JPH1147580A - Production of liposome dispersion - Google Patents

Abstract

PROBLEM TO BE SOLVED: To produce a liposome dispersion excellent in long-term stability in an industrial scale in the liposome dispersion using saturated phospholipid as membrane lipid. SOLUTION: In a liposome dispersion using saturated phospholipid as membrane lipid, a rough dispersion of liposome is ejected as a single jet stream and the jet stream is directionally converted by a counterposed bottom surface to be allowed to flow backward along a side wall so as to surround the jet stream to apply finely pulverizing treatment to the liposome rough dispersion by the shearing force generated at the interface of the jet stream and the back flow to obtain liposome with an average particle size of 70 nm or less and the max. particle size of 300 nm or less.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a method for producing a liposome dispersion having excellent stability in a liposome dispersion using a saturated phospholipid as a membrane lipid of the liposome.

[0002]

2. Description of the Related Art A liposome is a closed vesicle having a bilayer made of lipid and having an aqueous phase therein. Due to such a structure of the liposome, the water-soluble substance can be retained in the internal aqueous phase of the liposome, and the fat-soluble substance can be retained between the bilayer membranes. It has been studied as a kind of. In the field of cosmetics, blending into cosmetics has been attempted with expectation of an emollient effect of lipids themselves and a water retention effect due to liposomes having an internal aqueous phase.

[0003] On the other hand, phospholipids are mainly used as membrane lipids of liposomes. Among them, saturated phospholipids are generally used from the viewpoint of improving membrane permeability and oxidative stability of liposomes. I have. Production of liposome dispersions on an industrial scale involves preparing a crude liposome dispersion, and then preparing the crude dispersion with a micronizing device, for example, a high-pressure homogenizer such as a Gorin type, an ultrasonic irradiator, or a polycarbonate membrane filter. It is manufactured by a method of processing by high pressure filtration or the like.

However, a liposome is not a state in which an emulsifier is fixed around a fat globule like an emulsion, but a lateral diffusion movement in which a lipid molecule moves laterally in a membrane or a lipid molecule between bilayer membranes. The membrane lipids make various movements in the liposome, such as flip-flop movement, which is the movement of the liposome, and the structure of the liposome is hardly thermodynamically stable. For this reason, even if the liposome dispersion is stable immediately after preparation, liposomes will subsequently aggregate and fuse together, resulting in appearance changes such as the generation of precipitates and the precipitation of insolubles. The value of was lost. In particular, when a saturated phospholipid is used as a membrane lipid, since the molecule itself has a cylinder-type molecular structure, it is difficult to say that structurally maintaining closed vesicles is thermodynamically stable. Things tend to occur easily.

[0005] As a method for preventing such liposome aggregation and fusion, liposome membrane lipids are partially charged with a charged substance such as phosphatidylserine, dicetyl phosphate, phosphatidic acid, phosphatidylinositol or the like to increase the surface potential. Although a method of preventing agglomeration or the like by using an electrostatic repulsion force by this has been tried, it is still hard to say that it is sufficient. Therefore, there is a need for a liposome dispersion having excellent long-term stability even when a saturated phospholipid is used as a membrane lipid.

[0006]

Therefore, an object of the present invention is to provide a liposome dispersion using a saturated phospholipid as a membrane lipid so that a liposome dispersion having excellent long-term stability can be produced on an industrial scale. It is an object of the present invention to provide a method for producing a liposome dispersion liquid.

[0007]

Means for Solving the Problems The present inventors have conducted intensive studies to achieve the above object, and as a result, completed the present invention. In other words, the present invention provides (1) a method for injecting a crude liposome dispersion as a single jet stream in a liposome dispersion using a saturated phospholipid as a membrane lipid of the liposome, The jet stream is changed in direction and is caused to flow back along the side wall so as to wrap the jet stream, whereby the coarse liposome dispersion is subjected to micronization treatment by a shear force generated at the interface between the jet stream and the back stream, and the average particle size is reduced. A method for producing a liposome dispersion having a diameter of 70 nm or less and a liposome having a maximum particle diameter of 300 nm or less, (2) a liposome having an average particle diameter of 20 to 70 nm and a maximum particle diameter of 10
(1) The method for producing a liposome dispersion of (1) having a thickness of 0 to 300 nm.

