KR100795221B1 - Heparin coated liposomes containing Amphotericin B and preparation method of the same - Google Patents

Abstract

The present invention relates to liposomes encapsulated with amphotericin B and whose surface is modified with heparin, and more particularly, a certain amount of cationic lipid is contained in lipids forming liposomes and encapsulated amphotericin B. It relates to a liposome characterized in that the surface of the liposome is modified with heparin and a method for producing the same. The liposome of the present invention increases the encapsulation efficiency of amphotericin B, which is poorly soluble in water phase, by using cationic lipids, and encapsulates highly potent amphotericin B in liposomes having high biocompatibility, thereby preventing toxicity to normal cells. In addition to reducing, heparin formulated to increase the body's circulation time and have a sustained release effect.

Amphotericin B, liposomes, heparin, cationic lipids, circulation time, sustained release

Description

Heparin coated liposomes containing Amphotericin B and preparation method of the same} containing amphotericin Β and its surface modified with heparin

FIG. 1 shows the pharmacokinetic results of fungus zone (Fungozone ^® ), Ambisome (AmBisome ^® ) and amphotericin B, and the surface of heparin-modified liposomes (HCL-AmB) in rat blood. At this time,-■-is puncture zone,-●-is ambisosome,-▲-is HCL-AmB.

The present invention relates to a liposome in which amphotericin B is encapsulated and its surface is modified with heparin, and more particularly, a certain amount of cationic lipid is contained in lipids forming liposomes, and amphotericin B is encapsulated. Liposomes characterized in that the surface of one liposome is modified with heparin, and a method for producing the same. The liposome of the present invention increases the encapsulation efficiency of amphotericin B, which is poorly soluble in water phase, by using cationic lipids, and encapsulates highly potent amphotericin B in liposomes having high biocompatibility, thereby preventing toxicity to normal cells. In addition to reducing, heparin formulated to increase the body's circulation time and have a sustained release effect.

Ampotericin B, a polyene antifungal agent used in the present invention, has a therapeutic effect against almost all fungal infections. In particular, since amphotericin B has an excellent therapeutic effect against systemic fungi, it can effectively treat life-threatening serious infections in cancer patients, bone marrow transplant patients, neutrophil reduction patients, immunocompromised patients, or immunodeficiency patients. Ampotericin B is a polyene antifungal agent and antibiotic that binds to ergosterol, a membrane sterol of fungi, and relocates to ion channels, thereby preventing osmotic control of fungal cells and treating them against fungi.

However, amphotericin B is toxic to normal cells and tissues by binding to normal cells cholesterol by the same mechanism as ergosterol during intravenous injection, accompanied by side effects such as chills, fever, tissue necrosis, nephrotoxicity. In particular, amphotericin B requires restriction and caution due to the difficulty in releasing the urinary tract by hemodialysis, and especially when used in children, the elderly, and patients with weak immunity. .

In addition, amphotericin B is insoluble in water at pH 6-7 and has a very low solubility of 0.1 mg / ml at pH 2 or pH 11, so that salts, micelles, emulsions, nanoparticles, or Soluble in the form of liposomes.

US Pat. No. 4,822,777 prepared amphotericin B in the form of a salt to improve solubility. In the patent, 100-400 nm particles were prepared using only amphotericin B and cholesterol sulfate, and the solubility in aqueous phase was improved by forming salts with amphotericin B. However, the patent is prepared in the form of amphotericin B simply salt, solubility was improved, but it is not formed into a dosage form, the body circulation time is short, the dosage is limited and toxicity to normal cells.

US Patent No. 5,059,591 uses amphotericin B and cholesterol-polyethyleneglycol (PEG) to reduce the toxicity of amphotericin B. In this patent, amphotericin B is combined with cholesterol-PEG in the form of a salt, wherein the circulation time in the body is increased by using the properties of PEG bound with cholesterol.

US Pat. No. 4,981,690 describes a method for preparing liposomes encapsulated with amphotericin B. The patent describes a method for preparing a multi-membrane liposome using phospholipids and cholesterol.

