JPH03101614A - Production of liposome - Google Patents

Abstract

PURPOSE:To easily obtain a liposome useful for basic and application researches as a biomembrane model or a drug carrier for drug delivery, without using an organic solvent and a surfactant, by dispersing a liposome-membrane component substance in an aqueous solution and heating the mixture at a specific temperature. CONSTITUTION:A liposome dispersion is produced by dispersing 1 pt.wt. of a liposome-membrane component substance (e.g. phosphatidyl-choline in 3-100 pts.wt. of water, physiological saline water, buffer solution or an aqueous solution of sugar using an agitator. The dispersion is heated at or above the pretransition temperature(Tp) and below the main transition temperature(Tm) and held at the temperature for 5-20min. The treated dispersion is finally heated at >=Tm to obtain the objective liposome. The particle size can be uniformized by carrying out the above heat-treatment under irradiation with ultrasonic wave and the incorporation into the liposome membrane can be promoted by the use of 1 membrane component substance having high melting point in combination with the above substance.

Description

[Detailed description of the invention] (Industrial application field) The present invention relates to a method for producing ribosomes.

Ribosomes are closed vesicles with bilayer membranes composed mainly of amphiphilic substances such as phospholipids, and are used in basic and applied research as biological membrane models or drug carriers for drug delivery. (Background of the Invention) Many methods for preparing ribosomes are already known, such as “Methods of Biochemical A
“analysis” vol.33 (1988Lp.3
It is described in reviews such as 37. Most of these ribosome preparation methods involve chloroform, ether, ethanol, etc. 8 medium is used as a solvent for membrane component substances, or a surfactant such as Triton X-100 or deoxycholic acid is used as a solubilizing agent.

Therefore, there are problems with residual chemicals, process safety, and technical difficulties in industrial production. Methods for preparing ribosomes without using organic solvents or surfactants include JP-A-57-82310 and JP-A-57-8.
Freeze-drying method shown in No. 2311, JP-A-60-793
The heating method shown in No. 3 and the mechanical kneading method shown in No. 60-7934 have been developed. All methods involve hydration by mixing membrane constituent substances (mainly phospholipids) and an aqueous solution at a temperature above Tm; in the heating method, the temperature is adjusted to increase the temperature of the membrane constituent substances in order to accelerate the stagnation rate. In addition, in the mechanical kneading method, hydration is promoted by breaking the lipid particles using a mortar or the like in the mechanical kneading method. However, these methods are not necessarily satisfactory for industrial production. That is, in the heating method, the ribosome and the compound contained therein are heated to a temperature higher than Tα (e.g., to about 75°C in the case of dipalmitoylphosphatidylcholine (DI'PC)), so the type of the contained compound is heated. In some cases, it may change due to heat. Furthermore, both the heating method and the mechanical kneading method involve mechanical operation at a high temperature of Tc or higher (or Tα or higher), making the apparatus complicated to operate.

These problems can be avoided if a membrane component material with a low Tc is used, but membrane component materials with a high Tc are often used due to other problems such as leakage of encapsulated compounds.

(Problems to be Solved by the Invention) Therefore, the object of the present invention is to provide a method for preparing ribosomes without using an 1a'fJ medium or a surfactant, that is, a method for dispersing a membrane-specific substance in an aqueous solution. The goal is to further improve the method and develop a method that is as low-temperature, quick, and easy to operate. (Means for Achieving the Li! Subject) As a result of intensive study in line with the above purpose, the inventors have found that I
By making an aqueous solution dispersion of l2 precept substance powder and keeping it at a temperature above Tρ and below Tm for several minutes or more, the dispersed powder absorbs water and swells, and then when heated above Tm, myelination occurs immediately. They have discovered that ribosomes are formed through a similar structure, and have completed the present invention.

