JPH0773668B2 - Method for preparing liposome and its precursor - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial field of use] The present invention relates to a novel method for producing a liposome and a precursor thereof which have a high capture rate of a substrate and are stable.

Liposomes, which are lipid vesicles, are membrane materials for sensors,
Attempts are being made to apply it to reaction fields such as photochemical reactions and oxygen reactions, and to artificial cells. Further, in recent years, attention has been paid to the use of a drug as a carrier for drug delivery that allows it to reach a target tissue only or regulate its absorption into a living body.

[Conventional technology]

As a method for preparing a liposome in which a substrate is incorporated, for example, (1) phospholipid is dissolved in chloroform, chloroform is removed to form a thin film of phospholipid, and an aqueous solution of the substrate is added thereto, Mechanical vibration (vortex swing) is applied at a temperature above the phase transition temperature (Tc) of the used phospholipid to obtain an emulsion liposome. The liposome at this time is a multilamellar liposome (MLV) (Voltex swing method). The multilamellar liposomes are then further sonicated to give small unilamellar vesicles (SUVs) (ultrasound method).

(2) Forming mixed micelles with phospholipids, water-soluble substrates, surfactants or sodium (deoxy) cholate, and then removing the surfactants and cholates by dialysis, gel filtration, ultracentrifugation, etc. To obtain unilamellar vesicles (detergent removal method).

(3) An aqueous solution of a substrate is dispersed in a low boiling point organic solvent solution of phospholipid to form a W / O type emulsion, from which the organic solvent is distilled off to gel.

Then, this is dispersed in an aqueous dispersion medium to obtain liposomes (reverse phase evaporation method). In addition to these, many methods for preparing liposomes have been reported. (For example, Kikuchi, Inoue,
Cell Engineering 2 No. 9, 1136 (1983)) [Problems to be Solved by the Invention] The preparation of liposomes by these known methods involves many steps and complicated operations. In addition, it is difficult to prepare a large amount of the liposome dispersion, and the long-term storability of the obtained liposome dispersion is poor, and, for example, aggregation of liposome particles and leakage of contents occur.

Furthermore, freeze-drying of the liposomes has been attempted in order to improve storage stability, but when the freeze-dried product is redispersed in an aqueous medium, aggregation of particles is observed (poor redispersibility).
In addition, there is a problem in quality and manufacturing such that a part of the liposome is disintegrated and the capture rate of the substrate does not return to the original value.

[Means for solving problems]

As a result of intensive studies to solve these problems, the present inventors have found a novel method for preparing liposomes and completed the present invention.

That is, the present invention comprises: (1) forming a uniform water-in-oil emulsion comprising a lipid, an organic solvent in which the lipid is soluble, a water-soluble substrate to be encapsulated, and water, and lyophilizing the emulsion to give water and an organic solvent. A method for preparing a freeze-dried liposome precursor characterized by removing a solvent, (2) a uniform water-in-oil emulsion comprising a lipid, an organic solvent in which the lipid is soluble, a water-soluble substrate to be encapsulated, and water. The present invention relates to a method for preparing a novel liposome, which comprises forming, removing water and an organic solvent from the emulsion by lyophilization to prepare a lyophilized liposome precursor, and dispersing the lyophilized liposome precursor in an aqueous medium.

