JP5126874B2 - Method for producing liposome preparation - Google Patents

Description

  The present invention relates to a method for producing a liposome preparation having a regulated particle size distribution, and a liposome preparation obtained by the method.

  Liposomes are closed vesicles of bilayer membranes (liposome membranes) formed mainly by phospholipids and have similar structures and functions as biological membranes, and thus have been used as various research materials. Since this liposome can construct a so-called capsule structure that retains a water-soluble medicinal component in the aqueous phase inside and an oil-soluble medicinal component in the bilayer membrane, it can be used for diagnosis, treatment, makeup, etc. It has been used in various fields. Furthermore, in recent years, application to drug delivery systems (DDS) has been actively studied.

  Liposomes encapsulating such medicinal ingredients usually form a lipid film at the bottom of the flask under the rotation of a round bottom flask in a thermostat using a rotary evaporator or the like, and introduce an aqueous solution containing the inclusions therein. And formed under reduced pressure conditions (Non-Patent Document 1). And the liposome obtained in this way has a wide particle size distribution, and the liposome of the target diameter was recovered through treatment such as gel filtration. However, in this method, the recovered liposome is only about 1% of the input raw material, and an increase in production cost has been a big problem.

As a method for producing a liposome using a microchannel, there is a method such as Andreas Jahn (Non-patent Document 2). In this method, the flow path has a cross-shaped structure, and a mixture of inclusions, lipids, and an organic solvent flows in the central flow path, a buffer solution flows from the flow paths on both sides, and the mixing speed (dilution process) of the two adjusts the liposome. There is a technique to prepare. However, in this method, the organic substance remains in the liposome preparation, and an unfavorable influence on the living body due to the organic solvent becomes a problem.
J. Mol. Biol., 13,238 (1965): Bangham method J.Am.Chem. Soc. 2004, 126, 2674-2675 (Controlled Vesicle self-assembly in microfluidic channels with hydrodynamic focusing)

  An object of the present invention is to provide a method for efficiently preparing liposomes having a uniform particle size in a method for producing a liposome preparation, and to provide a liposome preparation obtained by the method.

  In the production method of the liposome preparation of the present invention, when producing a liposome using a microchannel, a phospholipid film is formed at one end in the microchannel, and then the inclusion target is introduced, and the flow rate and the channel inner diameter are determined. A liposome preparation having a uniform particle diameter is prepared by adjusting to form liposomes.

That is, this invention consists of the following.
1. A phospholipid dissolved in an organic solvent is introduced into a microchannel having an inner diameter of 100 to 1000 μm, and the organic solvent is dried to form a phospholipid thin film on the wall of the microchannel. A method for producing a liposome preparation having a uniform particle diameter, including a step of flowing an aqueous solution containing an inclusion target at a flow rate of 5 m / second, peeling off a thin film, and generating liposomes.
2. 2. The method for producing a liposome preparation according to item 1 above, wherein the temperature during liposome formation is 15 to 70 ° C.
3. 3. The method for producing a liposome preparation according to item 1 or 2, wherein the organic solvent is distilled off under reduced pressure to form a phospholipid thin film on the wall of the microchannel.
4). 4. The method for producing a liposome preparation according to any one of items 1 to 3, which comprises a step of centrifuging the liposome discharged from the microchannel.

  According to the method for producing a liposome preparation of the present invention, it is possible to efficiently obtain liposomes having a desired particle size, and since the method is simple, an excellent method for preparing liposomes at an industrial level. provide.

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

  In the method for producing a liposome preparation of the present invention, a phospholipid dissolved in an organic solvent is introduced into a microchannel, the organic solvent is dried to form a phospholipid thin film on the wall of the microchannel, and the flow rate is adjusted. Below, it is a method including the process of flowing the aqueous solution containing the inclusion object, peeling off the thin film, and producing liposomes.

  The liposome preparation of the present invention can be prepared as an aqueous dispersion preparation or lyophilized preparation of liposome. A suitable preparation can be prepared as an aqueous dispersion in which the inclusion target is a water-soluble drug or the like and liposomes encapsulating the water-soluble drug are dispersed in an aqueous solvent. In the aqueous dispersion of liposomes, stabilizers, chelating agents, antioxidants, viscosity modifiers, buffers, pH adjusters, and the like may be dissolved or dispersed. Various methods are known for preparing such an aqueous dispersion of liposomes, and it is appropriately selected according to the purpose, characteristics, use and the like.