Lipid powder is usually commercially available in a dry state of monohydrate, so its phase transition temperature (Tα) is high; for example, L-α
- In the case of Dival Yodo Toylphosphazylcholine (DPPC), it is 60 to 70 when measured by differential scanning calorimetry (DSC).
It is around °C. However, when DPPC is hydrated with 10 or more molecules of water per molecule (approximately 20% or more water by weight), the main transition temperature (Tm) decreases to 4FC. Therefore, if the lipid powder can absorb 20wL% or more of water in advance, ribosomes can be rapidly prepared at temperatures slightly above 41°C. The present inventors investigated methods for making lipid powder absorb water, and found that there were two methods: leaving it in high-humidity air to absorb moisture, and water (<TP).
), water absorption occurs only very slowly, and rapid water absorption occurs only when the aqueous lipid dispersion is heated to a temperature above Tp and below Tm, and the powder swells. It was discovered that practically sufficient water absorption can be completed in about a few minutes. The present invention has been made based on this knowledge. When this sufficiently water-absorbed dispersion was heated above Tm, ribosomes could be formed immediately. Furthermore, by irradiating the prepared ribosome dispersion with ultrasound, the particle size became smaller and more uniform. Typical membrane-binding substances used in the present invention include natural and synthetic phospholipids such as phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, and phosphatidic acid, and mixtures thereof, hydrogenated lecithin, etc. Processed natural phospholipids can also be preferably used. Moreover, cholesterol, derivatives thereof, gangliosides, alkylamines, fatty acids, dicetyl phosphate, etc. may be added to these as stabilizers for the membrane structure.

As the aqueous solution for dispersing the crocodile, water, physiological saline, a buffer solution, an aqueous solution of saccharide, and a mixture thereof are preferably used. The appropriate ratio of the membrane component material to the aqueous solution is about 3 to 100 parts by weight of the aqueous solution to 1 part by weight of the membrane component material. Compounds to be encapsulated in the ribosomes of the present invention are not particularly limited, but include sugars such as glucose and dextran, amino acids, bebutides, proteins, DNA, etc. ．． RNA and other nucleic acids. In addition to vitamins, penicillin G is used as a drug.
Antibiotics such as these, anticancer drugs such as methotrexate, etc. can be used, and these are generally used in the form of an aqueous solution.

Next, the procedure for preparing ribosomes according to the present invention will be described in detail. If Tp and Tm in the permanent state of the membrane component substance are known from literature, those values can be used, but if unknown, they can be determined by experiment. Differential scanning calorimetry (DSC) is generally convenient. A good way to prepare a sample for DSC measurement is to disperse membrane substance powder in water and directly measure it using a solution cell. ．． Depending on the type of membrane-enhancing substance such as hydrogenated lecithin, Tp may not be clearly observed, but in that case Tm-5°C
is treated as Tp. In addition, if the membrane material is a mixture of several types, it is desirable to match the phase transition temperature of the main material. Next, powder of a membrane-enhancing substance is added to an aqueous solvent containing a compound to be encapsulated in ribosomes, and dispersed using a stirrer such as a homogenizer, a homomixer, or a propeller mixer. The solution temperature at this time is usually room temperature, which is convenient, but for example, dipalmitoylphosphatidylcholine (DMP)
When using a lipid with a low Tm (23°C) as in C), be careful not to exceed that temperature. Subsequently, the dispersion liquid is heated to -t-Tp or more and Tm or less, and maintained at that temperature. The holding time may be between 1 minute and 60 minutes, but 5 minutes to 20 minutes is usually preferred. At this time D
Even when TpSTm is low and the temperature at the time of dispersion is Tp or higher, such as MPC, the same process is applied. Next, ribosomes are obtained by heating this dispersion to a temperature higher than the Tm, but it is usually heated at a temperature 5 to 20°C higher than the Tm for 3 to 60 minutes, more preferably 10 to 30 minutes. At this time, it is preferable to use ultrasonic irradiation in combination, as this has the effect of uniformizing the particle size, and when a membrane-enhancing substance with a high melting point is used in combination, it also has the advantage of promoting uptake into the ribosomal membrane. You can be there. Ribosomes can be obtained by the operations described above, but in order to make the particle size more uniform, it is also possible to use pressure filtration using a microfilter, such as Nuclepore@. The ribosomes obtained in this way may be used as they are, or they may be used after separating and removing compounds that are not encapsulated in the ribosomes by means such as centrifugation, gel gelation, or dialysis. (Effects of the Invention) The advantages of the method of the present invention compared to known ribosome preparation methods are as follows. 1) Since no organic solvents or surfactants are used, there is no adverse effect on performance due to residual organic solvents, and there is no problem in terms of process safety. 2) The maximum operating temperature is low, just above Tm, and there is no need for stirring at high temperatures, so there are few problems in terms of safety. In addition, it does not require special equipment and is easy to operate, so it can easily be used with scale amplifiers. 3) Since the operating temperature does not need to be as high as in known heating methods, there is less risk of changes in the encapsulated compound due to heat.