U.S. Patent No. 5,965,156 relates to a method for preparing liposomes encapsulated with amphotericin B. The patent encloses amphotericin B in liposomes and ion-bonds amphotericin B with anionic phosphatidylglycerol in an acidic organic solvent to increase the inclusion rate and reduce body toxicity when formed into a formulation. However, the above patent has a disadvantage in that the use of an acidic solution for ionic bonding of phosphatidylglycerol and amphotericin B promotes the decomposition of amphotericin and increases the loss rate. In addition, the stability of the formulation itself is also weakened due to the decomposition of the acid catalyst, and the rapid release of amphotericin B has a limitation in frequent administration and administration concentration.

Therefore, there is a need for a new method for improving the aqueous solubility of amphotericin B and reducing the toxicity of drugs to increase the sustained release and circulation time in the body as well as mass production.

Accordingly, the present inventors have made efforts to solve the problems of the above patents, and as a result, the encapsulation efficiency of amphotericin B is increased by using cationic lipids, and the highly toxic amphotericin B is encapsulated in a liposome having excellent biocompatibility. In addition to reducing toxicity to normal cells, he invented liposomes modified with heparin and amphotericin B having a sustained release effect, and a surface-modified liposome modified with heparin and a preparation method thereof.

The present invention provides a liposomal injection in which amphotericin B in which amphotericin B is encapsulated is modified with heparin.

The present invention also provides a method for preparing liposomes in which amphotericin B is encapsulated and whose surface is modified with heparin.

The present invention is a liposome injection containing amphotericin B, wherein the surface of the liposome encapsulated with amphotericin B is modified with heparin.

In another aspect, the present invention (1) mixing phosphatidylcholine, cationic lipids and cholesterol in a weight ratio of 40 ~ 70: 5 ~ 20: 10 ~ 40 dissolved in an organic solvent; (2) dissolving amphotericin B in a straight or branched alcohol having 1 to 6 carbon atoms; (3) mixing the solutions of steps (1) and (2) in a volume ratio of 1: 1 to 1: 9; (4) dispersing the mixed solution of step (3) in a water phase in a volume ratio of 2: 1 to 1:10 to form liposomes; (5) distilling the liposome solution of step (4) under reduced pressure at 20 to 50 ° C. to remove the organic solvent, and filtering to prepare a cationic liposome encapsulated with amphotericin B; (6) mixing the liposome solution and the heparin solution of step (5) such that the weight ratio of lipid to heparin to form a total liposome is 100: 2.5 to 100: 20; And (7) removing heparin or its derivatives from which liposomes are not modified in the mixed solution of step (6); and encapsulating amphotericin B, including the method of producing liposomes modified with heparin on its surface. do.

When explaining the invention in more detail as follows.

Lipids that form liposomes in the present invention are not particularly limited and may be known lipids. Examples of the lipid include phospholipids, glycolipids, fatty acids, dialkyl dimethylammnonium, polyglycerol alkyl ethers, polyoxyethylene alkyl ethers, alkylglycosides, alkylmethylglucamides, alkyl sucrose esters, Long-chain alkylamines or long-chain fatty acid hydrazides such as amphiphilic block copolymers such as dioxypolyoxyethylene ether and dialkyl polyglycerol ether, and polyoxyethylene-polylactic acid.

