Method and apparatus for preparing novel liposome

Abstract

Disclosed is a method for preparing a liposome formulation. In the disclosed method, a lipid fraction is dissolved in an organic solvent. The solution including a bioactive component and the lipid fraction, together with a carrier, is put in a reaction vessel, and a supercritical fluid is introduced thereto, so as to prepare particles coated with the bioactive component-lipid. The supercritical fluid is discharged by compression to obtain proliposome particles, and then the proliposome particles are hydrated by an aqueous solution including water so as to form a liposome solution. Preferably, the formulation may include one or more bioactive components. As required, the liposome formulation may be further processed by methods such as particle size reduction, removal of organic solvent, and freeze-drying. The preparation method can be easily carried out at a laboratory scale. Furthermore, the same method can be employed in liposome formulation preparation in mass production, or at a commercial scale.

Claims

1. A method for preparing liposome formulation, the method comprising the steps of: (a) dissolving a lipid fraction comprising at least one kind selected from phospholipid and sterol, and a bioactive substance in an organic solvent; (b) putting an organic solvent solution of the step (a) together with a saccharide carrier into a reaction vessel and preparing proliposome particles coated with a lipid-bioactive substance mixture by introducing a supercritical fluid thereto, wherein the supercritical fluid and the organic solvent solution of step (a) are mixed together until reaching equilibrium, wherein, after reaching equilibrium, an additional supercritical fluid is added, the saccharide carrier comprising one or more components selected from the group consisting of lactose, sucrose, maltose, trehalose, dextrose, sorbitol, mannitol, and xylitol; (c) discharging the supercritical fluid in step (b) by decompression so as to obtain the proliposome particles; and (d) hydrating the proliposome particles in step (c) by an aqueous solution comprising water at a temperature in the range of 65 to 95 C. so as to form a liposome solution, wherein the organic solvent including the lipid fraction is divided into a bioactive-component dissoluble solvent and lipid-fraction dissoluble solvent and the bioactive-component dissoluble solvent is at least one kind selected from the group consisting of dimethylacetamide, dimethylformamide and dimethylsulfoxide, and wherein the bioactive substance is amphotericin B.

2. A method as claimed in claim 1, further comprising the step of reducing a liposome particle size by passing the liposome solution in step (d) through a microfluidizer.

3. A method as claimed in claim 1, wherein the phospholipid is at least one kind selected from the group including phosphatidyl glycerols, phosphatidyl cholines, phosphatidyl ethanolamines, phosphatidylserines, phosphatidylinositols, yolk lecithin, soybean lecithin, N-[1-(2,3-Dioleoyloxy)propyl]-N,N,N-trimethylammonium methylsulfate (DOTAP), N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), dioctadecyldimethylammonium bromide (DODAB), 2,3-dioleyloxy-N [2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminium trifluoroacetate (DOSPA), dioleoyl phosphatidylethanolamine (DOPE), 1,2-dimyristyloxypropyl-3-dimethyl-hydroxy ethyl ammonium bromide (DMRIE), didodecyldimethylammonium bromide (DDAB), 3-[N(N,N-dimethylaminoethane)-carbamoyl]cholesterol (DC-Chol), dioctadecylamidoglycylspermine (DOGS), N,N-[bis(2-hydroxyethyl)]-N-methyl-N-[2,3-di(tetradecanoyloxy)propyl] ammonium iodide, [N,N,N,N-tetramethyl-N,N-bis(2-hydroxyethyl)-2,3-di(oleoyloxy)-1,4-butanediammonium iodide], diethylaminoethyl cellulose (DEAE-C), N,N,N,N-tetrameth N,N,N,N-tetrapalmitylspermine, dioleoyl phosphatidylethanolamine, N-t-butyl-N-tetradecyl-3-tetradecylaminopropionamidine, and diethylaminoethyl dextran (DEAE-D).

4. A method as claimed in claim 1, wherein the sterol is at least one kind selected from the group including cholesterol, cholesterol hexasuccinate, ergosterol, and lanosterol.

5. A method as claimed in claim 1, wherein the supercritical fluid is selected from the group including supercritical carbon dioxide, supercritical nitrogen monoxide, supercritical acetylene, supercritical trifluoromethane, supercritical propane, supercritical ethylene, supercritical chlorofluocarbon and supercritical xenon.

6. A method as claimed in claim 1, further comprising the step of sterile-filtrating the prepared liposome.

7. A method as claimed in claim 1, further comprising the step of freeze-drying the prepared liposome.

Description

BRIEF DESCRIPTION OF DRAWINGS

(1) The foregoing and other objects, features and advantages of the present invention will become more apparent from the following detailed description when taken in conjunction with the accompanying drawings in which:

(2) FIG. 1 is a mimetic diagram illustrating an apparatus for preparing amphotericin B-containing Liposome, according to one embodiment of the present invention.

(3) FIG. 2 is a schematic diagram illustrating the method for preparing liposomes according to one embodiment of the present invention.

EXPLANATION OF SYMBOLS

(4) 1: reaction vessel 2: fluid storage vessel 3: supercritical fluid supplying tube 4: supercritical fluid supply amount adjusting pump 5: reactive material introducing part 6: pressure meter 7: supercritical fluid discharging tube 8: pressure-reducing valve 9: magnetic bar 10: magnetic agitator 11: back pressure regulator

MODE FOR THE INVENTION

(5) Hereinafter, the present invention will be described in more detail with reference to Examples below. However, the Examples do not limit the scope of the present invention in any way but are only to help the understanding of the present invention.

