KR101142866B1 - Method of preparing liposome using shirasu porous glass membrane - Google Patents

Abstract

The present invention relates to a method for producing a liposome using a siras porous glass membrane, and more particularly, to a method for preparing a liposome that can adjust the size of the liposome particles prepared according to the number of passages and the extrusion pressure of the siras porous glass membrane of the aqueous liposome solution. . In the present invention, according to the characteristics of the durable, semi-permanent Siras porous glass can not only reduce the cost of the manufacturing process compared to the conventional film emulsification method using a polycarbonate membrane, but also by preparing liposome particles of various sizes, Liposomal particles usable as mass carriers in the medical, cosmetic and food fields, such as injectable preparations, contrast agents, detection reagents, and the like, can be provided.

Description

Method of preparing liposomes using a Siras porous glass membrane {Method of preparing liposome using shirasu porous glass membrane}

The present invention relates to a method for preparing liposomes using a siras porous glass membrane.

Liposomes are W / O / W-type emulsions [ J. Mol . Biol ., 13 , 238 (1965)], in which amphiphilic phospholipids are self-arranged by hydrophobic forces in the water phase. Phospholipid membrane constituting the liposome is the same as the structure of the cell, it is easy to enter the cell, and the space that can accommodate the water-soluble material in the interior compared to other structures, the carrier of the water-soluble material [ Eur. J. Pharm . Biopham . , 62, 110 (2006)] and carriers [ Nat . Rev. Drug Discov . , 145, 4 (1979).

Methods for preparing such liposomes include an ultrasonic method, a high speed rotation method, an emulsion diffusion method and a film emulsification method, and among these, a film emulsification method using a polycarbonate membrane is most widely used. In the film emulsification method using a polycarbonate membrane, a liposome having a predetermined size can be manufactured by passing a polycarbonate membrane by applying a constant pressure to an aqueous solution in which phospholipid is dispersed. However, the film emulsification method using the polycarbonate membrane is not suitable for mass production, and also has the disadvantage that the polycarbonate membrane is one-time. Thus, in order to supplement the disadvantages of the film emulsification method, recently, liposome preparation using a siras porous glass membrane has been attempted.

Shirasu Porous Glass Membrane (SPG Membrane) is a porous glass membrane obtained from volcanic ash. It is a new material developed in 1981 by Miyazaki Prefectural Industrial Test Center with abundant Syras of Southern Europe as the main raw material. It has a feature that can freely adjust the size of the pores. Therefore, by adjusting the pore size of the siras porous glass membrane as necessary and introducing various functional groups such as hydrophilic or hydrophobic functional groups on the surface thereof, it has been widely used in the manufacture of medical and food particles [ Colloid Surf . A- Physicochem . Eng . Asp ., 340, 20 (2009), J. Control . Release , 103, 31 (2005).

However, even in the case of manufacturing W / O type particles or O / W type particles using the Siras porous glass membrane, it is still difficult to manufacture micro-sized particles. In addition, when manufacturing the W / O type particles or O / W type particles by using a siras porous glass membrane, particles having a size larger than the pore size of the siras porous glass membrane is produced, to produce particles smaller than the pore size It is difficult. Accordingly, in order to commercialize liposomes having physical stability and various sizes, it is urgent to introduce a new production method which is cheaper than a conventional liposome preparation method and has a simple process.

It is an object of the present invention to provide a method for preparing liposomes using a siras porous glass membrane, and liposomes and mass carriers produced thereby.

The present invention provides a method for producing a liposome comprising the step of passing the aqueous solution of liposomes through a siras porous glass membrane as a means for solving the above problems.

The present invention provides another liposome particle produced by the production method according to the present invention as another means for solving the above problems.

The present invention is another means for solving the above problems, liposome particles according to the present invention; And it provides a mass carrier comprising a water-soluble material or a fat-soluble material supported on the inside of the liposome particles.

In the present invention, when the liposome aqueous solution is passed through the siras porous glass membrane, liposomes of various sizes can be prepared from several micrometers (μm) to several tens of nanometers (nm) by adjusting the extrusion pressure and the number of passages thereof. In addition, according to the present invention, the cost of the manufacturing process can be reduced compared to the conventional film emulsification method using a polycarbonate membrane according to the characteristics of the durable, semi-permanent Siras porous glass membrane.

