JP2010222282A - Method for producing liposome comprising fixing internal water phase - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a production method of a liposome that raises an inclusion rate of various water-soluble medicines than before and enhances stability of the liposome. <P>SOLUTION: The production method of the liposome comprises a first emulsifying step for preparing a W1/O emulsion by emulsifying a mixed liquid of an organic solvent (O), an aqueous solvent (W1), an additive (A) having an effect of substantially inhibiting flowability of W1 and mixed lipid components (F1) (and a substance to be included in the liposome may be further added to the mixed liquid), and other steps. Preferably, a gelling agent, a biocompatible polymer or a network polymer is employed as the additive (A). In the second emulsifying step subsequent to the first emulsifying step, an agitation emulsification method, a membrane emulsification method or a liquid droplet method is preferably employed. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to liposomes that can be used as drug carriers for pharmaceuticals, foods, and cosmetics.

  A liposome is a closed endoplasmic reticulum composed of a lipid bilayer mainly composed of phospholipid. Since it has a structure and function similar to a cell membrane, it is difficult to stimulate the immune system (low antigen) and is highly safe as a material. In addition, water-soluble drugs can be incorporated into the inner aqueous phase surrounded by lipid bilayer membranes, and fat-soluble drugs can be incorporated into lipid bilayer membranes. Can be retained. For these reasons, active research and development have been conducted on liposome preparations suitable for use in the fields of pharmaceuticals, cosmetics, foods, and the like, for example, drug delivery systems (DDS).

  As one of the research subjects related to liposomes, the inclusion rate of drugs contained in the inner aqueous phase (the total mass of drugs contained in the liposome suspension, in other words, the total mass of drugs used as a raw material for production) The ratio of the mass of drugs encapsulated in the body) may be improved. Since the lipid bilayer of liposomes is a weak bond just arranged, the encapsulated aqueous solution can go back and forth to some extent (however, ionic substances and polymers cannot go back and forth), and in addition the strength as particles Are not so high that the drugs are likely to leak out of the liposomes.

  Patent Document 1 discloses that an internal aqueous phase (W) in which a substance to be encapsulated is cooled or fixed is frozen and fixed by cooling the W / O emulsion, and an oil phase (O) maintaining a liquid state is removed. Then, a method for producing liposomes by adding an external aqueous phase after making a W / air (air) state is described. In this method, it is theoretically considered that the drugs in the frozen inner aqueous phase do not transfer to the outer aqueous phase, and the particles are not broken or aggregated due to the strength of ice. However, the encapsulation rate of the drugs in the inner aqueous phase in the examples is about 40% at the highest, and the purpose of improving the encapsulation rate has not been sufficiently achieved.

  On the other hand, a “microencapsulation method” is known in which liposomes are produced by forming a W / O emulsion and then dropping it into an aqueous solvent with stirring (for example, Patent Document 2). In the method of forming a W / O / W emulsion such as a stirring emulsification method, a membrane emulsification method, and a liquid suitability method used in this microencapsulation method, the inner aqueous phase (W1) can be in contact with the outer aqueous phase (W2). Therefore, it is thought that the drug in the inner aqueous phase should not be able to move to the outer aqueous phase, but in reality, the drug is almost always moved to the outer aqueous phase. As shown in the results of Examples in Patent Document 2, the encapsulation rate of drugs is not sufficiently increased (12 to 45%), and thus the microencapsulation method has not been put into practical use.

  In addition, there is an approach that uses the special properties of the drug itself contained in the liposome preparation to efficiently maintain it. For example, a liposome preparation “DOXIL” (registered trademark) marketed in Japan contains the anticancer drug doxorubicin whose solubility is pH-dependent. When the outer aqueous phase is neutralized while keeping the inner aqueous phase acidic in the production process, the drug that is weakly basic moves to the inner aqueous phase above the phase transition temperature of the liposome membrane to form an ammonium sulfate solution and salt, As a result, it is stabilized and encapsulated in liposomes at a high concentration. However, there are very limited drugs with special properties that can use such methods.

  In Patent Document 3, a liposome in which a labeling substance is encapsulated is dispersed in a liquid containing a compound such as gelatin, and a compound such as gelatin may be encapsulated in the liposome. A storage immunoassay reagent is described. However, this document only describes a method for producing such an immunoassay reagent by preparing a liposome in one step and then suspending it in a gelatin solution.

