KR100293572B1 - A method for preparation of polymerized liposome by controlling of PH - Google Patents

Abstract

The present invention relates to a method for producing a polymerized liposome having excellent stability by chemically bonding ends of hydrocarbon chains in a molecule constituting a liposome membrane by pH control, and a method for producing a polymerized liposome, Which can be usefully used as a drug delivery vehicle capable of slowly administering a drug.

Description

[0001] The present invention relates to a method for preparing a polymerized liposome by controlling pH,

The present invention relates to a method for producing a polymerized liposome, and more particularly, to a method for producing a polymerized liposome having excellent stability by binding chemically the end of a hydrocarbon chain in a molecule constituting a liposome membrane by pH adjustment The present invention relates to a drug delivery system which can be used effectively as a drug delivery system capable of slowly delivering a drug over a long period of time without reaching the site of an onset in a drug delivery system.

In general, the Drug Delivery System has begun to be put to practical use from slow-release drugs that slowly release the drug over a long period of time. However, the ultimate goal is to establish a targeting technique to reach the drug only at the site of onset. If you can take the medicine only at the site of the onset, you can make a big effect with a small amount of medicine without giving side effects to healthy tissue. Presently, drug targeting is promising as a way to seal the drug with liposomes, which is to put the liposomes into the blood to reach the onset area and release the drug contained in the liposomes.

However, since the constituent molecules of the liposome are very fluid, they do not maintain their original structure over time, but become bound together and become bulky and separated by precipitation in an aqueous solution, so that the drug contained in the liposome does not reach the onset area, . Therefore, a polymerized liposome has been proposed as a new substitute. This is because each vesicle, which is a liposome unit, is polymerized in it and can not be fused with other vesicles, so that the liposome can be stored for as long as it is initially made.

At present, there is a method of polymerizing the liposome by using light energy or increasing the temperature to increase heat energy. In particular, when a disulfide group is used, dithiothreitol (DTT) .

However, when polymerized by physical or chemical methods such as light, heat, DTT, etc., the drug contained in the liposome may be damaged.

Therefore, the inventors of the present invention have studied about a method of polymerizing more easily without physical and chemical impact, so that the drug contained in the liposome should be free of damage to use the polymerized liposome as a drug delivery system. As a result, Bis [12- (lipoyloxy) dodecanoyl] -1,2,3,4-tetrahydroisoquinolin-2-one prepared by the reaction of 1,2-bis (12- -sn-glycero-3-phosphocholine can be polymerized by itself in the weak alkaline state, it has been found that the liposome can be stably polymerized only by the pH control, and the present invention has been completed.

Accordingly, an object of the present invention is to provide a method for preparing a polymerized liposome, which can polymerize by adjusting the pH from 6.4 to 8.4, and partially lowering the pH to 6.4 after partial polymerization, .

Another object of the present invention is to provide a liposome preparation method and a liposome preparation method in which the polymerized liposome obtained by the above method is stable to temperature change, chemical substances, etc. and is not reactive with a substance contained in the liposome, And the use of the polymerized liposome as a drug delivery system is provided.

1 is a transmission electron microscopy photograph of a dried sample of microspheres formed of a polymerized liposome.

FIG. 2 shows the result of measuring the phase change of the polymerized liposome of the present invention and the known liposome membrane through heat absorption by differential scanning calorimetry.

FIG. 3 shows the results of measurement of the phase change of the polymerized liposome of the present invention and the known liposome according to the temperature using fluorescence polarization.

FIG. 4 shows the results of measurement of the stability of the polymerized liposome of the present invention and surfactants (SDS) of known liposomes using turbidity.

FIG. 5 is a graph showing that fluorescein isocyanated dextran contained in a polymerized liposome is gradually released. FIG.

FIG. 6 is a graph showing that bovine serum albumin contained in a polymerized liposome is slowly released. FIG.

7 is a graph showing that the fluorescent dextran contained in the polymerized liposome is slowly released in the presence of SDS.

8 is an electrophoresis photograph showing the degree of lysozyme sealing of a polymerized liposome and a known liposome.

Lane 1; Molecular weight marker

Lane 2; Egg white albumin solution

Lane 3; Polymerized liposome containing albumin albumin

Lane 4; Non-polymerized liposome containing albumin albumin

Lane 5; Bovine serum albumin solution

Lane 6; Polymerized liposome containing bovine serum albumin

Lane 7; Non-polymerized liposome containing bovine serum albumin

FIG. 9 is a graph showing the sealing efficiency of a polymerized liposome containing carboxyfluorescein, a substance having a small molecular weight, and a polymerized liposome mixed with a polymerized liposome and SDS.