1 is a graph showing the particle size of liposomes prepared according to the pore size and the extrusion pressure of each Shiras porous glass membrane (SPG membrane).
2 is a view showing the form and structure of liposomes prepared according to the present invention.
3 is a graph showing the transit time of liposomes according to the pore size of each Shiras porous glass membrane and the number of passages of the Shiras porous glass membrane.
Figure 4 is a graph showing the size of the liposome particles according to the number of passages of the siras porous glass membrane of the liposome aqueous solution.

The present invention relates to a method for producing liposomes comprising passing a liposome aqueous solution through a siras porous glass membrane.

Hereinafter, the liposome preparation method of the present invention will be described in detail.

In the present invention, the method for preparing liposomes may include passing the aqueous liposome solution through a siras porous glass membrane (SPG membrane).

The liposome aqueous solution of the present invention comprises the steps of mixing phospholipids and cholesterol; Dissolving the mixture in a solvent and evaporating the solvent to form a lipid membrane; And hydrating the lipid membrane in distilled water.

In preparing the liposome solution of the present invention, first, the phospholipid and cholesterol component may be mixed. The mixing ratio of the phospholipid and cholesterol is not particularly limited, but preferably, 10 parts by weight to 30 parts by weight of phospholipid and 1 part by weight to 10 parts by weight of cholesterol may be mixed. In the present invention, by controlling the content of the phospholipid and cholesterol at the weight ratio, while the cholesterol is located inside the phospholipid membrane, by strengthening the phospholipid membrane, it can have the effect of increasing the formulation stability of liposomes.

In the present specification, "parts by weight" means "weight ratio" unless otherwise specified.

The phospholipids of the present invention may be natural phospholipids or synthetic phospholipids. Specific types of the phospholipids that can be used in the present invention are not particularly limited, and for example, phosphatidylcholine (PC) series, phosphatidylinositol (PI) series, phosphatidylethanolamine (PE) series and phosphatidylserine (phosphatidylserine, PS) may be one or more selected from the group consisting of.

In the present invention, for example, 1,2-distearoyl-sn-glycero-3-phosphocholine (1,2-distearoyl-sn-glycero-3-phosphocholine, DSPC) of the phosphatidylcholine (PC) series ), But is not limited thereto.

In the present invention, the specific kind of the cholesterol is not particularly limited, and general ingredients that can be used in this field may be used.

In preparing the liposome solution of the present invention, after the mixing step, by dissolving the mixture of the phospholipids and cholesterol in a solvent, by removing the solvent by evaporation using a rotary evaporation condenser, a thin lipid membrane can be formed. .

The specific kind of the solvent used in the lipid film forming step of the present invention is not particularly limited, and any material that can dissolve the mixture appropriately and can be removed by evaporation can be used without limitation. In the present invention, for example, chloroform solution may be used as the solvent, but is not limited thereto.

In the present invention, the content of the solvent is not particularly limited, and in consideration of the content of the liposome aqueous solution to be prepared, a solvent having a volume equal to the volume of the aqueous liposome solution to be prepared may be used.

 Liposome aqueous solution can be prepared by hydrating the prepared thin lipid membrane in distilled water in the present invention. Phospholipids are composed of hydrophilic heads and hydrophobic tails, while phospholipids form lipid membranes, with hydrophilic heads facing outwards, and hydrophobic tails gathered inwards to form double membranes. .

In the present invention, the hydration of the lipid membrane in distilled water may be performed by adding distilled water to a round flask in which the lipid membrane is formed under atmospheric pressure and causing rotation using a rotary condensation evaporator.

In the present invention, the content of the distilled water used for the hydration is not particularly limited, it is possible to select an appropriate amount to hydrate the lipid membrane, for this purpose the volume of the solvent used to dissolve the mixture of lipid and cholesterol Volume amounts equal to or greater than may be used. In the present invention, when the volume of the distilled water used for hydration is less than the volume of the solvent used, there is a fear that all the thin lipid membranes formed in the round flask cannot be hydrated.