As the phospholipid, for example, phosphatidylcholine (soy phosphatidylcholine, egg yolk phosphatidylcholine, bovine phosphatidylcholine, dilauroylphosphatidylcholine, dimyristoylphosphatidylcholine, dipalmitoylphosphatidylcholine or distearoylphosphatidylcholine, etc.), phosphatidylethanolamine Lauroyl phosphatidyl ethanolamine, dimyristoyl phosphatidyl ethanol amine, dipalmitoyl phosphatidyl ethanol amine or distearoyl phosphatidyl ethanol amine, dioleoyl phosphatidyl ethanol amine, etc.), phosphatidylserine (dilauroyl phosphatidyl serine, di Myristoylphosphatidylserine, dipalmitoylphosphatidylserine or distearoylphosphatidylserine, etc.), phosphatidic acid, phosphatidylglycerol (dilauroylphosphatidylglycerol, dimyristoylphosphatidylglycerol, dipalmitoylphosphatidylglycerol or distea in Ylphosphatidylglycerol, etc.), phosphatidyl inositol (dilauroylphosphatidyl inositol, dimyristoyl phosphatidylinositol, dipalmitoyl phosphatidylinositol or distearoylphosphatidyl inositol, etc.), risphophosphidylcholine, sphingomyelin, yolk lecithin, soybean lecithin Or natural or synthetic phospholipids such as hydrogenated phospholipids.

As said glycolipid, glycerol glycolipid, sphingolipid glycolipid, etc. are mentioned, for example. Examples of the glycerol glycolipids include digalactosyldiglycerides (digalactosyldilauroylglycerides, digalactosyldimyristoylglycerides, digalactosyldipalmitoylglycerides or digalactosyldistearoylglycerides, and the like) or galactosyldi Glycerides (galactosyl dilauroyl glyceride, galactosyl dimyristoyl glyceride, galactosyl dipalmitoyl glyceride, galactosyl distearoyl glyceride, etc.) etc. are mentioned. Examples of the sphingoglycolipids include galactosyl selecbroside, lactosyl selecbroside, strong glosside and the like.

Examples of the sterols include cholesterol, cholesterol hexasuccinate, 3β- [N- (N '(N', N'-dimethylaminoethane) carbamoyl] cholesterol, ergosterol, lanosterol and the like.

Moreover, as said dialkyl dimethyl ammonium, dioctadecyl dimethyl ammonium (bromide salt), dialkyl dimethyl ammonium propane, dialkyl trimethyl ammonium propane, etc. are mentioned, for example.

The liposome forming lipids can be used alone or in combination of two or more thereof.

In particular, for the preparation of liposomes encapsulated with amphotericin B of the present invention, it is preferable to use phosphatidylcholine as a liposome-forming lipid, soybean phosphatidylcholine, egg yolk phosphatidylcholine or bovine phospholipid can be used, and more preferably hydrophobic tail carbon number. Has an alkyl chain having 16 to 18, and for example, dipalmitoylphosphatidylcholine or distearoylphosphatidylcholine can be used. Even if the carbon number is less than 16, it forms a strong bond with the amphiphilic amphotericin B. However, the phase transition temperature is lower than the body temperature, thereby lowering the stability of the liposomes, and when the carbon number is higher than 18, the binding force with the amphotericin B becomes weak. This is because the encapsulation efficiency of amphotericin B into the liposomes decreases and the size of the liposome particles increases. Phospholipid carbon number is an important determinant of HLB (hydrophilic hydrophobic equilibrium) value and phase transition temperature (Tc), and as the carbon number increases, the hydrophobicity increases and the phase transition temperature increases, while when the carbon number decreases, the hydrophilicity increases and the phase transition It is characterized by a decrease in temperature. In particular, 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine having 16 carbon atoms has the highest binding force with amphotericin B and at the same time, the phase transition temperature is higher than the body temperature at 41 ° C, which increases stability when forming liposomes. Do.

Since liposomes encapsulated with amphotericin B in the present invention should modify heparin on the surface, cationic liposomes capable of increasing the ionic binding force between the heparin and liposomes are preferable. For this purpose, dialkyldimethylammonium, dioleoylphosphatidylethanolamine, dioleoyldialkyltrimethylammoniumpropane or dioleoyldialkyldimethylammonium propane as cationic lipids are included in the liposome forming lipids. It is preferably included in 5 to 20% by weight. If the content of the cationic lipid is out of the lower limit, there is a problem such as weakening of the heparin coating on liposomes and increasing the size of the liposome itself. If the amount is out of the upper limit, the particle size is increased due to the aggregation of liposomes and heparin. there is a problem.

