temperature of 500 mbar, and then stirred for about 18 hours under a reduced pressure room temperature of 180 mbar. Was volatilized. A suspension of fine liposome particles was obtained, and it was confirmed that calcein was contained in the particles. The obtained liposomes had an average particle size at room temperature of 140 nm and a calcein encapsulation rate of 53%, and multivesicular liposomes were not confirmed.

Comparative Example 3
In the secondary emulsification step, a cylindrical SPG membrane having a diameter of 10 mm, a length of 125 mm, and a pore diameter of 10.0 μm was used, and the same operation as in Example 11 was performed except that the pressure required for membrane emulsification was about 5 kPa. Went. A suspension of fine liposome particles was obtained, and it was confirmed that calcein was contained in the particles. The obtained liposomes had an average particle size at room temperature of 134 nm and a calcein encapsulation rate of 65%, but the number of multivesicular liposomes was as large as 220.

Example 12
The same operation as in Example 1 was performed except that cytarabine (4 mM) was dissolved in the aqueous dispersion phase (W1) instead of calcein (0.4 mM). The obtained liposomes had an average particle size at room temperature of 168 nm and an encapsulation rate of 32%, and multivesicular liposomes were not confirmed.

Example 13
The same operation as in Example 2 was performed except that cytarabine (4 mM) was dissolved in the aqueous dispersion phase (W1) instead of calcein (0.4 mM). The average particle diameter of the obtained liposome at room temperature was 200 nm, the encapsulation rate was 33%, and multivesicular liposomes were not confirmed.

Comparative Example 4
The same operation as in Comparative Example 2 was performed except that cytarabine (4 mM) was dissolved in the aqueous dispersion phase (W1) instead of calcein (0.4 mM). The average particle diameter of the obtained liposomes at room temperature was 195 nm and the encapsulation rate was 30%, but the number of multivesicular liposomes was as large as 201.

*: In Example 10, after obtaining a W1 / O / W2 emulsion from a W1 / O emulsion by membrane emulsification with an SPG membrane (pore size 5.0 μm), an SPG membrane (pore size 0.5 μm) was used again. The W1 / O / W2 emulsion was subjected to membrane treatment. In other examples and comparative examples, a W1 / O / W2 emulsion was produced from a W1 / O emulsion by a single treatment using an SPG film.

Claims

A method for producing a liposome, comprising the following steps (1) to (3):
(1) Primary emulsification step: a step of preparing a W1 / O emulsion by emulsifying the organic solvent (O), the aqueous solvent (W1), and the mixed lipid component (F1);
(2) Secondary emulsification step: The W1 / O emulsion obtained in the above step (1) and the aqueous solvent (W2) are emulsified using a membrane having a pore diameter of 0.1 μm or more and 5.0 μm or less, Preparing a W1 / O / W2 emulsion;
(3) A step of preparing a liposome suspension by removing the organic solvent contained in the W1 / O / W2 emulsion obtained by the step (2);
However, you may perform the said primary emulsification process, after adding the substance further included in a liposome.
In the step (2), the W1 / O emulsion is passed through a membrane having a pore diameter of 0.1 μm or more and 5.0 μm or less and dispersed as droplets in the aqueous solvent (W2), whereby W1 / O / The method for producing a liposome according to claim 1, which is a step of preparing a W2 emulsion.
The step (2) further includes a membrane treatment step of allowing a W1 / O / W2 emulsion obtained by emulsification using the membrane to pass through a membrane having a pore diameter of 0.1 μm or more and 5.0 μm or less. The method for producing a liposome according to claim 1 or 2, characterized in that:
The method for producing a liposome according to any one of claims 1 to 3, wherein an SPG membrane is used as the membrane in the step (2).
5. The liposome according to claim 1, wherein a medical drug is used as the substance to be encapsulated in the liposome, and the average particle size of the obtained liposome is 50 nm to 200 nm. Production method.
In the step (2), in addition to the aqueous solvent (W2) and the W1 / O emulsion, at least one of a water-soluble emulsifier that does not break the liposome lipid membrane and the mixed lipid component (F2) is used. The method for producing a liposome according to any one of claims 1 to 5.
The method for producing a liposome according to any one of claims 1 to 6, wherein the liposome is a single-cell liposome.