The lipid used in the present invention is an amphipathic lipid.
Preferred hydrophilic groups of lipids include, but are not limited to, phosphate groups, carboxyl groups, sulphate groups, amino groups, glycidyl groups, and preferred hydrophobic groups are saturated and unsaturated hydrocarbon groups. However, the present invention is not limited to these. Preferred amphipathic lipids are those with phospholipids and close chemical structures. Examples of these are lecithin (phosphatidylcholine), phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid and their lyso forms, cardiolipin, sphingomyelin, cerebrosides, It is not limited to these. Particularly preferred are egg yolk lecithin, soybean lecithin, and hydrogenated products thereof, dipalmitoylphosphatidylethanolamine, dipalmitoylphosphatidylserine, brain sphingomyelin, dipalmitoylphosphatidylcholine, dimyristoylphosphatidyl. Choline, distearoylphosphatidylcholine, dioleoylphosphatidylcholine, dilinoleylphosphatidylcholine, dipalmitoylphosphatidylglycerol, dioleoylphosphatidylglycerol, dipalmitoylphosphatidic acid, dimyristoylphosphine Attic acid, dicetyl phosphate, stearyl amine, cetyl amine and the like can be mentioned. Further, in order to strengthen the lipid membrane, steroid compounds such as cholesterol, cholestanol, cholestane, etc., and α-tocopherol, butylhydroxytoluene, butylhydroxyanisole, etc. as antioxidants can be used together. When a compound having an acidic hydrophilic group (phosphate group, sulphate group, etc.) is used, the obtained liposome becomes anionic, and when a compound having a basic group such as an amino group is used, it is cationic. To obtain liposomes.

The liposome of the present invention can entrap any water-soluble substrate such as a physiologically active compound, a dye, a water-soluble polymer and a mixture thereof. At this time, as the physiologically active compound, nucleic acids, polynucleotides, antibacterial active compounds, antiviral compounds, antifungal compounds, anthelmintic compounds, antitumor compounds, proteins, toxins, Enzymes, hormones, neurotransmitters, glycoproteins, immunoglobulins, anti-inflammatory agents, anti-glaucoma agents, local anesthetics, etc.As dyes, acid dyes, basic dyes and fluorescent dyes, etc., Examples of water-soluble polymers include polysaccharides such as hyaluronic acid, polyvinyl alcohol, polyvinylpyrrolidone, and polyacrylic acid (soda). However, the water-soluble substrate used in the present invention is not limited to these and may be any compound desired to be encapsulated in liposomes.

The method for preparing the liposome according to the present invention is as follows.

First, dissolve the amphipathic lipid or lipid mixture in an organic solvent.
It Organic solvents dissolve lipids and are immiscible with water.
Any of them can be used, but among them, diethyl ether
Ethers, ethers such as dipropyl ether, dichloro ether
Chlorinated hydrocarbons such as methane and chloroform, hexa
Aliphatic hydrocarbons such as heptane and heptane and mixtures thereof
The thing is preferable. Then try to entrap it in liposomes
Add an aqueous solution of the substrate and add an ultrasonic disperser or microfluidizer.
Iser , Extruder Well dispersed by
To form a water-in-oil (W / O) emulsion. here,
The ratio of water solubility (aqueous phase) to lipid solution (oil phase) of the substrate is
Phase / oil phase is 1/1 to 1/100, preferably 1/3 to 1/50.
The amount of lipid is the amount required to coat the aqueous solution (aqueous phase).
If enough to exceed 15 mg of lipid per ml)
Good. This upper limit is limited only by cost-effectiveness practicability
To be done.

Then, the obtained emulsion is freeze-dried by a conventional method. For example, it is frozen with a refrigerant such as dry ice-acetone or liquid nitrogen, and the solvent is removed under vacuum using a commercially available freeze dryer.

As described above, a freeze-dried liposome precursor that can be stored in a sealed container can be obtained.

The liposome precursor obtained in the present invention is stored as it is, and when it is necessary or when the precursor is prepared, it is dispersed in an aqueous medium (such as physiological saline) to entrap the substrate. The liposome can be obtained. The particle size of the obtained liposome is between about 50 nm and 1000 nm, and is determined according to the composition of the lipid, the amount used, the type of substrate to be included, and the degree of W / O emulsion dispersion.

〔Example〕

The present invention will be described below with reference to examples, but the present invention is not limited thereto.