Heparin for modifying the liposome surface of the present invention can be separated and extracted from various animal tissues, or it is also possible to use recombinant heparin (recombinant heparin). Most of the heparin presently used for this purpose is separated from porcine mesangial membranes, and the separation process includes hydrolysis of the mucosa and extraction of heparin. In particular, the heparin of the present invention is to use heparin having a weight average molecular weight in the range of 500 to 50,000, and more preferably to use heparin having a weight average molecular weight of 1,000 to 5,000. If the molecular weight is less than the above range, there is a problem that the characteristics of heparin (increase in the circulation duration) is reduced, and if the molecular weight exceeds the above range, aggregation of liposomes through the network of heparin between liposomes, structural stability of liposomes There is a problem such as a decrease. Particularly, when using heparin having a molecular weight of several tens to millions of particles, the aggregation of the particles occurs rapidly, so that the formation of the formulation is not easy, and thus the measurement of particle size, encapsulation rate, and zeta potential is impossible. As used herein, "heparin" is meant to include the heparin, fragments of the heparin, salts of the heparin or derivatives of the heparin.

In the present invention, the surface of liposomes is modified with heparin to provide "in-circulation sustained" liposomes showing anionicity with improved stability and circulation time in blood. As used herein, "surface modification" means that the surface of the liposome is coated with a strong anionic heparin on the surface through ionic bonds. In addition, the "in vivo circulation sustained-type" liposome used herein is a liposome that heparin is modified on the surface of the liposome, unlike the usual liposome that disappears in the blood stream within several hours after administration, and the liposome is usually maintained for more than 60 hours in the blood stream after administration. Means liposomes. The heparin is 10 to 100 parts by weight, preferably 20 to 40 parts by weight, based on 100 parts by weight of the lipid forming liposome. If heparin is present below the above range, the anionicity of the surface of the liposome is not sufficient, so there is concern about the adsorption of plasma proteins in the blood, and the effect of improving stability and circulation time is not great, and the surface area of the liposome is limited even if the above range is exceeded. As a result, heparin no longer increases the body circulation time.

The average particle diameter of liposomes encapsulated with amphotericin B of the present invention and whose surface is modified with heparin is 50 to 300 nm, preferably 100 to 150 nm. If the mean particle size of the liposome exceeds the above range, uptake occurs by the reticuloendothelial system in the organ, ie, the liver or spleen during circulation, and if the liposome is below the above range, the amount of drug delivered to the target site ( drug payload) is insufficient.

Hereinafter, a method for preparing liposomes in which amphotericin B is encapsulated and whose surface is modified with heparin will be described.

In step (1), liposome-forming lipids, phosphatidylcholine, cationic lipids and cholesterol, are mixed in a weight ratio of 40 to 70: 5 to 20: 10 to 40. If the phosphatidylcholine used is less than the above range, the formation of liposomes is not easy, and if exceeded, the stability of liposomes is reduced. If the content of the cationic lipid is out of the lower limit, there are problems such as weakening of the heparin coating on liposomes, increase in the size of liposome itself particles, agglomeration between liposomes, and, if the cationic lipid is out of the upper limit, agglomeration of liposomes and heparin. There is a problem of increase in particle size. In addition, when the content of cholesterol is out of the lower limit, there is a problem that the encapsulation efficiency of the drug is reduced, when the content is out of the upper limit, there is a problem that the stability of the liposome particles is reduced. In addition, as the weight ratio of cholesterol increases, the encapsulation rate of the drug increases, but in the above range, the size of the particles increases, so it is preferable to prepare the above-mentioned range. The liposome forming lipid is dissolved in an organic solvent capable of dissolving the lipid, such as chloroform, methanol, toluene, and the like.