  Examples of water-soluble drugs encapsulated in liposomes include contrast agents, antioxidants, antibacterial agents, anti-inflammatory agents, blood circulation promoters, whitening agents, skin roughening agents, anti-aging agents, hair growth promoters, moisturizers, hormone agents Vitamins, pigments, and proteins.

Liposomes prepared by the production method of the present invention include multilamellar vesicles (MLV), single-layer LUV liposomes having a large particle size (Large unilamellar veislcles), particles having a particle size of less than 50 nm. Liposomes (small unilamellar vesicles) composed of small single-film SUVs may be mentioned, and these may be mixed.
Suitable liposomes are usually formed from lipid bilayers. As a component of the lipid membrane, usually phospholipid is preferably used.

  The raw material phospholipids used in the liposomes prepared by the production method of the present invention are neutral phospholipids such as lecithin, lysolecithin and / or hydrogenated products and hydroxide derivatives obtained from soybeans, egg yolks, etc. Can be mentioned. These may be used alone or in combination. Other phospholipids include egg yolk, soybean or other phosphatidylcholine, phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, phosphatidylethanolamine, sphingomyelin, synthetically obtained phosphatidic acid, dipalmitoyl phosphatidylcholine (DPPC), distearoyl Phosphatidylcholine (DSPC), Dimyristolphosphatidylcholine (DMPC), Dioleylphosphatidylcholine (DOPC), Dipalmitoylphosphatidylglycerol (DPPG), Distearoylphosphatidylserine (DSPS), Distearoylphosphatidylglycerol (DSPG), Dipalmitoylphosphatidylinositol (DPPI) ), Distearoyl phosphatidylinositol (DSPI), dipalmi Yl lysophosphatidic acid (DPPA), distearoyl lysophosphatidic acid (DSPA), and the like.

  These phospholipids are usually used alone, but may be used in combination of two or more. When two or more kinds of charged phospholipids are used, it is desirable to use them between negatively charged phospholipids or between positively charged phospholipids from the viewpoint of preventing liposome aggregation. When neutral phospholipids and charged phospholipids are used in combination, the weight ratio is usually 200: 1 to 3: 1, preferably 100: 1 to 4: 1, and more preferably 40: 1 to 5: 1.

  If desired, other components in addition to the lipid component can be added as membrane constituents of the liposome. Examples thereof include sterols such as cholesterol and cholesterol esters as membrane stabilizers or anchors for introducing polyalkylene oxide groups, and dialkyl phosphates such as dicetyl phosphate which is a charged substance.

In the manufacturing method of the present invention, the microchannel is a channel having an inlet and an outlet having a length of 2 mm to 5 m, and an inner diameter of the channel is 100 to 1000 μm, preferably 200 to 530 μm. The inner diameter is adjusted according to the desired liposome particle diameter. A phospholipid is dissolved in an organic solvent such as chloroform or alcohol (isopropyl alcohol) and introduced into the microchannel. The mixing ratio of the organic solvent and the phospholipid is not particularly limited as long as it is a sufficient amount for dissolving the phospholipid, but is generally adjusted to a concentration of 2 to 30 mg / ml and used. The amount of the phospholipid-containing organic solvent introduced into the microchannel is such that the channel is sufficiently filled according to the volume of the microchannel. After being introduced into the flow path, the organic solvent is completely blown out of the flow path by rotation, drying, etc., and a thin film is formed on the inner wall of the flow path. In order to fly the organic solvent, a uniform thin film is formed at a temperature of 15 to 40 ° C. for about 500 to 4000 minutes. The thickness of the thin film is about 2 to 20 μm.
After the thin film is formed, it is sufficiently dried (for example, over 500 to 1500 minutes), and then an aqueous solution containing inclusions, such as physiological saline, is introduced into the microchannel to form liposomes. At this time, the introduction speed of the aqueous solution is such that the flow rate is 0.01 to 0.5 m / second, preferably 0.05 to 0.3 m / second, and more preferably 0.1 to 0.2 m / second. The aqueous solution temperature in the flow path is 15 to 70 ° C, preferably 50 to 65 ° C, more preferably 55 to 62 ° C.
Under this condition, the thin film is peeled off by the aqueous solution, and the liposome is discharged from the channel. The formed liposome is recovered as a precipitate by means such as centrifugation. The collected liposome is subjected to filtration for particle size adjustment, addition treatment for formulation, sterilization, etc., if desired, and is prepared as a freeze-dried and / or aqueous solution formulation as desired.