When the lipid membrane formed as described above is hydrated in distilled water, self-array occurs due to hydrophobic force, and the hydrophilic head portion is brought into contact with water, and the hydrophobic tail portions may be gathered inward to form a lipid membrane layer. . Accordingly, an aqueous liposome solution in which a liposome of a W / O / W type in which an aqueous solution is carried therein can be prepared.

In the present invention, liposomes of a desired size can be prepared by passing the prepared liposome aqueous solution through a siras porous glass membrane.

The pore size of the siras porous glass membrane of the present invention may be 0.1 μm to 20 μm, preferably 0.1 μm to 10 μm, and more preferably 0.1 μm to 5 μm. In the present invention, by adjusting the pore size of the siras porous glass membrane within the above range, it is possible to produce a liposome of the desired size according to the intended use of the liposome.

In the present invention, the size of the liposome prepared by passing through the siras porous glass membrane may be smaller than the pore size of the siras porous glass membrane. In the prior art, when manufacturing W / O type particles or O / W type particles using the Siras porous glass membrane, the size of the particles produced using the Siras porous glass membrane is 3 to 50 times larger than the pore size of the Siras porous glass membrane. it is known that there can be large (Journal of Food Engineering 92 , p. 244, (2009)). However, in the present invention, when the liposome is prepared by passing the siras porous glass membrane, the size of the prepared liposome is reduced than the pore size of the siras porous glass membrane, thereby making it possible to manufacture liposome particles having various sizes. In the present invention, the size of the liposome particles prepared using the siras porous glass membrane may be reduced to 25% to 81% of the pore size of the siras porous glass membrane.

In the present invention, in the step of passing the prepared liposome aqueous solution through the siras porous glass membrane, the extrusion pressure may be 0.1 Mpa to 1.0 Mpa, preferably 0.2 Mpa to 0.8 Mpa. In the present invention, if the extrusion pressure of the aqueous liposome solution is less than 0.1 Mpa, the pressure is inadequate and the aqueous liposome solution cannot pass through the siras porous glass membrane.

Attached FIG. 1 is a graph showing the particle size of liposomes prepared according to the pore size and extrusion pressure of each Shiras porous glass membrane. As shown in FIG. 1, when a certain pressure is applied, the smaller the pore size of the Siras porous glass membrane, the smaller the particle size of the prepared liposome. In addition, when the pore size of the siras porous glass membrane is constant, it can be seen that as the extrusion pressure applied to the liposome aqueous solution increases, the particle size of the prepared liposomes becomes smaller.

As described above, in the present invention, by adjusting the pore size of the Siras porous glass membrane and the extrusion pressure with respect to the liposome aqueous solution, liposomes of a desired size can be prepared.

In the present invention, the number of times of passing the prepared liposome aqueous solution through the siras porous glass membrane is not particularly limited, and may be, for example, one or more times, preferably three or more times, and more preferably five or more times.

In the present invention, the upper limit of the number of passages is not particularly limited, and may be preferably 50 times or less, more preferably 30 times or less, and most preferably 10 times or less.

3 is a graph showing the transit time of the liposomes according to the pore size and the number of passages of the siras porous glass membrane of each of the siras porous glass membranes (SPG membrane). As shown in FIG. 3, for each of the Siras porous glass membranes having pore sizes of 0.2 μm, 0.5 μm, 0.7 μm, 1 μm and 2 μm, the number of passages of the liposome aqueous solution was performed from 1 to 6 times. As the number of passages increases, the passage time of the siras porous glass membrane of the liposome aqueous solution is shortened. This increases the number of times the liposome aqueous solution is passed through the Siras porous glass membrane having a pore of a certain size, the particle size of the prepared liposome is gradually reduced, it can be expected that the passage time of the liposome aqueous solution accordingly.