Example 1 Dipalmitoylphosphatidylcholine (DPPC) 58.7 mg, dioleoylphosphatidylcholine (DOPC) 15.7 mg and cholesterol 9.6 m as lipids in a 20 ml glass vial.
Take g, and add 7.5 ml of diethyl ether to dissolve. Add carboxyfluorescein (CF) solution (10
Add 2.5 ml of mM Tris-HCl Buffer, pH = 7.5, 100 mmol /) and disperse with a bath type ultrasonic disperser to form a W / O type emulsion. The emulsion is frozen in a dry ice-acetone refrigerant and freeze dried in a freeze dryer.
As described above, a powdery liposome precursor mixture which can be stored in a sealed container until needed was obtained.

6 mM of 10 mM Tris-HCl Buffer (pH = 7.5) was added to the freeze-dried powder.
1 was added, and the mixture was shaken, vortexed, and, if necessary, dispersed by an ultrasonic disperser to obtain an emulsion liposome dispersion. The average particle diameter of the liposome particles was 950 nm as measured with an N-4 submicron analyzer (Coulter).

Then, the capture rate of CF in the liposome was measured. The CF was quantified by measuring the fluorescence intensity (excitation light 490 nm, fluorescence 510 nm). The obtained liposome dispersion was diluted 1000 to 10000 times with Buffer (HEPES, pH = 8.5), and A: the fluorescence intensity of the diluted solution as it was (concentration of untrapped CF), B; 10 in the diluted solution. The fluorescence intensity (total CF concentration) when adding% sodium dodecyl sulfate was measured, and the capture rate was calculated by the following formula. Capture rate = (B−A) / B × 100 As a result, 14% of CF was captured.

The vial containing the lyophilized liposome precursor obtained here was stored in a refrigerator and the capture rate of CF after 6 months and 12 months was examined. It was 13% after 6 months and 13% after 12 months. It was. That is, the storage stability of the liposome precursor is good.

Example 2 A powdery liposome precursor mixture was obtained in the same manner as in Example 1 except that the lipid composition was DPPC 58.7 mg, DOPC 15.7 mg, stearylamine (SA) 2.7 mg, and cholesterol 9.6 mg. Further, 6 ml of 10 mM Tris-HCl Buffer (pH = 7.5) was added to obtain a liposome dispersion. Average particle size is 730nm, C
The capture rate of F was 22%. After 12 months refrigerated storage, the capture rate was 22%.

Example 3 In a 100 ml eggplant-shaped flask, DPPC 167.2 mg, DOPC 48 mg, SA
 5.4 mg, cholesterol 48.6 mg, α-tocopherol 20
A solution consisting of 15 mg of diethyl ether and 15 ml of diethyl ether
5 ml (10 IU / ml) of aqueous solution was added to prepare a liquid mixture,
This is a microfluidizer At (primary pressure 2.3kg / c
m²Secondary pressure 500kg / cm²4 ° C) dispersed W / O type emulsion
To get Place the emulsion in two 20 ml glass vials.
Add 10 ml each and freeze it with dry ice-acetone.
And freeze-dry with a centrifugal freeze dryer. From the above operation,
Insulated powder that can be stored in a sealed container until needed
A phosphorus-containing liposome precursor mixture was obtained.

6 ml of water was added to the lyophilized powder, and the mixture was shaken, vortexed, and if necessary dispersed with an ultrasonic disperser, an emulsion of insulin-containing liposome dispersion was obtained. The average particle size of the liposome particles was 750 nm.

The liposome dispersion is subjected to centrifugation (100,000 g, 30 minutes), the liposome particles are allowed to settle, and the insulin in the supernatant is measured by the EIA method (using an EIA method insulin measuring kit) to capture the insulin to the liposome. I asked.
As a result, it was 22.5%. The liposome precursor had a capture rate of 22% after being refrigerated for 12 months and stored.

