KR101846090B1 - Preparation method of liposomal doxorubicin - Google Patents

Abstract

The present invention relates to a novel process for the preparation of povidized liposome preparations containing doxorubicin. According to the present invention, it is possible to prepare a pegylated liposome preparation containing doxorubicin having a narrow nano-sized particle size distribution. Further, according to the present invention, the drug encapsulation rate and storage stability of the liposome preparation are excellent, so that the productivity can be improved.

Description

PREPARATION METHOD OF LIPOSOMAL DOXORUBICIN [0002]

The present invention relates to a process for the preparation of a povidized liposome preparation containing doxorubicin.

Doxorubicin (doxorubicin hydrochloride) is one of the most widely used cytotoxic anthracycline antibiotics in cancer chemotherapy and is known to be active against a variety of tumors. The doxorubicin is useful for the treatment of numerous solid tumors and leukemia, and is particularly effective for the treatment of breast cancer associated with multiple therapies. In addition, doxorubicin is a protocol therapy for Kaposi's sarcoma associated with AIDS and shows excellent activity against ovarian cancer, lung cancer, prostate cancer, uterine cancer, head and neck cancer, estrogenic sarcoma and Ewing's sarcoma .

However, doxorubicin has problems indicating cytotoxicity such as cardiac toxicity and bone marrow toxicity, which cause congestive heart failure. Thus, a liposomal doxorubicin preparation in which doxorubicin is encapsulated in liposome is used for effectively reducing the toxicity of doxorubicin and effectively delivering and releasing it to a target organ to be treated.

A commercially available liposome preparation containing such doxorubicin is Caelyx ( ^R ). These liposome preparations are liposome preparations in which liposome surface is coated with polyethylene glycol (PEG) polymer. PEG improves the blood circulation time of the drug, has low nonspecific transport to normal tissues and excellent tumor specificity.

The literature on the preparation of the above doxorubicin-containing pegylated liposome formulations includes U.S. Pat. No. 4,927,571. The patent document discloses a Thin-Film Hydration Method in which a phospholipid and a cholesterol are dispersed in an organic solvent, and then the solvent is removed and re-dispersed in an aqueous solution containing doxorubicin, lactose, and a salt. However, according to this method, the particle size of the liposome is not uniformly controlled, and a high filling rate of 95% or more can not be achieved.

Accordingly, a novel preparation method of doxorubicin-containing pegylated liposome preparation having narrow particle size distribution, excellent storage stability, and high inclusion rate in order to enhance the pharmacological effect of the pegylated liposome preparation containing doxorubicin and ensure uniform quality It is necessary.

U.S. Patent No. 4,927,571, "Preparation of injectable Doxorubicin / liposome suspension"

It is an object of the present invention to provide a novel preparation method capable of producing a pegylated liposome preparation containing doxorubicin of excellent quality.

In order to achieve the above object,

S1) adding phospholipids, sterols and PEG-conjugated lipids to the lower alcohol to produce a lipid solution;

S2) dropping the lipid solution into an inorganic salt aqueous solution to prepare a liposomal dispersion solution;

S3) passing the co-liposome dispersion solution through an extruder to perform extrusion molding;

S4) performing a dialysis process on the resultant co-liposome solution; And

S5) encapsulating doxorubicin in the co-liposome to prepare a liposome preparation. The present invention also provides a method for producing a pseudo-liposome preparation containing doxorubicin.

According to the present invention, it is possible to prepare a pellifϊed liposome preparation containing doxorubicin having a nano size and a narrow particle size distribution. The liposome preparation thus prepared has a very high containment rate of doxorubicin and excellent storage stability.

Such a manufacturing method enables a mass production of a liposome preparation through stable supply of raw materials and securing of equipment by a method optimized for a pilot scale.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 schematically illustrates a method for preparing a pegylated liposome preparation containing doxorubicin according to the present invention.
2 is a graph comparing particle size distributions of coelom liposomes before and after dialysis.
3 is a photograph of an ampule containing the liposome preparation prepared in Example 1. Fig.
4 is an ultra-low temperature transmission electron micrograph of the liposome preparation and the control preparation of Example 1. Fig.
5 is a graph of particle size distribution of the liposome preparation and the control preparation of Example 1. Fig.
Fig. 6 is a graph comparing the elution characteristics of the liposome preparation and the control preparation of Example 1. Fig.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, exemplary embodiments of the present invention will be described in detail with reference to the accompanying drawings. The present invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein.