  In Patent Document 4, a gelling agent (gelatin, carrageenan, etc.) is dissolved in an aqueous solution at a temperature higher than the gel-sol phase transition temperature with slow stirring, and a lipid is mixed into this solution and slowly stirred to give an emulsion. Is described, followed by rapid stirring to produce a liposome dispersion with a gelled inner core. However, the liposome production method described in this document is also based on one-step emulsification.

  Patent Document 5 describes a method for producing water-soluble drug-encapsulating liposomes, which is prepared by mixing a natural moisturizing polymer and a water-soluble drug-mixed aqueous solution and a liposome wall material solution. However, this document (Example) also describes only a method for producing liposomes in one step, such as the Bangham method, supercritical carbon dioxide method, emulsification with an ultrasonic disperser, and the inclusion rate is determined by any method. It remains around 7%. Moreover, in the invention described in this document, when it is used with gelatin, which is a typical gelling agent (Comparative Production Example 2), the viscosity is so high that sufficient dispersion cannot be achieved, and a good water-soluble drug-encapsulating liposome is obtained. The result is that it is not possible.

  In any of the above Patent Documents 3 to 5, in a production method that undergoes a two-stage emulsification step such as the production method of the present invention described later, a water-soluble drug is first used by using a compound such as gelatin in the primary emulsification step. There is no description or suggestion that the effect of improving the encapsulation rate is obtained.

Japanese Patent No. 4009733 JP 2001-139460 A Japanese Patent No. 2780116 Japanese translation of PCT publication No. 2002-511077 JP 2008-133195 A

  An object of the present invention is to provide a method for producing a liposome capable of increasing the encapsulation rate of various water-soluble drugs than before and improving the stability of the liposome.

  The present inventor examined the method described in Patent Document 1 and the inclusion rate was not increased by this method as soon as the frozen inner water phase was hard and relatively stable but touched the outer water phase. It was inferred that this was because the state after removal of the oil phase did not conform to the theory as described above due to reasons such as being dissolved in water.

  Further, in the method described in Patent Document 2, since the W1 / O / W2 emulsion particles are soft, there are cases where the lipid film wrapping the inner aqueous phase (W1) is broken or the O phase is sheared by stirring. It was inferred that the fact that the aqueous phase (W1) could not exist stably was a major reason why the inclusion rate of the drugs did not increase.

  And further examination, by forming a W1 / O emulsion using various additives having the effect of substantially suppressing the fluidity of the aqueous solvent in the primary emulsification step (to fix the inner aqueous phase) The inventors have found that it is possible to improve the encapsulation rate of water-soluble drugs by creating a situation in which the drug in the inner aqueous phase cannot move to the outer aqueous phase, and the present invention has been completed.

  In addition, additives such as gelatin are also used in the liposome production methods described in Patent Documents 3 to 5, but in contrast to the present invention, in these methods, the immobilization of the inner aqueous phase and the outer water are performed. The phase immobilization is not considered at all, and it does not contribute to the improvement of the encapsulation rate of the water-soluble drug, apart from the stability when the liposome is stored as a suspension or powdered for a long period of time.

That is, the method for producing a liposome of the present invention comprises the following steps (1) to (3);
(1) Primary emulsification step: emulsifying a mixed solution containing an organic solvent (O), an aqueous solvent (W1), an additive (A) having an effect of substantially suppressing the fluidity of W1, and a mixed lipid component (F1) Preparing a W1 / O emulsion by:
(2) Secondary emulsification step: A W1 / O / W2 emulsion is prepared by emulsifying the aqueous solvent (W2), the mixed lipid component (F2), and the W1 / O emulsion obtained by the above step (1). Process;
(3) A step of preparing a liposome suspension by removing the organic solvent contained in the emulsion obtained in the step (2);
However, you may perform the said primary emulsification process, after adding the substance which should be further included in a liposome.

  In this production method, 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 to 1000 nm. The “average particle size” of the liposome in the present invention refers to the number average particle size. As the emulsification method in the step (2), it is preferable to use a stirring emulsification method, a membrane emulsification method, or a droplet method. Examples of the additive (A) include a gelling agent, a biocompatible polymer, or a reticulated polymer (preferably a reticulated polymer having a reticulated portion covering the surface of the liposome membrane and an anchor portion located inside the liposomal membrane. ) Is preferably used.