10 is a graph showing the difference in the degree of production of antibodies against a protein (lysozyme) contained in a polymerized liposome and a known liposome.

Hereinafter, the present invention will be described in more detail.

The monomer used in the liposome polymerization of the present invention is 1,2-bis [12- (lipoyloxy) dodecanoyl] -sn-glycero-3-phosphocholine (dodecanoyl] -sn-glycero-3-phosphocholine (hereinafter referred to as DLL), lipoic acid anhydride and 1,2-bis (12-hydroxydodecanoyl) It is obtained by reacting 3-phosphocholine. It is dissolved in chloroform or methylene chloride, sealed to prevent moisture, and stored at about 4 ° C for long-term storage. On the other hand, when the monomer is stored for a long period of time, polymerized lipids may be partially formed. However, since the polymerized lipids are not dissolved in the organic solvent and are precipitated, most of them are stored stably in the organic solvent.

The polymerized liposome obtained according to the method of the present invention to be described later is manufactured as a comparatively inexpensive reagent and has no possibility of binding with a vaccine or a drug due to the lack of reactivity, thereby increasing the efficiency of expensive imported drugs such as vaccine, hormone, and antibiotic It is very economical because it can reduce usage and reduce side effects. In addition, polymeric liposomes are stable to temperature changes and can be stored for a long period of time because they hardly cause sedimentation even after long-term storage for a year or more. When a chemical such as sodium dodecylsulfate (SDS) is added, the membrane is not collapsed The release rate of the drug contained in the liposome remains unchanged and the sealing efficiency is also excellent, making it ideal as a drug delivery vehicle.

On the other hand, in the case of the mixed polymerized liposome in which the polymerized liposome of the present invention is mixed with 10% SDS, since SDS lowers the permeability of the polymerized liposome membrane, the sealing efficiency of the drug contained in the liposome can be further increased, It is possible to prevent the drug from being decomposed before reaching the site.

Hereinafter, a method for preparing a polymerized liposome according to the present invention will be described in detail.

In order to prepare the polymerized liposome of the present invention, 1,2-bis [12- (lipoyloxy) dodecanoyl] -sn-glycero-3-phosphocholine (DLL) Lt; RTI ID = 0.0 > 1, < / RTI &

(A) reacting 12-hydroxydodecanoic acid dissolved in tetrahydrofuran with dihydropyran to obtain 12- (tetrahydropyranyl) dodecanoic acid;

(B) reacting 12- (tetrahydropyranyl) dodecanoic acid obtained in the above step (A) with glycerophosphoryl choline (GPC) to obtain 1,2-bis (12-hydroxydodecanoyl) -sn-glycero-3-phosphocholine;

(C) reacting a lipophosphoric acid with dicyclohexylcarbodiimide under methylene chloride to obtain an anhydrphoinic acid; And

(D) reacting the 1,2-bis (12-hydroxydodecanoyl) -sn-glycero-3-phosphocholine obtained in the step (B) with the anhydrpholeic acid obtained in the step (C) To give 1,2-bis [12- (lipoyloxy) dodecanoyl] -sn-glycero-3-phosphocholine (DLL).

(A)

(B)

(C)

(D)

More specifically, a method for producing the monomer DLL used in the production of the polymeric liposome of the present invention will be described.

(A) 12-Hydroxydodecanoic acid was dissolved in tetrahydrofuran, dihydropyran was added thereto, and the mixture was stirred for about 1 hour. After adding p -toluenesulfonic acid thereto for about 1 day, the solvent was removed , And performing silica gel chromatography to obtain 12- (tetrahydropyranyl) dodecanoic acid;

(B) A mixture of 12- (tetrahydropyranyl) dodecanoic acid, glycerophosphoryl choline (GPC) and 4- (dimethylamino) pyridine obtained in the step (A) , DMAP) and dicyclohexylcarbodiimide (DCC) were dissolved, and the mixture was stirred at room temperature under a nitrogen gas for about 2 to 3 days. Then, the solvent was removed, and the solvent was dissolved again in a 1/1 mixed solution of methanol and water (12-hydroxydodecanoyl) -sn-glycero-3-phosphine (AGP-50) resin, Obtaining a pocololine;