The present invention provides a method for producing a pegylated liposome preparation containing doxorubicin having a narrow particle size distribution, an excellent sealing rate and a high storage stability. PEGylated liposome preparations are liposome preparations in which a polyethylene glycol (PEG) chain is bonded to the surface, and can be protected from the immune system upon human administration, and exhibit excellent pharmacological effects with a long half-life in the body.

Specifically,

S1) adding phospholipids, sterols and PEG-conjugated lipids to the lower alcohol to produce a lipid solution;

S2) dropping the lipid solution into an inorganic salt aqueous solution to prepare a liposomal dispersion solution;

S3) passing the co-liposome dispersion solution through an extruder to perform extrusion molding;

S4) performing a dialysis process on the resultant co-liposome solution; And

S5) encapsulating doxorubicin in the co-liposome to prepare a liposome preparation. The present invention also provides a method for producing a pseudo-liposome preparation containing doxorubicin.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 schematically illustrates a method for preparing a pegylated liposome preparation containing doxorubicin according to the present invention.

Doxorubicin-containing liposome preparation gilhwa the page has been marketed, e.g., such as Doxil ^®, Caelyx ^®. These liposome formulations are required to have a high encapsulation rate due to the nature of doxorubicin, which exhibits cytotoxicity, and are required to have narrow particle size distribution and high storage stability in order to ensure uniform quality.

According to the present invention, by performing extrusion and dialysis on a co-liposome dispersion solution, uniform liposome particle size distribution can be obtained, purity can be increased, and a liposome preparation containing doxorubicin pegylated with high encapsulation rate and storage stability can be prepared .

Hereinafter, each step will be described in detail.

S1) lipid solution preparation step

First, in step S1, a lipid solution is prepared by adding phospholipids, sterols, and PEG-conjugated lipids to lower alcohols.

The phospholipid used in the present invention is a follicular lipid and may be phosphatidylcholine (PC), phosphatidyl acid (PA), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol Is not particularly limited as long as it is a lipid having a phosphate ester group such as fingo myelin (SM).

Specifically, it may be at least one selected from the group consisting of egg yolk lecithin, soybean lecithin, sphingomyelin, hydrogenated soybean phosphatidylcholine, yolk phosphatidylcholine, distearoylphosphatidylcholine, and hydrogenated lecithin, but is not limited thereto. Preferably, hydrogenated soybean phosphatidylcholine (HSPC) is used.

In the present invention, the sterol is added in order to control the flowability of the liposome membrane so that the liposome can maintain a more stable structure. As the sterol, at least one selected from the group consisting of cholesterol, cholesterol hexasuccinate, ergosterol, and lanosterol may be used, and cholesterol is preferably used.

The PEG-conjugated lipid means a lipid to which a polyethylene glycol (PEG) polymer, which is a hydrophilic polymer, is bonded at one end. A liposome preparation containing PEG-conjugated lipid as a constituent of the membrane reduces the adsorption between blood proteins and liposomes and thus has an effect of increasing the half-life of the body.

The PEG-conjugated lipids can be used either commercially or directly from phospholipids and PEG.

When PEG-conjugated lipids are used in direct preparation, it is preferred that the PEG used be capped at the end with methoxy, ethoxy or other unreactive groups and the other end with a reactive chemical group. Such PEG may be used by purchasing the PEG having a weight average molecular weight (Mw) of 500 to 20,000 g / mol, or may be used by direct preparation.

The lipid conjugated with the PEG is a follicular lipid, preferably having two hydrocarbon chains, typically an acyl chain and a polar head group. Examples of such lipids include phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidyl acid (PA), phosphatidylinositol (PI), and phosphatidylinositol (PI), which have two hydrocarbon chains, typically about C14 to 22, Myelin (SM), and is preferably phosphatidylethanolamine (PE).

The PEG may be activated at one end by reaction of a suitable activating agent such as cyanuric acid, diimidazole, anhydride, etc., and the activated PEG reacts with the follicular lipid to form a PEG-conjugated lipid. Alternatively, a method such as activating a lipid group instead of activating PEG may be used.

In the present invention, as a PEG-conjugated lipid, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-methoxy-poly (ethylene glycol- ), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-methoxy-poly (ethylene glycol-5000) (DSPE-mPEG-5000) , And more preferably DSPE-mPEG-2000 having a molecular weight in the range of 2650 to 3050 g / mol is used.