  In the liposome production method of the present invention, the flowability of the inner aqueous phase can be suppressed by using a specific additive in the primary emulsification step, and the strength of the liposome membrane can be increased. Even when a method, a membrane emulsification method, a liquid suitability method, or the like is used, the W / O / W emulsion particles are less likely to be destroyed, and a decrease in the encapsulation rate can be suppressed. As a result, it becomes possible to efficiently produce liposomes by improving the encapsulation rate of water-soluble drugs, and at the same time, the stability of the liposome particles can be improved.

− Production raw material −
-Additive (A) having an effect of substantially suppressing the fluidity of W1
In the present invention, the “additive having an effect of substantially suppressing the fluidity of W1” means that the fluidity is lowered by adding to the aqueous solvent W1, and the solvent itself or a drug dissolved or suspended. It is an additive that has the effect of suppressing the movement of the substance through the liposome membrane and improving the encapsulation rate of substances (water-soluble drugs) to be encapsulated in the liposome compared to when it is not used, Refers to a substance such as a gelling agent, biocompatible polymer, or network polymer described below.

(1) Gelling agent The “gelling agent” used in the present invention refers to a substance that suppresses the fluidity of W1 by having the property of gelling and solidifying the aqueous solvent W1. It is distinguished from other additives (A) such as a “thickening stabilizer” described later in terms of gel transition.

  As the gelling agent, various gelling agents added to pharmaceuticals, foods, etc., such as gelatin, agar, carrageenan, and pectin can be used. The amount of the gelling agent added is preferably 0.1 to 5% by weight, most preferably 1 to 2% by weight based on the aqueous solvent (W1), for example, in the case of gelatin. The other gelling agent is preferably added in such an amount that a gel having the same degree of hardness as that obtained when gelatin is added at the above-mentioned ratio.

(2) Biocompatible polymer The “biocompatible polymer” used in the present invention has a property of increasing the viscosity of the aqueous solvent W1 and a high molecular weight that cannot pass through the liposome membrane (considered to be about 10,000 or more). It is a biocompatible polymer that suppresses fluidity of phospholipids, and is distinguished from the “network polymer” described later in that it does not have a molecular structure that chemically interacts with or physically interacts with (arranges) phospholipids. It is.

  Examples of such biocompatible polymers include dextran, chitosan, sodium carboxymethylcellulose, xanthan gum, locust bean gum, guar gum, other polysaccharides, which are also known as thickeners / stabilizers, and pharmaceuticals. Amphiphilic polymer, polylactic acid, protein, nucleic acid, non-natural protein, non-natural nucleic acid, etc. The addition amount of the thickener / stabilizer in the biocompatible polymer is preferably 0.001 to 1% by weight, and 0.01 to 0.1% by weight with respect to the aqueous solvent (W1) in the case of dextran, for example. Most preferred. It is preferable that the amount of the other solvent is such that the viscosity of the aqueous solvent W1 is approximately the same as when dextran is added at the above ratio. Further, the addition amount of the biocompatible polymer other than the thickener / stabilizer such as an amphiphilic polymer is preferably 0.001 to 1% by weight with respect to the aqueous solvent (W1), and 0.01 to 0.1% by weight. % Is most preferred.

(3) Reticulated polymer The “reticular polymer” used in the present invention has a reticulated molecular structure that coats the surface of the liposome membrane (mainly the inner aqueous phase side), thereby suppressing the fluidity of the aqueous solvent W1. Refers to a molecule. For example, a compound such as pullulan derivative, PVLA (polyvinylbenzyllactone amide), P (VMA-co-VAL) (a copolymer of Np-vinylbenzyl-D-lactoneamide and 4-vinylbenzylhexadecanamide) is used. be able to. The addition amount of the reticulated polymer is preferably 0.001 to 10% by weight, and most preferably 0.1 to 2% by weight with respect to the aqueous solvent (W1).

  The reticulated polymer preferably has a reticulated portion covering the surface of the liposome membrane and an anchor portion located inside the liposomal membrane. For example, a cholesterol derivative in which a site having a network molecular structure (for example, a polysaccharide such as pullulan or a polymer) is introduced into the hydroxyl group at the 1-position of cholesterol, or a derivative in which a network molecular structure is introduced into an alkyl chain (for example, P (VMA- co-VAL)). It can be said that such a reticulated polymer constitutes a part of the liposome membrane together with the mixed lipid component (F1).

・ 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 membrane of the liposome, and the mixed lipid component (F2) used in the secondary emulsification step mainly constitutes the outer membrane. The mixed lipid components (F1) and (F2) may have the same composition or different compositions.