In step (2), amphotericin B is dissolved in a linear or branched alcohol having 1 to 6 carbon atoms. It is preferable to dissolve amphotericin B at 0.1 to 1 mg / ml in the alcohol. If the concentration of amphotericin B is out of the lower limit, the drug encapsulation concentration is lowered. If the concentration of amphotericin B is out of the upper limit, amphotericin B is not completely dissolved in alcohol. In addition, when using amphotericin B other organic solvents such as dimethyl sulfoxide (DMSO), dimethylformamide (DMF) and the like, there is a problem that the solvent is not easily removed and biocompatibility is reduced.

In step (3), the solutions of steps (1) and (2) are mixed at a volume ratio of 1: 1 to 1: 9. When the ratio of the solution (1) exceeds 50 (v / v)%, the concentration of the drug decreases, and when it is less than 10 (v / v)%, the amount of phospholipid is excessively reduced, making it difficult to form a formulation. There is this.

In step (4), the mixed solution of step (3) is dispersed in a water phase in a volume ratio of 2: 1 to 1:10, preferably 1: 1 to 1: 3 to form liposomes. The aqueous phase may be distilled water; Saline solution; Sugar solutions such as sucrose solution, maltose solution or mannitol solution; Buffer solutions such as phosphate buffer solutions; Or isotonic solution may be used. If the aqueous phase is less than the volume ratio range, the dispersed liposome particles aggregate to increase the particle size of the final liposome. If the aqueous phase exceeds the volume ratio, the solution may be diluted to add an unnecessary concentration process. In addition, it is preferable to disperse the mixed solution of step (3) in water using a syringe at a rate of 1-5 ml / min in tip sonication to form liposomes. If the rate of dispersion exceeds the above range, the size of the particles is increased, and even if less than the above range, it is difficult to form particles below a certain size, so the rate of dispersion of the above range is preferable.

In step (5), the liposome solution of step (4) is distilled under reduced pressure at 20 to 50 ° C. to remove an organic solvent, and filtered to prepare a cationic liposome encapsulated with amphotericin B. When the distillation under reduced pressure is less than 20 ℃, the removal time increases and it is difficult to completely remove the organic solvent, and if it exceeds 50 ℃ to maintain the above temperature because the liposomes formed and the drug can be denatured It is preferable. In addition, in order to completely remove the organic solvent, it is preferable to remove 0.5 to 5 times the volume of the organic solvent together with the organic solvent during the vacuum distillation. Purified liposome solution is prepared as a liposome solution having a uniform size distribution between 0.1 ~ 0.5 ㎛ using an injection molding machine. At this time, the filter membrane used in the injection molding machine is suitable for the pore size of 0.1 ~ 0.5 ㎛ the same as the particle size of the liposome. When the pore size exceeds 0.5 μm and the liposome exceeds 0.5 μm, the circulatory time is rapidly shortened due to capillary or reticuloendothelial cells during intravenous injection. There is a problem that the yield is drastically reduced by filtration.

(6) The liposome solution of step (5) and the heparin solution (concentration 0.3-5 mg / ml) are mixed in a weight ratio such that the weight ratio of lipid: heparin forming total liposomes is 100: 2.5-100: 20. When the weight ratio of heparin is less than the above range, a sufficient amount of heparin on the liposome surface is not easily modified, and when it exceeds the above range, aggregation occurs between liposome particles. In addition, the mixing speed of the liposome solution to the heparin solution is preferably 0.01 ~ 1 ㎖ / min, if the speed exceeds the rapid aggregation occurs due to heparin between the liposome particles, even if mixed below the above-mentioned coating effect The speed is preferable because it does not have a large effect.

(7) Remove the heparin unmodified liposomes from the mixed solution of step (6). Dialysis, gel filtration chromatography, or a high pressure filter may be used to remove heparin not bound to liposomes. In the case of dialysis, the molecular weight of the semipermeable membrane is preferably 50,000 to 500,000 for removing heparin, which is a linear polymer, and more preferably, gel filtration chromatography to remove phospholipids and amphotericin B that are not formed as liposomes together with heparin. It is more preferable to use.

Hereinafter, the present invention will be described in more detail based on the following examples, but the present invention is not limited thereto.