In preparing the lipid solution in step S1, the phospholipid, sterol, and PEG-conjugated lipid are preferably contained in a weight ratio of 3 to 9: 1 to 3: 1 to 3 in view of securing the stability of the liposome membrane.

As the lower alcohol in the step S1, at least one selected from the group consisting of ethanol, isopropanol and propanol may be used, and among them, ethanol is preferably used.

The operation temperature of step S1 is preferably from room temperature to 80 DEG C, more preferably from 60 to 80 DEG C, and it is more preferable to proceed under stirring for even dispersion of the lipid component.

S2) co-liposome dispersion solution preparation step

Step S2 is a step of dropping the ethanol solution in which the phospholipid, sterol, and PEG-conjugated lipid are dispersed in an inorganic salt aqueous solution to prepare a blank liposome dispersion solution.

The lipid bilayer follicle is formed by suspending the follicular lipid such as phospholipid in an inorganic salt aqueous solution, and the sterol is inserted into the liposome together with the phospholipid to give the characteristics such as the strength and permeability of the liposome membrane.

Examples of the inorganic salt that can be used in step S2 include an ammonium ion as a cation and include at least one selected from the group consisting of ammonium sulfate, ammonium chloride, ammonium acetate, ammonium formate, and ammonium carbonate, Ammonium sulfate is preferably used. The concentration of the inorganic salt aqueous solution in step S2 is preferably 200 to 300 mM.

The temperature for performing the step S2 is preferably from room temperature to 80 DEG C, more preferably from 60 DEG C to 80 DEG C, and is preferably carried out under stirring so that a liposome of uniform particle size can be produced.

The dropping rate of the lipid solution and the stirring time after dropping are not particularly limited, but the stirring time after dropping is preferably 20 minutes or more, more preferably 30 minutes to 1 hour.

S3) Extrusion step

In step S3, the co-liposome dispersion solution prepared in step S2 is passed through an extruder to perform extrusion molding.

The extrusion molding method is a method in which a co-liposome dispersion solution is injected into an extruder equipped with a membrane filter of an appropriate size, and a high pressure is applied to the liposome solution by passing the liposome solution through a nitrogen gas. At this time, the size and the particle size distribution of the co-liposome can be controlled by the membrane filter (pore) of the extruder and the extrusion pressure applied.

The extruder that can be used in the present invention is a LIPEX extruder manufactured by Northern lipids. The filter used herein may be a polycarbonate membrane filter having a pore diameter of 40 to 400 nm, and the extrusion pressure may be adjusted as required, but is preferably 350 to 850 psi.

When the diameter and the extrusion pressure of the filter voids are less than or equal to the above-mentioned range, it is difficult to produce co-liposomes having nano-sized diameters, and the particle size distribution of the co-liposomes becomes very broad.

The extrusion molding may be repeated several times until the average particle diameter (D90) of the liposome satisfies 150 nm or less. Herein, D90 means that 90% by weight of co-liposome among 100% by weight of co-liposome has a particle diameter of 150 nm or less.

In the present invention, the extrusion molding can be performed by replacing the diameter of the used filter with a smaller one, or by stacking several filters so that the diameter of the filter is reduced according to the advancing direction of the extrusion. At this time, It is preferably 40 to 50 nm. Further, during the extrusion molding, a process of increasing the extrusion pressure may be further performed if necessary.

S4) Dialysis step

In step S4, a dialysis process is performed on the co-liposome solution in which the extrusion process is completed. The dialysis process is performed to remove impurities such as inorganic salts remaining in the prepared co-liposome.

As the dialysis solution used in the dialysis process, a buffer solution containing white sugar is used.

White sugar (sucrose) is a iso-osmotic agent that does not react with liposomes and remains on the inner and outer surfaces of the liposome membrane to strengthen the membrane. In particular, the present invention is used as a low-temperature protective material to ensure long-term stability against the distribution period of the liposome preparation. In addition, there is an effect of reducing the leakage of the drug after the injection of doxorubicin. In order to ensure the above effect, the concentration of white sugar contained in the aqueous solution is preferably 5 to 15% (w / v), more preferably 10%.

As the buffer, glycine, phosphate, citrate, acetate and histidine are preferable, and histidine is more preferable.

Most preferably, the dialysis solution uses an L-histidine solution containing 10% white sugar.