4 is a graph showing the size of liposome particles according to the number of passages of the siras porous glass membrane of the liposome aqueous solution, which supports the contents of FIG. 3. In the accompanying FIG. 4, the liposome aqueous solution was passed through the siras porous glass membrane having a pore size of 1 μm under an extrusion pressure of 0.4 Mpa from one to five times, and in the case of the liposome particles passed through once, a diameter of about 0.85 μm In the case of particles of liposomes that were passed five times, the diameter was about 0.75 μm. As such, it can be seen that the number of passages of the siras porous glass membrane of the liposome aqueous solution is inversely proportional to the size of the prepared liposome particles.

The invention also relates to liposome particles produced by the production process according to the invention.

In the present invention, the liposome particles prepared by the above production method may have a diameter of 0.05 μm to 10 μm, preferably 0.05 μm to 5 μm.

The liposome particles of the present invention is a double membrane structure made of phospholipids, and may have a hollow portion capable of supporting a material therein.

The present invention also provides liposome particles according to the present invention; And it relates to a mass carrier comprising a water-soluble substance or a fat-soluble substance supported on the inside of the liposome particles. As described above, the liposome particle of the present invention is a bilayer structure made of phospholipid, and has a hollow portion capable of supporting a substance therein, and can carry a water-soluble substance containing a water-soluble substance or a fat-soluble substance.

In the present invention, if necessary, the hollow size of the liposome particles can be freely adjusted. Therefore, in the present invention, the ratio of the material to be supported may be freely adjusted depending on the situation in which the liposome particles are used.

In the present invention, a specific kind of water-soluble substance or fat-soluble substance that can be supported inside the liposome particles is not particularly limited. For example, pharmacologically active ingredients, contrast agents, luminescent substances, fluorescent substances, cosmetic preparations and functionalities One or more selected from the group consisting of food ingredients, but is not limited thereto.

In the present invention, the method of supporting the above water-soluble substance or fat-soluble substance in the inside of the liposome particles is not particularly limited, and for example, a method of mixing the liposome particles and the water-soluble substance or fat-soluble substance together in a suitable solvent, Liposomes may be supported using an encapsulation method of a water-soluble material or a fat-soluble material using a difference in concentration between the inside and the outside of the liposome, but is not limited thereto.

In addition, the use of the mass carrier of the present invention can be applied is not particularly limited, drug carriers containing water-soluble or fat-soluble drugs, carriers containing a functional material for cosmetics or carriers containing contrast substances used for MRI, etc. Can be mentioned. However, the use of the mass carrier of the present invention is not limited to the above uses, and various water-soluble substances or fat-soluble substances contained therein may be adjusted and used for various purposes.

[ Example ]

Hereinafter, the present invention will be described in more detail with reference to the following examples and comparative examples, but the scope of the present invention is not limited by the following examples.

Manufacturing example  One

A mixture of 95.7 mg of 1,2-distearoyl-sn-glycero-3-phosphocholine (1,2-distearoyl-sn-glcero-3-phosphocholine, DSPC) and 31.9 mg of cholesterol (CHOL) was purified to purity 99 After dissolving in 10 ml of% chloroform solution, the chloroform was removed by evaporation using a rotary evaporator condenser to form a thin lipid film. Thereafter, the lipid membrane was hydrated in 10 ml of distilled water to prepare an aqueous liposome solution.

Example  One

In order to confirm the change in the size of the prepared liposome particles according to the extrusion pressure of the liposome aqueous solution, liposome particles were prepared as follows.

For each Shiras porous glass membrane having pore sizes of 0.2 μm, 0.5 μm, 0.7 μm, 1 μm, 2 μm and 5 μm, the extrusion pressure was changed to 0.2 Mpa, 0.4 Mpa, 0.6 Mpa and 0.8 Mpa, respectively, 10 ml of the liposome aqueous solution prepared in Preparation Example 1 was passed through the respective Siras porous glass membranes at a temperature of 60 ° C.

Example  2

In order to check the size change of the prepared liposome particles according to the number of passages of the siras porous glass membrane of the liposome aqueous solution, liposome particles were prepared as follows.