(Examples) Next, the present invention will be further explained with reference to Examples, but the present invention is not limited to these. Commercially available α-dipalmitoyl phosphatidylcholine (
A bosome containing carboxyfluorescein (CF9) as a model compound was prepared using DPPC).
It is already known that PPC has a 'rp of 35°C and a Tm of 41'C. 100mM CF and 50mM N
aCj! 6 m M Trls buffer containing y one (
pH 7.0) 0.9g of L-α-DPPC powder in 30yd
was added and stirred for 3 minutes using a homogenizer at room temperature to finely disperse the powder. Next, use a constant temperature water bath to heat the temperature to 38°C, which corresponds to the Tp of DPPC or higher, for 10 minutes, and then
It was heated to 50° C. for 10 minutes above the Tm of PPC, and finally it was irradiated with ultrasonic waves at 50° C. for about 5 minutes using a bath-type ultrasonic irradiator rL (Model W-220R manufactured by Honda Electronics Co., Ltd.) and returned to room temperature.

The extraribosomal liquid of the obtained ribosome dispersion was centrifuged into 6mM TriS containing 150mM NaCl.
Changed to buffer 1- and removed C that was not encapsulated in ribosomes.
F was separated and removed. The encapsulated CFI is added to the ribosome dispersion using a surfactant (
Deoxycholic acid) was added to destroy the ribosomes to make a homogeneous solution, and then the fluorescence was measured using a fluorometer (492 nm excitation, 517 nm excitation).
The encapsulation efficiency was 7.5% when quantified using the following method. The particle size determined by light scattering (photon correlation spectroscopy) was approximately 1 μm, and uniform particle shape was observed by phase contrast optical microscopy. When a commercially available DPPC powder was directly measured by DSC, the phase transition temperature (Tα) of the powder was 69°C. 6mM containing 100mM CF and 50mM NaCl
Tris buffer y (pH 7.0) 30d to 75
Heat to ゛C, add 0.9g of DPPC powder to it and heat to 7
The mixture was stirred for 3 minutes using a homogenizer while maintaining the temperature at 5°C.

The obtained ribosome dispersion was washed by centrifugation, and the amount of encapsulated CF was quantified in the same manner as in Example 1. The encapsulation efficiency was 6.6%, and the average particle size was 2.5 to 3 μm. According to the results, the particle size distribution was somewhat broad. Temperature comparison test L The same formulation as in Example 1 was used, and after dispersing DPPC in the encapsulated liquid at room temperature, the temperature was increased to 50% above Tm without heating to 38°C.
It was heated at ℃ for 10 minutes. When a part of the sample was taken and observed under a phase contrast optical microscope, many coarse particles of several tens of microns remained, indicating that ribosomes were not completely formed. When the sample was further heated at 50°C for 5 hours and then subjected to ultrasonic irradiation at 50°C for 10 minutes, it was determined by optical microscopy that there were no coarse particles larger than 5 μm in diameter, and it was determined that the particles had almost become ribosomes. Centrifugation was carried out in the same manner as in Example 1, and the CF entrapment efficiency was measured and found to be 3.2%.

! { L-α-dimyristoyl phosphatidylcholine (DMPC) and L-α-dimyristoyl phosphatidylserine (DMPS) were used as a mixture as membrane components.

5mM phosphate buffer containing 0.28M glucose-p
0.8 g of DMPC powder and 0.2 g of DMPS powder were added to H7.4, 30- and dispersed for 3 minutes with a homogenizer at room temperature (20"C). The Tp of DMPC, which is the main component, is 14
℃, this dispersion was further allowed to stand at room temperature for IO minutes, and then heated at 30℃, which is above Tm (23℃), for IO minutes. After 5 minutes of ultrasonic irradiation at 30°C, a portion of the sample was taken and placed on a Sepharose 4B column (30cmφ x 2
Glucose not encapsulated in ribosomes was removed using 150mM NaCj! 5mM containing
Replaced with phosphate buffer pH 7.4. The obtained ribosomes were first disrupted with a surfactant, and then glucose was colorimetrically fixed, and the entrapment efficiency was 7.1%. Uniform granular ribosomes were observed by phase-contrast optical microscopy, and the average particle size by light scattering was 0.8μ. Procedural amendment

Claims (1)

[Claims] After dispersing the ribosome membrane component substance in an aqueous solution, it is first heated to a temperature above the pre-transition temperature (Tp) and below the main transition temperature (Tm) of the membrane component substance, and then heated to above Tm. Characteristic method for producing ribosomes.