Production Example  One

60 mg of phosphatidylcholine (PC) selected from dilauroylphosphatidylcholine (DLPC), dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC) and distearoylphosphatidylcholine (DSPC) and cholesterol (CHOL) ) 20 mg was dissolved in 2 ml of chloroform to prepare a lipid mixture solution.

Ampoterisin B (AmB) was dissolved in methanol at a concentration of 0.5 mg / ml, and 2 ml of the lipid mixture solution and 8 ml of amphotericin B solution were mixed to prepare 10 ml of the AmB-lipid mixed solution.

10 ml of the AmB-lipid mixed solution was dispersed in 20 ml of distilled water at a rate of 2 ml / min while tip sonicating with a syringe to form liposomes, and then using a distillation distillation apparatus, the total amount of the solution at 35 ° C. was increased to 10 10 ml of the organic solvent and 10 ml of distilled water are removed until it reaches ml, and the size distribution of the particles is uniformed by passing through a 0.2 µm semipermeable membrane using an extruder.

The particle size of the liposome prepared by the above process was measured in a particle analyzer (Electrophoretic light scattering spectrophotometer, ELS-8000, Osuka Electronics, Japan) and is shown in Table 1.

division Composition ratio (weight ratio) Particle size (nm) Phase transition temperature Sample 1 DLPC: CHOL = 60: 20 79 -1 ℃ Sample 2 DMPC: CHOL = 60: 20 85 23 ℃ Sample 3 DPPC: CHOL = 60: 20 117 41 ℃ Sample 4 DSPC: CHOL = 60: 20 289 55 ℃

Production Example  2: Cationic  Preparation of Liposomes

In preparing liposomes in the same manner as in Preparation Example 1, in order to increase the encapsulation rate of Sample 3 having a liposome particle of 150 nm or less and a Tg of 41 ° C. to 90% or more, diethyldiocta The cationic liposomes were prepared by adding decylammonium bromide salt (DDAB) and dioleoyltrimethylammoniumpropenemethyl salt (DOTAP).

Changes in liposome particle size, zeta potential, and encapsulation efficiency according to the content of DDAB and DOTAP were measured and shown in Table 2 and Table 3, respectively.

division Composition (weight ratio) Particle size (nm) Zeta Potential (mV) Encapsulation Efficiency (%) DPPC CHOL DDAB Sample 3 60 20 0 117.0 6.81 87.3 Sample 5 60 20 2.5 115.0 40.76 87.1 Sample 6 60 20 5 87.3 42.27 97.6 Sample 7 60 20 10 101.8 41.80 91.2 Sample 8 60 20 20 89.6 43.59 98.9

division Composition (weight ratio) Particle size (nm) Zeta Potential (mV) Encapsulation Efficiency (%) DPPC CHOL DOTAP Sample 3 60 20 0 117.0 6.81 87.3 Sample 9 60 20 2.5 149.8 28.34 85.4 Sample 10 60 20 5 152.7 30.65 89.8 Sample 11 60 20 10 154.7 38.79 90.5 Sample 12 60 20 20 150.3 37.59 95.5

Production Example  3: Preparation of Heparin-Modified Liposomes

Heparin was used as having a weight average molecular weight of 1,500 and a linear characteristic.

Liposomes prepared by the method of Sample 6 of Preparation Example 2 were mixed in a weight ratio of lipid: heparin forming liposomes in a weight ratio of 100: 0 ~ 100: 40, wherein heparin was dissolved and mixed in the same volume of water as the liposome solution. . The mixed solution was purified by gel permeation chromatography (GPC) to prepare heparin-modified amphotericin B liposomes, and the particle size and zeta-potential by weight parts of liposomes and heparin were measured and shown in Table 4 below.

division Liposomes: Heparin (weight ratio) Particle size (nm) Zeta Potential (mV) Sample 6 100: 0 87.3 42.27 Sample 13 100: 1.25 83.2 37.42 Sample 14 100: 2.5 107.1 31.10 Sample 15 100: 5 138.3 22.53 Sample 16 100: 10 186.0 9.19 Sample 17 100: 20 232.8 5.91 Sample 18 100: 40 Cohesion Not measurable