(C) stirring lipophosphoric acid and dicyclohexylcarbodiimide under methylene chloride, followed by filtration to obtain an anhydric acid; And

(D) 1,2-bis (12-hydroxydodecanoyl) -sn-glycero-3-phosphocholine obtained in the step (B), the anhydrinic acid obtained in the step (C) - (dimethylamino) pyridine were mixed and stirred at room temperature under nitrogen gas for about 1 day. Then, the solvent was removed, and the solution was dissolved again in a 1/1 mixed solution of methanol and chloroform. Next, the filtrate is dissolved again in an organic solvent and subjected to silica gel chromatography to obtain 1,2-bis [12- (lipoyloxy) dodecanoyl] -sn-glycero-3-phosphocholine (DLL) . On the other hand, in the step (D) for producing the monomer DLL, the organic solvent is not particularly limited, but methylene chloride may be used, for example.

The method for preparing the polymerized liposome using the DLL produced by the above-

1) dissolving the DLL in chloroform, placing it in a test tube, blowing nitrogen gas to remove the solvent, and drying it in a vacuum for about 1 hour to form a thin film;

2) adding an aqueous solution having a pH of 6.4 to a test tube having the thin film formed in the step (1) and vortexing for about 30 minutes to obtain a semi-transparent liposome solution;

3) passing the semi-transparent liposome solution of the step (2) through the extruder device maintained at about 40 ° C to a filter of a predetermined size at a pressure of nitrogen or argon gas about 10 times;

4) adjusting the pH of the solution of step (3) to 8.4, and then stirring at room temperature for about 1 day; And

5) bringing the pH of the solution of step (4) to 6.4;

.

On the other hand, if the DLL thin film is not uniformly dispersed in the aqueous solution in the step (2), it may be polymerized before the size adjustment to give irregular distribution of the liposome, and polymerization of the film state may occur, It should be confirmed that this solution has spread evenly. Ultrasonic waves can be applied for a short time in order to spread the thin film well in the aqueous phase. In step (3), the liposome solution in a semi-transparent state is passed through a filter having a predetermined size through an extruder device for size adjustment and unilamellarization of the membrane. For example, a filter having a size of 0.1 to 0.4 m Can be used.

Hereinafter, the present invention will be described more specifically by way of Examples and Test Examples. However, the present invention is not limited to these examples.

[Preparation Example 1] Synthesis of 1,2-bis [12- (lipoyloxy) dodecanoyl] -sn-glycero-3-phosphocholine

A. 12- (Tetrahydropyranyl) dodecanoic acid

10 g of 12-hydroxydodecanoic acid was dissolved in 100 ml of tetrahydrofuran, to which 9 g of dihydropyran was added. The completely dissolved solution was stirred for about 1 hour and 100 mg p -toluenesulfonic acid was added. The solution was then stirred for one day, then the solvent was removed and the remaining material was separated by silica gel chromatography to give 12 g of the title compound.

R _f = 0.5 (chloroform / methanol, 20/1, v / v)

_{^{1 H NMR (CDCl 3) δ1.25}} (s, 14 H, CH 2), 1.40~1.80 (s, 10 H, CH 2), 2.32 (t, 2H, CH 2 C = O), 3.38~4.10 ( _{m, 4H, CH 2 O)} , 4.6 (s, 1H, CH)

IR? _{C = 0} 1701 cm ^-1

B. Synthesis of 1,2-bis (12-hydroxydodecanoyl) -sn-glycero-3-phosphocholine

7 g of 12- (tetrahydropyranyl) dodecanoic acid of the step A, 2.7 g of glycerophosphorylcholine, 2 g of 4- (dimethylamino) pyridine and 3 g of dicyclohexylcarbodiimide were dissolved in a chloroform solution, And stirred for 3 days. Subsequently, the solvent was removed, and the remaining material was dissolved in a 50 ml mixed solution (methanol / water = 50/50), and this was mixed with AG MP-50 resin (Bio-Rad) and stirred. After filtration to remove the resin, the solvent was completely removed and the remaining material was separated by silica gel chromatography to obtain 2 g of the title compound.