[0008]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described below in detail.
In the present invention, “%” is all “% by weight”,
All particle diameters are particle diameters by “volume conversion”. In the present invention, the “phospholipid” refers to a substance having two or more hydrophobic groups comprising a phosphoric acid group and an acyl group and / or an alkyl group in a molecule, for example, phosphatidylcholine (P
C), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidylglycerol (PG), phosphatidic acid (PA), sphingomyelin (SP)
M), one or two of cardiolipin or derivatives thereof
And the like.

In the present invention, "saturated phospholipid"
The term “phospholipid” refers to a phospholipid in which the acyl group and / or alkyl group, which is a hydrophobic group, is a saturated type. Phospholipids are prepared according to the “Iodine value (IV)” of the oil and fat test method of the thirteenth revised Japanese Pharmacopoeia. When analyzed, those having IV = 5 or less are also included. Examples of such phospholipids include dipalmitoyl phosphatidylcholine (DPPC), egg yolk phospholipids, and those derived from plant-derived soybean phospholipids by hydrogenation, and the like.

In the present invention, "crude liposome dispersion"
The term refers to a liposome dispersion before subjecting to micronization treatment, and its preparation method is not particularly limited.However, as production on an industrial scale, for example, after dispersing a saturated phospholipid in water, Examples thereof include a method of heating to a temperature higher than the phase transition temperature and a method of dispersing a saturated phospholipid in water heated to a temperature of the phase transition temperature or higher.

Further, in the present invention, it is preferable that "a crude liposome dispersion liquid is jetted as a single jet stream, the jet stream is turned at a bottom face which is arranged oppositely, and a backflow is applied along a side wall so as to wrap the jet stream. Thereby subjecting the crude liposome dispersion to micronization by the shearing force generated at the interface between the jet stream and the backflow. " E. FIG. E. FIG. This refers to treating a crude liposome dispersion with DeBEE2000 manufactured by International Ltd. or an apparatus equivalent thereto. Hereinafter, the principle of the formation of fine particles will be described with reference to FIGS. The crude liposome dispersion liquid 1 is jetted from a nozzle 2 as a single jet stream 3, the jet stream 3 is changed in direction at an opposed bottom surface 4, and a side wall 5 is wrapped around the jet stream 3.
, The shearing force generated at the interface 7 between the jet stream 3 and the back stream 6 causes the coarse dispersion of the liposome to be atomized.

In the present invention, "a liposome having an average particle size of 70 nm or less and a maximum particle size of 300 nm or less" refers to a liposome particle size of a liposome dispersion obtained by the production method of the present invention, which is commercially available. NICOM Model37 manufactured by Pacific Scientific, a particle size distribution analyzer
0 or an equivalent particle size, the average particle diameter was 70 nm or less, and the maximum particle diameter was 300 n.
m or less. In particular, those having an average particle diameter of 50 nm or less, and a maximum particle diameter of 200 nm or less,
Even after long-term storage, the average particle size and the maximum particle size are almost the same as those immediately after preparation, and the appearance is hardly changed. On the other hand, if the average particle size is larger than 70 nm or the maximum particle size is larger than 300 nm, insolubles and the like are not observed immediately after preparation, but precipitates and the like are generated after storage, which is not preferable as a liposome dispersion. Also, other micronization devices,
For example, in a liposome of a liposome dispersion obtained by treatment with a Gaulin-type high-pressure homogenizer or the like,
Even if the average particle size is 70 nm or less and the maximum particle size is 300 nm or less, a precipitate or the like is generated after storage, which is not preferable as a liposome dispersion. Incidentally, the average particle diameter is smaller than 20 nm and the maximum particle diameter is 10 nm.
Since liposomes smaller than 0 nm are difficult to produce on an industrial scale, including the production method of the present invention, the liposomes obtained by the production method of the present invention have an average particle diameter of 20 to 70 nm, and Maximum particle size is 100
３００300 nm.