The dialysis membrane used herein has a molecular weight cut-off (MWCO) of 10,000 to 20,000 daltons, preferably 10,000 to 14,000 daltons, and the material thereof is not particularly limited.

For example, the dialysis membrane may be made of a material such as cellulose, ethylene-vinyl alcohol copolymer, polyacrylonitrile, polymethyl methacrylate, or the like.

Preferably, the dialysis is performed at 0 to 10 ° C., preferably at 3 to 5 ° C., and the dialysis solution is exchanged at intervals of 4 hours while being performed for 12 hours or more. Under such conditions, the purification effect can be obtained without affecting the structure, stability and particle size distribution of the liposome.

S5) Preparation step of liposome preparation (liposome preparation containing doxorubicin)

In step S5, doxorubicin is encapsulated into the liposome prepared and purified as described above using a pH gradient or chemical gradient.

It is preferable that doxorubicin hydrochloride is used as the doxorubicin to be encapsulated in the co-liposome in the step S5.

The inclusion of doxorubicin can be achieved by dissolving doxorubicin hydrochloride in a dialysis solution of S4 stage, mixing with a liposomal dispersion solution at 55 to 60 DEG C for 0.5 to 5 hours. Thereafter, the pH is adjusted to 6.0 to 7.0 using hydrochloric acid and sodium hydroxide to prepare a liposome preparation in which the liposome is pegylated with doxorubicin.

The doxorubicin-containing pegylated liposome preparation prepared through step S5 is stored at a low temperature of 2 to 8 占 폚.

The liposome formulations prepared according to the present invention exhibit excellent storage stability at low temperatures, including sterols. In this case, the storage stability means that the liposome preparation is prevented from being entangled due to the self-adhesion of the liposome during the storage period or the use period of the liposome preparation. The liposome preparation with improved storage stability prevents the liposome particles from becoming larger and maintains a uniform particle size distribution, thereby preventing vascular clogging during intravenous administration to the patient and securing a uniform quality.

It was confirmed that the doxorubicin-containing pegylated liposome preparation prepared according to the present invention had an average particle size (D50) of about 100 nm, a span value of about 0.03, and an inclusion rate of 96% or more.

The span value is a value indicating the degree of particle size distribution of the liposome. The smaller the value, the more uniform and narrow the particle size distribution of the liposome. The span value can be expressed by the following equation (1).

[Equation 1]

Span value = (D90 - D10) / D50

In the above formula (1), D90 and D10 mean particle diameters corresponding to 90% and 10% of the total volume when the particle size of liposome particles is measured and the volume is accumulated from small particles. Since the liposome preparation of the present invention has a uniform particle size distribution with a span value of 0.05 or less, it can exhibit predictable pharmacokinetics characteristics.

Thus, the pegylated liposome preparation containing doxorubicin prepared according to the present invention can be used as an anticancer agent with reduced human toxicity, and can be used for prevention and treatment of ovarian cancer or breast cancer.

The doxorubicin-containing pegylated liposome formulations prepared according to the present invention can be used for intravenous drug delivery, transdermal, expulsion, and delivery of drugs to the lungs. The techniques necessary for such formulation and pharmaceutically acceptable carriers, additives and the like may be widely known to those skilled in the art.

It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the spirit or scope of the inventions. , And that such changes and modifications are within the scope of the appended claims.

[Example]

Example 1: Preparation of liposome preparation

Glycero-3-phosphoethanolamine-N-methoxy-poly (ethylene glycol-2000) (DSPE-mPEG- 2000) were dispersed in 3.25 ml of ethanol and stirred at 65 캜. The lipid solution was added dropwise to 50 ml of a 250 mM ammonium sulfate solution, mixed and stirred at 65 DEG C for 1 hour to prepare a co-liposome. A 47 mm polycarbonate membrane filter was attached to an extruder (LIPEX extruder, 100 ml, Northern lipids), and the above-mentioned liposome dispersion solution was added and extruded at 500 psi pressure. The extrusion was repeated until the size (D90) of the liposome was 150 nm or less.

The liposome solution whose particle size distribution was adjusted to a desired size by the above extrusion was put into a dialysis membrane having a cut-off molecular weight (MWCO) of 12,000 daltons and loaded on an L-histidine solution containing 10% And dialyzed at 4 < 0 > C for 12 hours. FIG. 2 is a graph of the particle size distribution before and after dialysis. From this, it can be seen that the particle size distribution of the co-liposome is not different between before and after dialysis.