For each of the Siras porous glass membranes having pore sizes of 0.2 μm, 0.5 μm, 0.7 μm, 1 μm and 2 μm, the number of repeated passages of the liposome aqueous solution was performed from 1 to 5 times, prepared in Preparation Example 1 above. 10 ml of aqueous liposomes were passed through each of the siras porous glass membranes at a temperature of 60 ° C. and a pressure of 0.6 Mpa.

For liposome particles prepared in the examples, the size, shape and transit time of the liposome particles were measured by the following method.

One. Liposome  Particle size measurement

After 2 ml of the liposomes prepared in Examples 1 and 2 were collected, they were placed in a light scattering particle size analyzer (DLS, Nano-S, manufactured by Marlvern, UK), and the size of the liposome particles was measured.

2. Liposome  Measure the shape of the particles

After administering 2 μl of the liposomes prepared in Examples 1 and 2 to the copper grid, the liposomes were added to an ethanol / liquid nitrogen solution to keep the liposomes in a cooling crystal state. Thereafter, the liposomes maintained in the cooling crystal state were placed on an electron transmission microscope (cryo-TEM, LIBRA 200 MC, manufactured by Carl Zeiss, Germany), and irradiated with 80 Kv to observe the shape and structure thereof.

3. Liposome  Particle Syras  Measurement of passage time of porous glass membrane

The time taken for the liposome prepared in Example 2 to pass from the inlet to the outlet of the siras porous glass membrane was measured using a stopwatch.

The measurement results as described above are collectively described in FIGS. 1 to 4.

The size change of the prepared liposome particles according to the extrusion pressure of the liposome aqueous solution is shown in FIG. 1, and the size change of the prepared liposome particles according to the number of passages of the siras porous glass membrane of the aqueous liposome solution is shown in FIGS. 3 and 4. The shape and structure of the prepared liposome particles are shown in FIG. 2.

As can be seen from the accompanying Figures 1 to 4, the size of the prepared liposome particles while varying the number of passage of the glass siras porous membrane of the aqueous solution of liposomes at a constant temperature and a certain pressure condition, the more the number of passages, The size of the prepared liposome particles decreased, and as a result of observing the size of the prepared liposome particles while varying the extrusion pressure of the aqueous liposome solution in the Siras porous glass membrane having a predetermined temperature and a predetermined pore size, the higher the extrusion pressure, , The size of the prepared liposome particles was reduced. In addition, according to the pore size of the siras porous glass membrane, the size of the prepared liposome particles were all smaller than the pore size. As such, liposome particles of a desired size can be prepared by controlling the number of passages of the siras porous glass membrane and / or the extrusion pressure or the pore size of the siras porous glass membrane of the aqueous liposome solution.

Claims (12)

The liposome aqueous solution is passed through a siras porous glass membrane having a pore size of 0.1 μm to 20 μm at an extrusion pressure of 0.1 Mpa to 1.0 Mpa, wherein the size of the liposome prepared by passing through the siras porous glass membrane is greater than that of the siras porous glass membrane. Method for producing liposomes smaller than the pore size. The method of claim 1,
Liposome aqueous solution, the step of mixing the phospholipids and cholesterol;
Dissolving the mixture in a solvent and evaporating the solvent to form a lipid membrane; And
Liposome manufacturing method comprising the step of hydrating the lipid membrane in distilled water. The method of claim 2,
Method for producing a liposome comprising a mixture of 10 parts to 30 parts by weight of phospholipid and 1 to 10 parts by weight of cholesterol. The method of claim 2,
A method for producing liposomes, wherein the phospholipid is a natural phospholipid or a synthetic phospholipid. delete delete delete The method of claim 1,
A method for producing a liposome, wherein the number of passages of the siras porous glass membrane of the liposome aqueous solution is one or more times. A liposome particle prepared by the method according to claim 1 and having a diameter of 0.05 μm to 10 μm. delete Liposome particles according to claim 9; And
Mass carrier comprising a water-soluble substance or a fat-soluble substance carried on the inside of the liposome particles. The method of claim 11,
A water-soluble substance or a fat-soluble substance is at least one substance carrier selected from the group consisting of pharmacologically active ingredients, contrast substances, luminescent substances, fluorescent substances, cosmetic preparations and functional food ingredients.

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