Examples 1a to 1f and 2a to 2d

Preparation of Amphotericin B Liposome

(6) 84 mg of distearoyl phosphatidylcholine (DSPC) was dissolved in 1 ml of a 1:1 mixed solvent of chloroform and methanol at 65 C. 200 mg of ascorbic acid (VIt-C) was completely dissolved in 2 ml of N,N-dimethylacetamide (DMA) through ultrasonication for 10 minutes. 50 mg of amphotericin B was dissolved in the DMA-Vit C solution at 65 C., and the resulting solution was added with the DSPC solution. 213 mg of hydrogenated soybean phosphatidyl choline and 52 mg of cholesterol were dissolved in 1 ml of a 1:1 mixed solvent of chloroform and methanol at 65 C. The solution of hydrogenated soybean phosphatidyl choline and cholesterol was mixed with the amphotericin B-DSPG solution.

(7) The amphotericin B-lipid solution and 900 mg of lactose were received in a reaction vessel 1, followed by sealing. The temperature of the reaction vessel was maintained at 45, 55, and 65 C. Through the operation of a pump 4, supercritical carbon dioxide was injected from a gas storage vessel 2 to the reaction vessel 1 via a supercritical fluid supplying tube 3, and then a pressure meter 6 was used to maintain the pressure at 150, 200, 250, and 300 bar. Through the operation of an agitator 10, the reaction vessel was rotated for 40 minutes at 500 rpm, while the materials were mixed up to an equilibrium state. To the reaction vessel, additional supercritical carbon dioxide was injected so as to remove the remaining organic solvent. Then, the supercritical carbon dioxide was discharged through a supercritical fluid discharging tube 7 by opening a pressure-reducing valve 8 while the reaction vessel was gradually decompressed to atmospheric pressure. After the decompression, from the reaction vessel, lactose particles coated with drugs and lipids were obtained. The particles were added with an aqueous solution (including 10 ml of water) at 55, 65, 75, and 85 C., and were agitated for 30 minutes, so as to provide a liposome solution.

(8) The particle size of the liposome obtained as described above was measured by an electrophoretic light scattering spectrophotometer (ELS-8000), and the drug content was measured. The results of the Examples are shown as below.

(9) Example 1 relates to the characteristics of a liposome solution according to a temperature and a pressure of a reaction vessel in a supercritical process. Particle sizes and drug contents according to temperatures and pressures of the reaction vessels, in Examples 1a to 1f, are noted in Table 1. Example 2 relates to the characteristics of a liposome solution according to a hydration temperature when proliposome particles prepared at a reaction temperature of 45 C. and a pressure of 200 bar are hydrated by addition of an aqueous solution (including water) in a supercritical process corresponding to Example 1a. Particle sizes, drug contents, and formation/non-formation of liposome according to hydration temperatures in Examples 2a to 2d are noted in Table 2.

(10) TABLE-US-00001 TABLE 1 Particle sizes and drug contents according to temperatures and pressures of the reaction vessels, in Examples 1a to 1f, Reaction vessel in a supercritical process Particle Drug Example 1 Temperature( C.) Pressure(bar) size(nm) content(%) a 45 200 949.3 90.5 b 55 200 801.6 86.3 c 65 200 839.1 77.1 d 45 150 761.0 91.1 e 45 250 855.7 84.2 f 45 300 821.3 79.7

(11) TABLE-US-00002 TABLE 2 Particle sizes, drug contents, and formation/non-formation of liposome according to hydration temperatures in Examples 2a to 2d Hydration tem- Particle Drug Example 2 perature( C.) size(nm) content(%) etc a 55 N/A N/A Non Formation of liposome b 65 949.3 90.5 Formation of liposome c 75 866.2 88.1 Formation of liposome d 85 752.1 84.3 Formation of liposome

Experimental Example 1

Stability of Amphotericin B Containing Liposome Against Freeze-Drying

(12) In order to improve ease of storage of amphotericin B containing liposome according to the present invention, freeze-drying was carried out, and the stability was tested.

(13) From among the example conditions, liposome, which was prepared by preparing proliposome at 45 C. and 200 bar, and carrying out hydration at 65 C., was passed through a microfluidizer so as to reduce the particle size of the liposome. Then, the liposome was freeze-dried, and was reconstituted by using distilled water as a redispersion solution. Before and after the freeze-drying, the size, the zeta potential, the drug content, and the loading efficiency of each liposome were measured. The result is noted in brief in Table 3.

(14) TABLE-US-00003 TABLE 3 Before and after the freeze-drying, the size, the zeta potential, the drug content, and the loading capacity of each liposome Particle Drug Loading state size(nm) Zeta potential content(%) capacity(%) Before freeze- 137.35 42.49 91.6 89.2 drying After freeze- 146.85 43.64 90.2 83.5 drying

(15) According to the result, the liposome prepared by a supercritical process can maintain the average size, the zeta potential, the drug content, and the loading efficiency even after freeze-drying, and thus liposome formulation can be preserved for a long time by using freeze-drying.

INDUSTRIAL APPLICABILITY

(16) Although several exemplary embodiments of the present invention have been described for illustrative purposes, those skilled in the art will appreciate that various modifications, additions and substitutions are possible, without departing from the scope and spirit of the invention as disclosed in the accompanying claims.