[0013] The liposome dispersion obtained by the production method of the present invention is not particularly limited, but components used for pharmaceuticals and cosmetics, for example, carcinostatic agents such as cytosine arabinoside, methotoxate and adriamycin, amphotericin B, gentamicin , Antibiotics such as piperacillin, liver diseases such as glutathione, superoxide dismutase, enzymes such as glucoamylase, tumor necrosis factor (TNF),
Epidermal growth factor (EGF), physiologically active substances such as erythropoietin, prostaglandins, hormones such as steroids,
Immunostimulants such as muramyl dipeptide, lymphokine and lentinan, nucleic acids of DNA and RNA, vitamin A and its derivatives, vitamin B and its derivatives, vitamin C
And its derivatives, vitamin E and its derivatives, vitamins D, pantothenic acid, vitamins such as nicotinamide, hyaluronic acid, chondroitin sulfate, α-hydroxy acid, sorbitol, xylitol, humectants such as maltitol, propylene glycol, Polyhydric alcohols such as 1,3-butylene glycol and glycerin, evening primrose, avocado oil, olive oil, mink oil, fatty acids, sphingolipids, lysophospholipids, squalene, squalane, fats and oils such as sterols and derivatives thereof, polyvinyl alcohol, Thickeners such as methylcellulose, organic solvents such as ethanol, other organic acids, pH adjusters, preservatives, anti-inflammatory agents, chelating agents, natural extracts, fragrances and the like can be added.

Although it is unclear for what reason the liposome dispersion obtained by the production method of the present invention has excellent storage stability, it is necessary to control the average particle diameter and the maximum particle diameter according to the micronization principle of the present invention. It is speculated that thermodynamically stable liposomes are easily obtained among liposomes made from the membrane lipid used, which may contribute to storage stability.

Hereinafter, a typical production method of the present invention will be described, but it is not particularly limited to this production method. Preparation of crude dispersion of liposome Add saturated phospholipid to water heated above the phase transition temperature and disperse using a stirrer such as a homomixer, or add saturated phospholipid to water and add a homomixer And then heated to a phase transition temperature or higher. In addition, when it is desired to obtain a liposome dispersion in which a pharmaceutical or cosmetic component is encapsulated in the internal aqueous phase or bilayer of the liposome, if the component is a water-soluble component, an aqueous solution in which the water-soluble component is dissolved is used. Prepare a crude dispersion of liposomes instead of water as described above,
Alternatively, even if a water-soluble component such as a polyhydric alcohol dissolves a saturated phospholipid, a solution in which the saturated phospholipid is dissolved with the component is added to water, and the above method is used. A crude liposome dispersion may be prepared. Furthermore, when the component to be encapsulated is a fat-soluble component, the component is dissolved in an organic solvent together with a saturated phospholipid, and then the solvent is removed and added to water. When a dispersion is prepared or a component that dissolves a saturated phospholipid and a fat-soluble component such as a polyhydric alcohol is used as an organic solvent, the saturated phospholipid and a fat-soluble component are dissolved in the component. The resulting solution may be added to water, and a crude liposome dispersion may be prepared by the above method. In addition, the phase transition temperature of the saturated phospholipid is measured in advance by a differential scanning calorimeter (DSC).

[0016] The liposome dispersion liquid by the micronization treatment
Preparation A coarse dispersion of the above liposome is micronized using the micronization principle of the present invention, for example, a commercially available DeBE
Process at a pressure of 25000 psi or more using E2000.

Next, the present invention will be described based on Examples and Test Examples.
This will be described in more detail.

Example 1 Preparation of crude liposome dispersion Hydrogenated egg yolk phospholipid (IV = 4, lipid composition: PC = 8)
(5%, PE = 12%, SPM = 1%, LPC = 1%, other = 1%) 20 g is dissolved in 300 g of propylene glycol heated to about 75 ° C. On the other hand, 1680 g of purified water in which 2 g of methylparaben was dissolved was heated to about 75 ° C., and a homomixer (special machine chemical industry: TK homomixer) was used.
While stirring at 10,000 rpm, the above solution was gradually added, and the mixture was further stirred for 10 minutes to prepare a crude dispersion of liposomes. Preparation of liposome dispersion by micronization treatment The crude liposome dispersion was subjected to micronization treatment once using DeBEE2000 at a pressure of 30,000 psi to obtain a liposome dispersion. The resulting liposome dispersion is
The average particle size of the liposome was 46 nm, and the maximum particle size was 198 nm. In addition, 50 liposome dispersions were added.
After filling 50 mL into a vial bottle having a capacity of mL (milliliter) and sealing, it was stored at 40 ° C. for 6 months, but no precipitate or insoluble matter was observed.