R _f = 0.25 (chloroform / methanol / water, 65/25/4, v / v / v)

_{^{1 H NMR (CD 3 OD)}} δ1.30~1.65 (s, 36 H, CH 2), 2.32 (t, 4 H, CH 2 C = O), 3.22 (s, 9 H, N (CH 3) 3 ), 3.52 (t, 4 H , CH 2 O), 3.60~4.55 (m, 8 H, CH 2 O, N (CH 2)), 4.85 (s, 2 H, OH), 5.2 (m, 1 H , CH)

IR? _OH 3390,? _{C = 0} 1721 cm ^-1

C. persimmon

2 g of lipoic acid and 1.5 g of dicyclohexylcarbodiimide were dissolved in 100 ml of methylene chloride and stirred, followed by filtration to obtain 700 mg of the title compound.

R _f = 0.94 (Mussoorie Four acid), R _f = 0.73 (lipoic acid) (chloroform / methanol / water, 65/25/4, v / v / v )

D. Synthesis of 1,2-bis [12- (lipoyloxy) dodecanoyl] -sn-glycero-3-phosphocholine

1 g of 1,2-bis (12-hydroxydodecanoyl) -sn-glycero-3-phosphocholine in Step B and 1.5 g of anhydrous formic acid in Step C were mixed with 0.4 g of 4- (dimethylamino) Under nitrogen gas for 1 day. The reaction was completed while confirming the progress of the reaction by TLC. The reacted reaction solution was filtered and the solvent was removed at low pressure. This was dissolved in 20 ml of a mixed solution (methanol / chloroform = 50/50) and passed through an AG MP-50 resin. The filtrate was dissolved in chloroform and purified by silica gel chromatography to obtain 1.4 g of the title compound .

R _f = 0.45 (chloroform / methanol / water, 65/25/4, v / v / v)

_{^{1 H NMR (CDCl 3) δ1.25}} (s, 28 H, CH 2), 1.40-2.05 (m, 20 H, CH 2), 2.3 (t, 8 H, CH 2 C = O), 2.20-2.65 _{(m, 4 H, CH 2} -lipoic ring), 3.15 (t, 4 H, CH 2 SS), 3.40 (s, 9 H, N (CH 3) 3), 3.55 (m, 2 H, CHSS), 4.08 (t, 4 H, CH 2 OC = O), 3.80-4.6 (m, 8 H, CH 2 O, NCH 2), 5.20 (m, 1 H, CH (CH 2 O))

IR? _{C =} 1732,? _{N (CH3) 3} 970, 1050, 1090 cm ^-1

[Preparation Example 2] Preparation of high molecular weight liposome

Bis (12- (lipoyloxy) dodecanoyl] -sn-glycero-3-phosphocholine (DLL) prepared in Preparation Example 1 was dissolved in chloroform at a concentration of 30 mg / ≪ / RTI > Approximately 100 μl (3 mg) was taken from the solution, placed in a test tube, and nitrogen gas was blown to remove the solvent, followed by drying in a vacuum for 1 hour to form a thin film. 1 ml of an aqueous solution having a pH of 6.4 was added thereto, and after 30 minutes, the mixture was subjected to a boathrex-mixing to uniformly spread the thin film into an aqueous solution to obtain a semi-transparent liposome solution.

The translucent liposome solution was passed through a 0.1 탆 filter through an extruder at a pressure of nitrogen gas 10 times to obtain a transparent solution. On the other hand, the temperature of the extruder was maintained at 40 占 폚 using a water bath. The clear solution passed through the extruder was adjusted to pH 8.4 with 0.3M NaOH solution and shaken for 1 day. After the reaction was completed, the pH was lowered to 6.4 with 0.3 M HCl to obtain a polymerized liposome in an aqueous solution state. The R _f value of the monomer DLL is 0.45, while the R _f value of the obtained polymerized liposome is 0, and a transmission electron microscopy photograph thereof is shown in FIG. In addition, as the polymerisation process proceeds, the ring structure of the dithiolane of the DLL disappears, and its intrinsic absorption wavelength (333 nm) is gradually reduced.

[Test Example 1]

In order to measure the phase change of the polymerized liposome of Production Example 2 according to the temperature, it was found that the reaction was carried out in the same manner as in Production Example 2 except that DMPC (dimyristyl phosphatidylcholine, A), dipalmitoyl phosphatidylcholine (DPPC), distearyl phosphatidylcholine The phase change through heat absorption of DSPC: distearyl phosphatidylcholine (C) and the polymerized liposome (D) of Preparation Example 2 was measured using a differential scanning calorimeter (MAC Science DSC 3100, Japan). The results are shown in Fig.