Experimental Example

Sample 15 of Preparation Example 3, fungi zone (Fungozone ^® , Sample 19) and Ambisome (AmBisome ^® , Sample 20), which were intravenous antifungal drugs, were injected intravenously into mice at a concentration of 2 mg / kg for 24 hours. It is shown in Table 5 by comparing the area, the maximum concentration and the half-life of the retention concentration.

division AUC _{0 ~ 24h} C _max T _1/2 Sample 15 15.765 2.392 11.904 Sample 19 0.742 0.306 1.058 Sample 20 18.560 4.304 7.541

And AUC is the area (area under the curve) and C _max for the concentration of stay means the peak serum concentration, T _1/2 is the half-life.

Sample 15 of the present invention was significantly superior in all respects to the intravenous antifungal drug, Punjzon (Sample 19). The highest concentration is about half of the albisosome, which is less susceptible to side effects due to the toxicity of amphotericin B, and the half-life is about 57% longer than that of the albisome, which is much more advantageous as a circulating sustained liposome. In addition, the sample 15 exhibits the characteristics of circulating sustained liposomes in the body even though the zeta potential has a positive value unlike the amblysome, indicating that the above characteristics are obtained in the amblysomal and other mechanisms due to the use of heparin.

The present invention increases the encapsulation efficiency of amphotericin B, which is poorly soluble in water phase, using cationic lipids, and encapsulates highly potent amphotericin B in liposomes with high biocompatibility, thereby reducing toxicity to normal cells. Of course, modified heparin provides amphotericin B liposomes modified with heparin and increase the circulating time in the body and have a sustained release effect and a preparation method thereof.

Claims (13)

delete delete delete delete delete delete delete (1) mixing phosphatidylcholine, cationic lipids and cholesterol in a weight ratio of 40 to 70: 5 to 20: 10 to 40 and dissolving them in an organic solvent; (2) dissolving amphotericin B in a straight or branched alcohol having 1 to 6 carbon atoms; (3) mixing the solutions of steps (1) and (2) in a volume ratio of 1: 1 to 1: 9; (4) dispersing the mixed solution of step (3) in a water phase in a volume ratio of 2: 1 to 1:10 to form liposomes; (5) distilling the liposome solution of step (4) under reduced pressure at 20 to 50 ° C. to remove the organic solvent, and filtering to prepare a cationic liposome encapsulated with amphotericin B; (6) mixing the liposome solution and the heparin solution of step (5) such that the weight ratio of lipid to heparin to form a total liposome is 100: 2.5 to 100: 20; And (7) removing the heparin or its derivatives not modified liposomes in the mixed solution of step (6); containing amphotericin B containing a method for producing a liposome modified with heparin surface. The method according to claim 8, In the step (2), amphotericin B is dissolved in a straight or branched alcohol having 1 to 6 carbon atoms at a concentration of 0.1 to 1 mg / ml, and the amphotericin B is encapsulated and the surface is modified with heparin. Method for preparing liposomes. The method according to claim 8, Encapsulating the amphotericin B in step (4) by dispersing the mixed solution of step (3) using a syringe at a rate of 1 to 5 ml / min using tip sonication. Method for producing a liposome modified with this heparin. The method according to claim 8, The method of preparing liposomes encapsulated with amphotericin B, wherein the surface is modified with heparin, characterized in that (5) removes 0.5 to 5 times the volume of the organic solvent with the organic solvent. The method according to claim 8, Method for producing a liposome, characterized in that the filtration membrane using a filtration membrane having a pore size of 0.1 ~ 0.5 ㎛ in step (5) to uniformize the size distribution of the particles. The method according to claim 8, Method (7) the method of producing a liposome encapsulated amphotericin B, characterized in that to remove the unmodified heparin in liposomes using dialysis, gel filtration chromatography or a high pressure filter.

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