  EXAMPLES Hereinafter, although an Example demonstrates this invention further more concretely, this invention is not limited to these Examples.

(Production Example 1)
Preparation of liposomes
After adding 10 ml of chloroform to 0.3 g of egg yolk lecithin and 20 mg of dicetyl phosphate, the mixture was stirred until completely dissolved. Keeping the temperature at 60 ° C., a sufficient amount was introduced into a microchannel having an inner diameter of 200, 320 and 530 μm and a length of 1.5 m, chloroform was distilled off under reduced pressure for 1000 minutes, and a phospholipid thin film was removed. Formed on the inner wall. Next, physiological saline at a temperature of 60 ° C. was introduced from the inlet of the microchannel at a flow rate of 0.05 to 0.25 m / sec to obtain a dispersed aqueous solution of liposomes formed from the outlet of the microchannel. This aqueous solution was centrifuged at 12000 rpm, and multilamellar liposomes were collected as a precipitate. The precipitate was lyophilized to prepare a dry liposome preparation.

(Experimental example 1)
About the liposome obtained by the method shown in the Examples, measurement of the influence of the inner diameter of the microchannel, measurement of the influence of the flow rate of the aqueous solution introduced into the channel, measurement of the influence of temperature, measurement of the particle size distribution, And yield evaluation [amount of liposome having a width of ± 25 nm from the peak value of particle diameter (mg) / amount of egg yolk lecithin (mg) in channel tube × 100] was performed.

FIG. 1 shows the particle diameter of a liposome when a microchannel having an inner diameter of 320 μm is used and physiological saline is introduced into the channel at a flow rate of 0.25 m / sec under the condition of 60 ° C. to form liposomes. This is a comparison between the distribution and the particle size of liposomes produced by the Bangham method (Non-patent Document 1). In the figure, the horizontal axis indicates the particle diameter (nm), the vertical axis indicates the intensity (%) (probability density), the dotted line indicates the present invention, and the solid line indicates the Bangham method. According to the method of the present invention, the distribution is one peak, but according to the Bangham method, there are two peaks.
Fig. 2 shows the formation of liposomes by introducing physiological saline into the channel at a flow rate of 0.05 to 0.25 m / sec under conditions of 60 ° C using microchannels with an inner diameter of 200, 320 and 530 µm. The average particle diameter of the liposomes when measured was measured. In the figure, the horizontal axis indicates the flow rate of physiological saline introduced into the microchannel, the vertical axis indicates the particle size of the obtained liposome, the one-dot chain line is based on the Bangham method, and □ indicates the inner diameter of the microchannel is 320 μm. Indicates the average particle diameter of liposomes obtained by the microchannel having an inner diameter of 530 μm, and the rhombus indicates the average particle diameter of the liposome obtained by the microchannel having an inner diameter of 530 μm. In the method of the present invention, it is shown that the particle size can be adjusted according to the flow rate.
Table 1 shows the yield results and shows that the yield is affected by the inner diameter of the microchannel and the introduction rate of the aqueous solution into the microchannel. Since the recovery rate by the Bangham method is 0.82%, 320 μm, 60 ° C., 0.25 m / second, 530 μm, 60 ° C., 0.25 m / second, 530 μm, 60 ° C., 0.15 m / second, 530 μm, It can be confirmed that a preferable recovery rate is obtained at 60 ° C. and 0.05 m / sec.

The particle size distribution of the liposome preparation of the present invention is shown. The influence of the flow rate and the inner diameter of the microchannel in the production method of the liposome preparation of the present invention is shown.

Claims (4)

A phospholipid dissolved in an organic solvent is introduced into a microchannel having an inner diameter of 100 to 1000 μm, and the organic solvent is dried to form a phospholipid thin film on the wall of the microchannel. A method for producing a liposome preparation having a uniform particle diameter, including a step of flowing an aqueous solution containing an inclusion target at a flow rate of 5 m / second, peeling off a thin film, and generating liposomes. The method for producing a liposome preparation according to claim 1, wherein the temperature at the time of liposome formation is 15 to 70 ° C. The method for producing a liposome preparation according to claim 1 or 2, wherein the organic solvent is distilled off under reduced pressure to form a thin film of phospholipid on the wall of the microchannel. The manufacturing method of the liposome formulation as described in any one of Claims 1-3 including the process of centrifuging the liposome discharged | emitted from the microchannel.