  The composition of these mixed lipid components is not particularly limited. 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. 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. The aqueous solvent (W1) and the organic solvent (O) used in the primary emulsification step form an aqueous phase and an oil phase of the W1 / O emulsion, respectively. The aqueous solvent (W2) used in the secondary emulsification step is W1 / O. / The outer water phase of the W2 emulsion is formed. Examples of the aqueous solvent include pure water containing other solvents mixed with water as necessary, salts and sugars for adjusting osmotic pressure, buffers for adjusting pH, and the like as organic solvents. Examples thereof include those composed of a compound that is not mixed with an aqueous solvent such as hexane (n-hexane) and chloroform.

-Substances to be encapsulated In the present invention, substances to be encapsulated in liposomes (collectively referred to as drugs) are not particularly limited, and are known in the fields of pharmaceuticals, cosmetics, foods, etc. depending on the use of liposomes Various substances 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 agent, antioxidant agent, anti-inflammatory agent, 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 cutin preparation (one having a toxoid such as tetanus as an antigen; one having a virus such as diphtheria, Japanese encephalitis, polio, rubella, mumps, hepatitis; a DNA or RNA vaccine), Pharmaceutical aids such as dyes / fluorescent dyes, chelating agents, stabilizers, preservatives and the like can be mentioned.

− Method for producing liposome −
The manufacturing method of the liposome of this invention has the following process (1)-(3), and can combine another process suitably as needed.

(1) Primary emulsification step In the primary emulsification step, a mixed solution containing an organic solvent (O), an aqueous solvent (W1), an additive having an effect of substantially suppressing the fluidity of W1, and a mixed lipid component (F1). This is a step of preparing a W1 / O emulsion by emulsification.

  The method for preparing the W1 / O emulsion in the primary emulsification step is not particularly limited, and a method using an apparatus such as an ultrasonic emulsifier, a stirring emulsifier, a membrane emulsifier, or a high-pressure homogenizer can be used.

  When a gelling agent is used as the additive (A), the primary emulsification step is performed at a temperature higher than the sol-gel phase transition temperature of the gelling agent, and at an appropriate stage after the W1 / O emulsion is formed, It is necessary to lower the temperature to cause gelation.

  In the present invention, in order to encapsulate the water-soluble drug in the liposome, (i) the water-soluble drug is dissolved or suspended in advance in the aqueous solvent (W1) of the primary emulsification step, and at the end of the secondary emulsification step. A method for obtaining a liposome encapsulating it, (ii) after obtaining a (empty) liposome that does not encapsulate a water-soluble drug, adding a water-soluble drug to the dispersion of the liposome, or once freezing Any method of adding a water-soluble drug to the liposome when it is redispersed in an aqueous solvent and stirring it, for example, can be used.

  In the above method (i), the water-soluble drugs are already immobilized in the inner aqueous phase (W1) in a predetermined manner at the stage where the W1 / O emulsion is formed in the primary emulsification step. Leakage is suppressed. On the other hand, in the method (ii) above, after the inner aqueous phase (W1) having a predetermined function is formed, the added water-soluble drugs are taken into the liposome and fixed in the inner aqueous phase at that stage. And leakage to the outside is suppressed thereafter.

  In addition, fat-soluble drugs are also added to the liposome in advance by adding them at the time of the primary emulsification step as described above (i) or after obtaining empty liposomes as described above (ii). Can be included.

(2) Secondary emulsification step In the secondary emulsification step, W1 / O / W2 is obtained by emulsifying the aqueous solvent (W2), the mixed lipid component (F2), and the W1 / O emulsion obtained in the above step (1). It is a step of preparing an emulsion.

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

  As a method for preparing the W1 / O / W2 emulsion in the secondary emulsification step, a stirring emulsification method, a membrane emulsification method, a liquid suitability method and the like used in the microencapsulation method are suitable. As described above, in the present invention, the additive (A) is added only to the inner aqueous phase in the primary emulsification step to immobilize the inner aqueous phase, so that the W1 / O / W2 emulsion particles are less likely to be destroyed and the secondary Even if the above-described stirring emulsification method or the like is used in the emulsification step, the encapsulation rate can be maintained well. That is, while enjoying the original advantage of the stirring and emulsification method that is relatively simple and easy to scale up, liposomes can be produced with a higher encapsulation rate than in the past.