Next, doxorubicin encapsulation was performed. To 5 ml of 10% white sugar-containing L-histidine solution used for dialysis, 20 mg of doxorubicin hydrochloride was dissolved. Then, the liposome dispersion solution purified by dialysis was mixed and allowed to stand at 65 ° C for 1 hour to enclose doxorubicin in the liposome Respectively. The pH of the liposome solution that had been sealed was adjusted to 6.0 to 7.0. The finished liposome preparation was stored at 2-8 [deg.] C in an ampoule state as shown in Fig.

Experimental Example 1: Analysis of liposome preparation

In order to evaluate the physicochemical properties of the liposome preparations prepared in Example 1, characteristics such as constellation, osmotic pressure, particle size distribution, and the content of encapsulated doxorubicin were analyzed. In this experimental example, a Caelyx ^® injection of Janssen, one of the commercially available doxorubicin-containing pegylated liposome preparations, was used.

(1) Statistical analysis

The properties of the liposome preparations prepared in Example 1 were observed using a cryo-transmission electron microscope (Tecnia F20 G2 from FE). At this time, dilute the undiluted liposome preparation stock to 1/5 concentration, place 3 μl on a quantifoil ^® grid, freeze in liquefied ethane using a bitrobot, Were used.

Referring to FIG. 4, it can be confirmed that the liposome preparation of the present invention has a spherical shape with a particle size of 100 nm or less like the control preparation. In addition, it can be seen that the particle size of the liposome preparation of the present invention is more uniform than that of the Celix injection.

(2) Osmotic pressure analysis

Osmotic pressure of the liposome preparation of Example 1 was measured using a freezing point osmolality meter (Osmomat 3000, Gonotec), and the average value was 365 mOsmol / kg, which was similar to the control formulation (362 mOsmol / kg).

(3) Particle size analysis

The particle size of liposomes can affect the pharmacokinetics of the body. In other words, if the particle size is too small, the disappearance rate of the body is too fast to exhibit the desired drug efficacy. Conversely, if it is too large, it may affect the capillary blood vessels of the kidney or other organs.

The particle size of Example 1 and the control formulation was analyzed using a Zetasizer Nano ZS particle size analyzer from Malvern. The particle size analyzer analyzes the particle size of the nanoparticles by calculating the Brownian motion of the nanoparticles as an equation, and the particle size is larger than that of the scanning electron microscopic analysis. The results are shown in Fig.

As a result of particle size analysis, the average particle size (D50) of the preparation of Example 1 and the control preparation was found to be about 100 nm, and the span value of the preparation of Example 1 was 0.07 and that of Example 1 was 0.03, Of the particles exhibit a narrower particle size distribution. From these results, it can be confirmed that the liposome preparation produced by the production method of the present invention is superior to conventional Celix injection in terms of particle size uniformity.

(4) Measurement of inclusion rate

In order to measure the content of doxorubicin encapsulated in the liposome, HPLC analysis was performed by the test method described in Korean Pharmacopoeia. The analysis conditions are as follows.

* Column: Stainless steel pipe having an internal diameter of 4.6 mm and a length of 50 mm was charged with 5 탆 octysilyl silica gel

* Mobile phase: 0.02 M ammonium acetate buffer (pH 4.0): 0.02 M ammonium acetate TS = 50: 50 (volume ratio)

* Detector: Ultraviolet absorptiometer (measuring wavelength: 254 nm)

* Flow rate: 1.0 ml / min

As a result of HPLC analysis, the chromatograms of the comparative preparation and the preparation of Example 1 were confirmed to be similar. The contents of doxorubicin hydrochloride were 101.3% and 99.7%, respectively, I could.

On the other hand, supernatant was obtained by centrifugation at 100,000 G for 2 hours using an ultrahigh-speed centrifuge, and the supernatant and the stock solution of the liposome preparation before centrifugation were analyzed using an ultraviolet spectrometer, from which the inclusion rate was calculated. The results are shown in Table 1.

Before centrifugation
Doxorubicin content Supernatant
Doxorubicin content Inclusion rate Example 1 100.2% 4.1% 96.1% Control formulation 100.4% 5.7% 94.7%

As shown in Table 1, the formulation of Example 1 had an incorporation rate of 96.1%, which was higher than that of the control formulation. As the inclusion rate is increased, the liposome preparation produced by the present invention can be expected to reduce the toxicity in vivo and thereby increase the safety.