From FIG. 2, non-polymerized liposomes, DMPC, DPPC or DSPC, undergo temperature-sensitive phase changes at a constant temperature, but the polymerized liposomes of the present invention do not undergo phase changes even at high temperatures, .

[Test Example 2]

(A), DPPC (B), DSPC (C), and the polymer of Production Example 2 were measured using diphenylhexatriene, which is a indicating substance showing the phase change of the film, to observe the phase change of the polymerized liposome Fluorescence polarization of the phar- liposome (D) was measured using a LS-50B Luminescence Spectrometer from Perkin-Elmer. The results are shown in Fig.

From FIG. 3, it can be seen that the fluorescence polarity value of the non-polymerized liposomes is decreased, but the fluorescence polarization value of the polymerized liposome is almost unchanged. Therefore, it can be seen that the polymerized liposome of the present invention is a very stable film.

[Test Example 3]

In order to measure the stability of the polymerized liposome by the chemical, sodium dodecyl sulfate (SDS) known to dissolve the membrane structure in the polymerized liposome of Preparation Example 2 and 3 mg of DMPC was added in an aqueous solution at different concentrations, The turbidity was measured using a Hewlett-Packard HP8453 Diode-Array Spectrophotometer. The result is shown in Fig.

From FIG. 4, it can be seen that the addition of a very small amount of SDS to DMPC, which is a non-polymerized liposome, causes the structure to collapse and becomes transparent. However, even when the concentration of SDS is increased to a considerable extent in the polymerized liposome, the stability of the film is maintained, Able to know. Therefore, it can be seen that the polymerized liposome of the present invention is stable against chemicals such as SDS.

[Production Example 3]

An aqueous solution of polymerized liposome encapsulated with fluorescent dextran was obtained in the same manner as in Preparation Example 2 except that 1 ml of an aqueous solution of pH 6.4 in which 0.5 mg of fluorescent dextran having a molecular weight of 4,400 was dissolved was put into the DLL thin film.

[Production Example 4]

An aqueous solution of polymerized liposome encapsulated with bovine serum albumin was prepared in the same manner as in Preparation Example 2 except that 1 ml of an aqueous solution of pH 6.4 in which 1 mg of bovine serum albumin was dissolved was added to the DLL thin film.

[Test Example 4]

In order to examine the release rate of the substances contained in the polymerized liposome, the polymerized liposome aqueous solutions of Preparation Examples 3 and 4 were placed in a dialysis membrane (Spectrapore # 2, 2 cm x 5 cm) and immersed in 500 ml of buffer solution. Then, the fluorescence intensity of the solution in the dialysis membrane was periodically measured using a LS-50B Luminescence Spectrometer of Perkin-Elmer, and the results are shown in FIG. 5 and FIG. 5 and 6, it can be seen that the substance contained in the polymerized liposome of the present invention is gradually released.

[Test Example 5]

In order to observe the change in the rate of release of the substance contained in the liposome by a chemical substance such as SDS, 0.1% SDS was added to the aqueous solution of the polymerized liposome encapsulated in the fluorescent dextran of Production Example 3, and after 1 hour, And placed in a dialysis membrane (Spectrapore # 2, 2 cm x 5 cm), and immersed in 500 ml of buffer solution. Then, the fluorescence intensity of the solution in the dialysis membrane was periodically measured using a Perkin-Elmer LS-50B Luminescence Photometer, and the result is shown in FIG.

From FIG. 7, it can be seen that the release rate of the substance contained in the liposome does not change greatly with addition of SDS.

[Production Example 5]

An aqueous solution of a polymerized liposome encapsulated with egg white albumin was prepared in the same manner as in Preparation Example 2 except that 1 ml of an aqueous solution of pH 6.4 in which 1 mg of ovalbumin was dissolved was put into the DLL thin film.

[Comparative Production Example 1]

Dymylstilphosphatidylcholine (DMPC) was dissolved in chloroform at a concentration of 30 mg / ml, and about 100 μl (3 mg) was taken into a test tube. The solvent was removed by blowing nitrogen gas and dried in a vacuum for 1 hour Thin film was made. 1 ml of a solution containing 1 mg of albumin albumin was added thereto, followed by boathrex-mixing to allow the thin film to spread evenly through the aqueous solution. Then, the mixture was passed through a 0.1-μm filter 10 times with a nitrogen gas pressure through an extruder, To obtain an aqueous non-polymerized liposome solution in which albumin was sealed.