  In the secondary emulsification step, the ratio of the mixed lipid component (F2) added to the aqueous solvent (W2), the volume ratio of the W1 / O emulsion to the aqueous solvent (W2), and other operating conditions are as follows. It can be adjusted as appropriate while considering the application.

(3) Solvent removal step The solvent removal step is a step of preparing a liposome suspension by removing the organic solvent (O) contained in the W1 / O / W2 emulsion obtained in the above step (2). is there. Examples of the solvent removal method include a method of evaporating with an evaporator and a submerged drying method, but are not particularly limited.

  The in-liquid drying method is a method in which the organic solvent (O) contained in the W1 / O / W2 emulsion is evaporated and removed by collecting the W1 / O / W2 emulsion, transferring it to an open container and allowing it to stand or stir. Yes, a lipid membrane composed of the mixed lipid membrane components (F1) and (F2) is formed around the inner aqueous phase, and liposomes can be obtained. At this time, evaporation of the solvent can be promoted by heating or decompression.

  Although the temperature conditions in the submerged drying method are influenced by the solvent type to be used, a condition range in which the solvent does not bump is set, a range of 0 to 60 ° C is preferable, and 0 to 25 ° C is more preferable. When a gelling agent is used as the additive (A), the temperature condition should be lower than the sol-gel phase transition temperature of the gelling agent. 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 suddenly boil. For example, when a chemical that is weak against heat is used, it is preferable that the solvent is distilled off at a lower temperature and under reduced pressure.

Further, the solvent removal by the submerged drying method does not require the stirring of the W1 / O / W2 emulsion, but the solvent removal proceeds more uniformly by stirring. After preparing the W1 / O / W2 emulsion by the stirring emulsification method in the above step (2), the steps (2) and (3) may be continuously performed so that stirring is further continued to remove the solvent. it can.
The average particle size of the liposome finally obtained by the production method of the present invention as described above is preferably 50 to 1000 nm, which is suitable for a liposome preparation for medical use, for example.

(4) Other processes As other processes performed as needed, a freeze-drying process is mentioned, for example. After the liposome suspension is obtained, it is desirable to further freeze-dry it to make the liposome suitable for storage until use. Freeze-drying can be performed using the same means and apparatus as in the case of producing conventional liposomes. For example, according to an indirect heating freezing method, a refrigerant direct expansion method, a heat medium circulation method, a triple heat exchange method, an overlapping refrigeration method, and the like (temperature: −120 to −20 ° C., pressure: 1 to 15 Pa, time: It may be lyophilized in 16 to 26 hours).

(Measurement method of calcein inclusion rate)
The fluorescence intensity (F _total ) of the entire liposome aqueous solution (3 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 outer aqueous phase 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.

(Measuring method of particle size distribution)
The aqueous liposome solution was diluted 10 times with a chloroform / hexane mixed solvent (volume ratio: 4/6, the same specific gravity as the inner aqueous phase), and a dynamic light scattering nanotrack particle size analyzer (UPA-EX150, Nikkiso Co., Ltd.) ) Was measured, and based on this, the average particle size (number average) was calculated.

Example 1 : Production by stirring emulsification of calcein-encapsulated liposomes using gelatin as an additive In a 100 mL beaker, 2 g of egg yolk lecithin (PL-100M: Kewpie Co., Ltd.), 20 mL of dichloromethane, and calcein (0.1 mmol / L) And 5 mL of an aqueous solution containing gelatin (S-511-5, Nippi Corporation, pig-derived acidic type) (1% by weight), and stirred at high speed for 30 minutes at a temperature of 25 ° C. and a rotational speed of 24,000 rpm, uniformly A W / O emulsion solution was obtained by dispersing.

  Into a separately prepared 300 L flask, put 5 mL of the obtained W / O emulsion solution and 50 mL of Tris-HCl buffer (pH 9, 10 mmol / L), and stir for 30 minutes at a temperature of 15 ° C. and a rotation speed of 800 rpm. To obtain a W (immobilized) / O / W emulsion solution.

  Next, this solution was subjected to solvent removal with an evaporator at 15 ° C. under a reduced pressure of 300 mbar to obtain a liposome solution. The average liposome particle size at room temperature was 555 nm, and the calcein encapsulation rate was 37.8%.

Comparative Example 1
Calcein-encapsulating liposomes were produced by stirring emulsification in the same manner as in Example 1 except that gelatin was not added. The average particle size of the liposomes was 434 nm, and the calcein encapsulation rate was 13.9%.