(5) Measurement of inclusion rate after ultrasonic treatment

In order to confirm the stability of doxorubicin encapsulated in the liposome, the inclusion rate after treatment with weak ultrasonic waves (20 kHz) was measured using the test method described in the FDA guidance of the US. The results are shown in Table 2.

Incorporation rate before ultrasonic treatment Rate of inclusion after ultrasonic treatment Example 1 96.1% 93.2% Control formulation 94.7% 92.1%

After the ultrasonic treatment, the preparation of Example 1 showed a high packing rate of 93% or more. From this, it can be confirmed that the liposome preparation of the present invention has little leakage of drug encapsulated in ultrasonic treatment, and maintains stability.

(6) Dissolution test

In order to confirm the in vitro release characteristics of the liposome preparation prepared in Example 1, a dissolution test was conducted using a USP-4 dissolution tester of Sotax. The dissolution test was carried out in such a manner that the liposome preparation was loaded on a phosphate buffer solution of pH 6.5 and the amount of elution was measured at 37 캜 for 4 days.

6 is a graph showing the results of the dissolution test. The liposome preparation of Example 1 exhibits a similar amount of elution to that of conventional Celix injection, and the release characteristics in the body can be judged to be similar.

Experimental Example 2: Analysis of cholesterol content

Cholesterol is the most important ingredient in the stability of liposomes. That is, it is important to confirm that the content of cholesterol has the greatest influence on the release and storage stability of the liposome-encapsulated doxorubicin outside the liposome, so that the content of cholesterol contained in the liposome preparation falls within the ideal range. Thus, HPLC was performed to confirm whether the liposome preparation prepared in Example 1 contained an appropriate amount of cholesterol. The analysis conditions are as follows.

* Column: ODS charge of 3 ㎛ in stainless steel pipe with internal diameter of 4.6 mm and length of 50 mm

* Mobile phase: methanol: tetrahydrofuran: Formic acid buffer = 45: 3: 2 (volume ratio)

(Preparation of Formic Acid Buffer: 184 g of formic acid was weighed and dissolved in 900 ml of water, adjusted to pH 3.0 with ammonia water, and water was added to make 1 L)

* Detector: Differential refraction detector (40 ℃)

* Flow rate: 1.0 ml / min

The optimal cholesterol content of doxorubicin-containing pegylated liposome formulations ranges from 15% to 20%. The liposome preparation prepared in Example 1 had a cholesterol content of 17.7%, which satisfied the above-mentioned optimum content range. Accordingly, it can be judged that the liposome preparation produced according to the production method of the present invention shows excellent storage stability.

According to the method for preparing a pegylated liposome preparation containing doxorubicin of the present invention, it is possible to produce a liposome having a nano-size and narrow particle size distribution through a series of steps of preparation of a co-liposome, extrusion molding and dialysis, It is possible to prepare a pegylated liposome preparation containing doxorubicin excellent in stability and encapsulation. Accordingly, the liposome preparation produced according to the present invention can be used as an anticancer agent with reduced human toxicity, and can be used for prevention and treatment of ovarian cancer or breast cancer.

Claims (12)

S1) Hydrogenated soybean phosphatidylcholine, cholesterol and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-methoxy-poly (ethylene glycol-2000) (DSPE-mPEG- : In a weight ratio of 1: 1, to ethanol to prepare a lipid solution;
S2) dropping the lipid solution into 250 mM ammonium sulfate to prepare a co-liposome dispersion solution;
S3) passing the co-liposome dispersion solution through an extruder under a pressure of 350 to 850 psi to perform extrusion molding so that the span value of the liposome is 0.03 or less and the average particle diameter (D90) is 150 nm or less;
S4) performing a dialysis process so that the particle size distribution (PDI) of the liposome particles is 0.03 or less in the obtained coenzyme liposome solution; And
S5) encapsulating doxorubicin in a co-liposome to prepare a liposome preparation,
Wherein the cholesterol is contained in an amount of 15 to 20% by weight based on the whole liposome preparation,
The dialysis was performed at 4 ° C for 12 hours using a dialysis solution in a dialyzer equipped with a dialysis membrane having a molecular weight cut-off (MWCO) of 12,000 daltons, and the dialysis solution was exchanged at intervals of 3 to 5 hours However,
Wherein the dialysis solution is a buffer solution containing 10% by weight of white sugar. delete delete delete delete delete delete delete delete delete delete delete

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