[Comparative Production Example 2]

Except that 1 mg of bovine serum albumin dissolved in DMPC was added to the DMPC membrane to obtain a non-polymerized liposome encapsulated with bovine serum albumin.

[Test Example 6]

Polymerized liposomes of Production Examples 4 to 5, non-polymerized liposomes of Comparative Production Examples 1 to 2, egg albumin solution and bovine serum albumin solution were electrophoresed to measure the sealing efficiency of the protein of the polymerized liposome. The results are shown in Fig.

8, it can be seen that the sealing efficiency of the polymerized liposome is much higher than that of the non-polymerized liposome.

[Production Example 6]

An aqueous solution of a polymerized liposome encapsulated with carboxyfluorescein was obtained in the same manner as in Preparation Example 2 except that 1 ml of an aqueous solution of pH 6.4 in which 0.1 mg of carboxyfluorescein was dissolved was added to the DLL thin film.

[Production Example 7]

Bis (12- (lipoyloxy) dodecanoyl] -sn-glycero-3-phosphocholine (DLL) prepared in Preparation Example 1 was dissolved in chloroform at a concentration of 30 mg / 4 < / RTI > Approximately 100 μl (3 mg) was taken from this solution, placed in a test tube, added with 10 μl of 10% SDS chloroform solution, and then nitrogen gas was blown to remove the solvent, followed by drying in vacuum for 1 hour to form a thin film. Then, 1 ml of an aqueous solution of pH 6.4, in which 0.1 mg of carboxyfluorescein was dissolved, was added to the mixture, and the mixture was subjected to boathrex-mixing for 30 minutes to uniformly spread the thin film in an aqueous solution of carboxyfluorescein to obtain a translucent liposome solution.

The translucent liposome solution was passed through a 0.1 탆 filter through an extruder at a pressure of nitrogen gas 10 times to obtain a transparent solution. On the other hand, the temperature of the extruder was maintained at 40 占 폚 using a water bath. The clear solution passed through the extruder was adjusted to pH 8.4 with 0.3M NaOH solution and shaken for 1 day. After the reaction was completed, the pH was lowered to 6.4 with 0.3 M HCl to obtain a polymerized mixed liposome aqueous solution sealed with carboxyfluorescein.

[Test Example 7]

In order to measure the sealing efficiency of the polymerized liposome of Preparation Example 7 and the mixed polymerized liposome of Polymerized Liposome of Preparation Example 8 and SDS, the fluorescence intensity was measured using a LS-50B Luminescence Spectrometer manufactured by Perkin-Elmer. The results are shown in Fig.

From FIG. 9, it can be seen that the sealing efficiency of the mixed polymerized liposome to which SDS is added is higher than that of the polymerized liposome. In other words, it can be seen that SDS has an effect of lowering the permeability of the polymerized liposome membrane.

[Production Example 8]

A denatured lysozyme was dissolved at a concentration of 20 mg / ml, and a solution of the modified liposome-encapsulated polymerized liposome was prepared in the same manner as in Preparation Example 2 except that 1 ml of the 6.4 acceptor solution was added to the DLL thin film. .

[Comparative Production Example 3]

A denatured lysozyme-encapsulated non-polymerized liposome was obtained in the same manner as in Comparative Preparation Example 1, except that 1 ml of a solution prepared by dissolving denatured lysozyme at a concentration of 20 mg / ml was added to the DMPC thin film.

[Test Example 8] Antibody generation test

The polymerized liposome of Preparation Example 8, the non-polymerized liposome of Comparative Preparation Example 3, and the lysozyme solution were injected into mice by 100 쨉 l. Since the encapsulation efficiency is better than that of polymerized liposomes, the amount of non-polymerized liposomes was added three times more to equal the amount of protein encapsulated. Blood was collected by cutting the rats' tail every two weeks after injection. The collected blood was left at room temperature for 30 minutes and centrifuged at 12,000 × g for 20 minutes. The supernatant was transferred to a new test tube and sodium azide (NaN ₃ ) was added to a final concentration of 5-10 mM and stored at -80 ° C. Serum was obtained at 0, 2, 4, 6, 8, 10 and 12 weeks before the injection. At 2 weeks, blood was boosted by injecting the same volume of lysozyme solution.