Example 2 Production of Calcein-Encapsulated Liposomes Using Gelatin as an Additive by SPG Film Emulsion A W / O emulsion solution was obtained in the same manner as in Example 1 except that dichloromethane was changed to hexane. The outlet side of the SPG membrane (pore diameter: 5 μm) was filled with Tris-hydrochloric acid buffer solution (pH 8, 50 mmol / L) containing 3% sodium casein as an outer aqueous phase solution to a temperature of 15 ° C. / O emulsion was supplied to produce a W / O / W emulsion.

  Next, the W / O / W emulsion was transferred to an open glass container without a lid and allowed to stand at 15 ° C. for about 20 hours to volatilize hexane. The average particle size of the liposomes at room temperature was 339 nm, and the calcein encapsulation rate was 61.0%.

Comparative Example 2
Calcein-encapsulated liposomes were produced by SPG membrane emulsification in the same manner as in Example 2 except that gelatin was not added. The average particle size of the liposomes was 322 nm, and the calcein encapsulation rate was 50.2%.

Example 3: Use of gelatin as an additive, except for changing prepared egg yolk lecithin by sessile drop of calcein containing liposomes (PL-100M) to hydrogenated soybean phospholipid (NC-50, NOF Corporation) Example In the same manner as in Example 2, a W / O type emulsion solution was obtained.

  To a separately prepared 100 L beaker, 20 mL of Tris-HCl buffer solution (pH 8, 50 mmol / L) containing 3% sodium caseinate and 20 mL of hexane were added to obtain two layers, and the temperature was adjusted to 15 ° C. The W / O emulsion was slowly supplied to the upper hexane layer and allowed to stand for 1 day. When the aqueous phase containing the lower liposome was separated, the average particle size of the liposome at room temperature was 920 nm, and the calcein encapsulation rate was 39.0%.

Comparative Example 3
Calcein-encapsulating liposomes were produced by the droplet method in the same manner as in Example 3 except that gelatin was not added. In this case, the formation of liposomes was not observed.

(Discussion)
As described above, in Examples 1 to 3 to which gelatin was added, the calcein encapsulation rate was improved as compared with Comparative Examples 1 to 3 in which gelatin was not added. This is thought to be due to the fact that the gelatin gels at 15 ° C. and the inner aqueous phase is fixed, so that calcein present in the inner aqueous phase cannot move to the outer aqueous phase and remains. .

Example 4 : Production by stirring and emulsification of calcein-encapsulating liposomes using dextran as an additive In a 50 mL beaker, 2 g of egg yolk lecithin (PL-100M: Kewpie Co., Ltd.), 10 mL of chloroform, calcein (0.1 mmol / L) and Add 5 mL of Tris-hydrochloric acid buffer solution (pH 8, 10 mmol / L) containing dextran (molecular weight 10,000, 0.01 mmol / L), treat at a temperature of 25 ° C. for 15 minutes with an ultrasonic disperser, and disperse uniformly To obtain a W / O emulsion solution.

  Into a separately prepared 300 L flask, put 15 mL of the obtained W / O emulsion solution and 150 mL of Tris-HCl buffer (pH 8, 10 mmol / L), and stir with a mechanical stirrer at a rotation speed of 850 rpm for 60 minutes. It was dispersed to obtain a W (immobilized) / O / W type emulsion solution.

  Next, the solvent was removed from the solution with an evaporator at a reduced pressure of 300 mbar to obtain a liposome solution. The obtained liposomes had an average particle size of 241 nm and a calcein encapsulation rate of 18.8%.

Example 5
Calcein-encapsulated liposomes were produced by stirring emulsification in the same manner as in Example 4 except that a reagent having a molecular weight of 40,000 was used as dextran. The average particle size of the liposomes was 202 nm, and the calcein encapsulation rate was 29.8%.

(Discussion)
As described above, in Examples 4 to 5 to which dextran was added, the use of a polymer having a large molecular weight resulted in an improvement in the calcein encapsulation rate. This is considered to be due to the effect that the portion of the inner aqueous phase occupied by the polymer is improved, so that calcein present in the inner aqueous phase cannot move to the outer aqueous phase and remains.