Example 4 Egg yolk lecithin 75 mg, cholesterol 9.6 mg, α-
Tocopherol 7.5 mg, aqueous phase is bleomycin aqueous solution
A bleomycin-containing liposome precursor mixture was obtained in the same manner as in Example 1 except that the amount was 2.5 ml (10 mg / ml). Further, 6 ml of water was added thereto to obtain a bleomycin-containing liposome dispersion. Average particle size of liposome particles 850n
The capture rate of m and bleomycin was 15.5%. After the liposome precursor was stored in the refrigerator for 12 months, the capture rate of bleomycin was 15%.

Example 5 Egg yolk lecithin 150 mg, cholesterol 19.8 mg, α
-Tocopherol 15 mg, aqueous phase 5 ml cisplatin solution
A cisplatin-containing liposome precursor mixture was obtained in the same manner as in Example 3, except that the amount was 1 mg / ml in a 5% glucose aqueous solution. In addition, 6m of 5% glucose solution
l was added to obtain a cisplatin-containing liposome dispersion.
The average particle size of the liposome particles was 750 nm, and the capture rate of cisplatin was 9.5%. The liposome precursor had a capture rate of cisplatin of 9.0% after being refrigerated for 12 months.

Hereinafter, a comparison will be made with the reverse phase evaporation method, which is said to obtain the highest capture rate among the conventional preparation methods.

Comparative Example 1 DPPC 58.7 mg, DOPC 15.7 mg, in a 100 ml eggplant-shaped flask,
Take 9.6 mg of cholesterol and 7.5 ml of diethyl ether,
Furthermore, CF aqueous solution (Tris-HCl Buffcr (pH = 7.5) 100 mmol / m
l) Add 2.5 ml and disperse with a bath type ultrasonic disperser, and remove diethyl ether with an evaporator to form a gel. 6 ml of Tris-HCl Buffer (pH = 7.5) was added to this, and the gel was dispersed while rotating with an evaporator, and CF-containing liposomes (Reverse-phase Evaporation Vesicle)
Got As in Example 1, the average particle size of the liposome particles,
The CF capture rates were 1100 nm and 4.5%, respectively. It was 14% with the method according to the invention.

Comparative Example 2 The lipid composition was DPPC 58.7 mg, DOPC 15.7 mg, SA 2.7 mg, cholesterol 9.6 mg, and the aqueous phase was 2.5 ml of insulin solution.
Insulin-containing liposome (REV) was obtained in the same manner as in Comparative Example 1 except that the amount was (10 IU / ml).

In the same manner as in Example 3, the average particle size of the liposome particles and the capture rate of insulin were determined, and were 960 nm and 1 respectively.
It was 2.5%.

22.5% with the method according to the invention.

Then, 5 ml of the obtained liposome dispersion was placed in a 20 ml vial and freeze-dried. 5 ml of water was added to the lyophilized product for redispersion, and the product was returned to the liposome dispersion. The capture rate of insulin at this time was reduced to 3.5%.

〔The invention's effect〕

The method for preparing liposomes according to the present invention has a smaller number of preparation steps than other methods and can be stored for a long period of time because it is in the form of freeze-dried powder. Furthermore, by dispersing in an aqueous medium by an appropriate method, a liposome having a high inclusion amount of a substrate can be obtained, which contributes to promotion of liposome production and application.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (56) References JP-A 51-26213 (JP, A) JP-A 60-58915 (JP, A) JP-A 61-103822 (JP, A) JP-A 55- 118415 (JP, A)

Claims (2)

[Claims] 1. A uniform water-in-oil emulsion comprising a lipid, an organic solvent in which the lipid is soluble, a water-soluble substrate to be encapsulated, and water is formed, and the emulsion is lyophilized to remove the water and the organic solvent. A method for preparing a freeze-dried liposome precursor, which comprises: 2. A uniform oil-in-water emulsion comprising a lipid, an organic solvent in which the lipid is soluble, a water-soluble substrate to be encapsulated and water is formed, and water and the organic solvent are removed from the emulsion by lyophilization, A novel method for preparing a liposome, which comprises preparing a freeze-dried liposome precursor and dispersing the precursor in an aqueous medium.