Example 1 Preparation of crude liposome dispersion 1960 g of purified water in which 2 g of methylparaben was dissolved was heated to about 75 ° C., and hydrogen was stirred at 10,000 rpm using a homomixer (Toki Hikami Kogyo: TK homomixer). Added egg yolk phospholipids (IV = 1, lipid composition: PC = 8
8%, PE = 10%, SPM = 0.5%, LPC = 0.
5%, other = 1%) 40 g gradually added,
The mixture was stirred for 10 minutes to prepare a crude liposome dispersion. Preparation of Liposomal Dispersion by Micronization Treatment The above crude liposome dispersion was micronized four times at 45,000 psi using DeBEE2000 to obtain a liposome dispersion. The resulting liposome dispersion is
The average particle size of the liposome was 23 nm, and the maximum particle size was 104 nm. In addition, 50 liposome dispersions were added.
After filling 50 mL into a vial bottle having a capacity of mL (milliliter) and sealing, it was stored at 40 ° C. for 6 months, but no precipitate or insoluble matter was observed.

[0019]

[Test example] Test example 1 Test method In the subsequent micronization treatment, the liposome dispersion shown in Table 1 before storage was prepared by using the crude liposome dispersion of Example 1 and changing the processing pressure, the number of treatments and the micronization apparatus. did. 50 mL (milliliter) of this liposome dispersion
After filling 50 mL into a vial with a capacity and sealing,
The dispersion was stored for months, and the state of the dispersion was visually compared with the average particle diameter and the maximum particle diameter of the liposome before and after the storage, and the storage stability of each dispersion was evaluated. The particle size was measured under the following conditions. Particle size distribution analyzer: NICOMP Model370 (Pacific Sc
Measurement mode: Solid Particles (converted to volume) Integration time: 10 minutes or more Residual: 2.0 or less

[0020]

[Table 1]

Table 1 shows the test results. Product No. of the present invention 1
-4 and the control product No. 1 and 2, the average particle diameter of the liposome dispersion prepared by the micronization principle of the present invention was 70%.
nm or the maximum particle size is 300n
When the average particle diameter is larger than 70n, a precipitate or an insoluble matter is generated after storage, which is not preferable.
m and a maximum particle diameter of 300 nm or less can be understood to be excellent in storage stability without generation of precipitates and the like even after storage. In particular, the product No. 1-2
Is less than 50 nm, and those having a maximum particle size of 200 nm or less have almost no change in both the average particle size and the maximum particle size before and after storage, and the state of the dispersion is also the state before storage. It is understood that they are retained and are more excellent in storage stability.

Also, the product No. of the present invention. 1-4 and control product N
o. From 3 to 4, even if the average particle size is 70 nm or less, and even if the maximum particle size is 300 nm or less, unless prepared according to the micronization principle of the present invention, a precipitate or an insoluble matter is preferably generated after storage. It is understood that there is no.

[0023]

As described above, a liposome dispersion having excellent long-term stability, which has not been observed in the conventional production method, can be obtained. Therefore, the liposome dispersion can be obtained not only in the fields of medicine and cosmetics but also in other fields. Is also expected to expand applications.

[0024]

[Brief description of the drawings]

FIG. 1 is a side sectional view for explaining the principle of forming fine particles of the present invention.

FIG. 2 is a cross-sectional view taken along the line AA of FIG.

[Explanation of symbols]

 DESCRIPTION OF SYMBOLS 1 Crude liposome dispersion 2 Nozzle 3 Jet flow 4 Bottom surface 5 Side wall 6 Reverse flow 7 Interface

Claims (2)

[Claims] In a liposome dispersion using a saturated phospholipid as a membrane lipid of a liposome, a crude dispersion of the liposome is jetted as a single jet stream, and the jet stream is jetted at a bottom face arranged opposite to the liposome. By changing the direction and flowing back along the side wall so as to envelop the jet flow, the coarse liposome dispersion is subjected to micronization treatment by the shear force generated at the interface between the jet flow and the back flow, and the average particle diameter is 70 nm or less. And the maximum particle size is 300
A method for producing a liposome dispersion, characterized in that a liposome having a diameter of at most nm is obtained. 2. The liposome has an average particle size of 20 to 70 n.
2. The method for producing a liposome dispersion according to claim 1, wherein m and the maximum particle diameter are 100 to 300 nm.