Example 6 : Production by stirring emulsification of calcein-encapsulating cationic liposomes using membranous polymer as an additive In a 50 mL beaker, 2 g of egg yolk lecithin (PL-100M: Kewpie Co., Ltd.), 10 mL of chloroform, and calcein (0.1 mmol) / L) and P (VMA-co-VAL) (Seradix Co., Ltd.) 25 mg of Tris-HCl buffer solution (pH 8, 10 mmol / L) containing 25 mg is added, and treated at a temperature of 25 ° C. for 15 minutes with an ultrasonic disperser. And uniformly dispersed to obtain a W / O type emulsion solution.

  Into a separately prepared 300 L flask, 15 mL of a W / O emulsion solution added with 0.5 g of cationic lipid and 150 mL of Tris-HCl buffer (pH 8, 10 mmol / L) were added, and the rotation was performed at 850 rpm for 60 minutes. The mixture was stirred with a mechanical stirrer and dispersed uniformly to obtain a W (immobilized) / O / W emulsion solution. Stirring was further continued for 3 hours to remove the solvent to obtain a liposome solution. The obtained liposomes had an average particle size of 213 nm and a calcein encapsulation rate of 53.8%.

Comparative Example 4
Calcein-containing cationic liposomes were produced by stirring emulsification in the same manner as in Example 6 except that P (VMA-co-VAL) was not added. The average particle size of the liposomes was 197 nm, and the calcein encapsulation rate was 12.8%.

(Discussion)
As described above, in Example 6 to which the membranous polymer was added, the calcein inclusion rate was improved as compared with Comparative Example 4 in which the film-like polymer was not added. This is presumably because the presence of membrane-like polymer on the liposome surface has the effect that calcein present in the inner aqueous phase stays blocked by the movement path to the outer aqueous phase.

Example 7 : Production by stirring and emulsification of calcein-encapsulating liposomes using gelatin and membrane polymer as additives In a 100 mL beaker, 2 g of egg yolk lecithin (PL-100M: Kewpie Co., Ltd.), 20 mL of dichloromethane, and calcein (0. 1 mmol / L), 5 mL of an aqueous solution containing gelatin (S-511-5, Nippi Co., Ltd., pig-derived acidic type) (1 wt%) and P (VMA-co-VAL) (Seradix Co., Ltd.) 25 mg, The mixture was stirred at a high speed for 30 minutes at a temperature of 25 ° C. and a rotation speed of 24,000 rpm, and uniformly dispersed to obtain a W / O emulsion solution.

  Into a separately prepared 300 L flask, put 5 mL of the obtained W / O emulsion solution and 50 mL of Tris-HCl buffer (pH 9, 10 mmol / L), and stir for 30 minutes at a temperature of 15 ° C. and a rotation speed of 800 rpm. To obtain a W (immobilized) / O / W emulsion solution.

  Next, this solution was subjected to solvent removal with an evaporator at 15 ° C. under a reduced pressure of 300 mbar to obtain a liposome solution. The obtained liposomes had an average particle size at room temperature of 315 nm and a calcein encapsulation rate of 49.9%.

(Discussion)
As described above, in Example 7 in which gelatin and a film-like polymer were added, the calcein inclusion rate was improved as compared with Example 1 in which no film-like polymer was added. This is thought to be due to the synergistic effect of the two additives.

Claims (5)

A method for producing a liposome, comprising the following steps (1) to (3);
(1) Primary emulsification step: emulsifying a mixed solution containing an organic solvent (O), an aqueous solvent (W1), an additive (A) having an effect of substantially suppressing the fluidity of W1, and a mixed lipid component (F1) Preparing a W1 / O emulsion by:
(2) Secondary emulsification step: A W1 / O / W2 emulsion is prepared by emulsifying the aqueous solvent (W2), the mixed lipid component (F2), and the W1 / O emulsion obtained by the above step (1). Process;
(3) A step of preparing a liposome suspension by removing the organic solvent contained in the emulsion obtained in the step (2);
However, you may perform the said primary emulsification process, after adding the substance which should be further included in a liposome.   The method for producing a 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 to 1000 nm.   The method for producing a liposome according to claim 1 or 2, wherein a stirring emulsification method, a membrane emulsification method, or a droplet method is used as the emulsification method in the step (2).   The method for producing a liposome according to any one of claims 1 to 3, wherein a gelling agent, a biocompatible polymer, or a network polymer is used as the additive (A).   The method for producing a liposome according to claim 4, wherein a reticulated polymer having a reticulated portion covering the surface of the liposome membrane and an anchor portion located inside the liposomal membrane is used as the additive (A).