To carry out the ELISA analysis, 200 쨉 l of the lysozyme was added to each well of the 96-well plate so that the final concentration of lysozyme was 5 / / ml (10 mM Tris-HCl, pH 7.4), and the mixture was allowed to stand at 4 째 C for 10 hours. The lysozyme solution was removed from the plate and washed with 20 μl of 0.05% Tween-20 in wells. Blocking buffer was added to the wells in an amount of 200 ㎕, and the mixture was allowed to stand at 37 캜 for 1 hour. Rat serum was diluted 100-fold and added to each well in an amount of 100, and left for 2 hours. The cells were washed three times with 200 μl of 0.05% Tween-20, and 75 μl of the secondary antibody solution was diluted 4,000 times. Each well was left at 37 ° C. for 1 hour, and then 200 μl of 0.05% Tween- Washed it once. 100 쨉 l of p- nitrophenyl phosphate solution was added, and the mixture was wrapped with aluminum foil, followed by reaction at 37 캜 for 30 minutes. The reaction was terminated by adding 30 μl of 0.5 M NaOH solution, and the absorbance was measured at 405 nm using an ELISA reader. Seventy-two absorbance values were obtained from six rat sera of each experimental group. Twenty times of absorbance values were obtained from the mice. Least Significant Difference, Tukey's Studentized Range, and Duncan's multiple range test Duncan's Multiple Range). The results are shown in Fig.

From FIG. 10, it can be seen that when the lysozyme is sealed with the polymerized liposome of the present invention, the production of the antibody is continuously exhibited, but that of the non-polymerized liposome is gradually decreased. Furthermore, considering that the sealing efficiency of the polymerized liposome is about three times or more as compared with the known non-polymerized liposome, it can be seen that about three times more antibodies are produced than differences in the table.

As described above, the polymerized liposome produced by the present invention is a substance which is very stable to temperature change, chemical substances and the like and does not react with the drug contained in the liposome, It can release the drug contained in the liposome slowly over a long period of time without changing the release rate, and thus can be very useful as a drug delivery system in the drug delivery system. In addition, when the present invention is applied to a vaccine, the use of the polymerized liposome of the present invention can increase the efficacy of the antibody by continuously producing antibodies, thus reducing the amount of expensive imported drugs such as vaccines, hormones, and antibiotics.

Claims (3)

A method for producing a polymerized liposome, 1) 1,2-bis [12- (lipoyloxy) dodecanoyl] -sn-glycero-3-phosphocholine -phosphocholine is dissolved in chloroform, the solution is put into a test tube, nitrogen gas is blown to remove the solvent, and this is dried in a vacuum for about 1 hour to form a thin film; 2) adding an aqueous solution having a pH of 6.4 to a test tube having the thin film formed in the step (1) and vortexing for 30 minutes to obtain a translucent liposome solution; 3) passing the liposome solution in the semi-transparent state of step (2) through the extruder device kept at 40 ° C for 10 times under a nitrogen or argon gas pressure to a filter of a predetermined size; 4) adjusting the pH of the solution of step (3) to 8.4, and then stirring at room temperature for 1 day; And 5) bringing the pH of the solution of step (4) to 6.4; Wherein the liposome is a liposome. The method of claim 1, wherein the step of dissolving 1,2-bis [12- (lipoyloxy) dodecanoyl] -sn-glycero-3-phosphocholine in chloroform in step (1) ≪ / RTI > further comprising the step of adding sodium dodecyl sulfate. The method of claim 1, wherein the 1,2-bis [12- (lipoyloxy) dodecanoyl] -sn-glycero-3-phosphocholine is (A) reacting 12-hydroxydodecanoic acid dissolved in tetrahydrofuran with dihydropyran to obtain 12- (tetrahydropyranyl) dodecanoic acid; (B) 12- (tetrahydropyranyl) dodecanoic acid obtained in the step (A) is reacted with glycerophosphoryl choline to obtain 1,2-bis (12-hydroxydodecanoyl) -sn -Glycero-3-phosphocholine; (C) reacting a lipophosphoric acid with dicyclohexylcarbodiimide under a salt methylene to obtain an anhydrphoinic acid; And (D) reacting the 1,2-bis (12-hydroxydodecanoyl) -sn-glycero-3-phosphocholine obtained in the step (B) with the anhydrpholeic acid obtained in the step (C) To obtain 1,2-bis [12- (lipoyloxy) dodecanoyl] -sn-glycero-3-phosphocholine; Lt; RTI ID = 0.0 > of: < / RTI >