KR20000032803A - Manufacturing method of human body affinitive liposome and the composition thereof - Google Patents

Abstract

PURPOSE: A manufacturing method of human body affinitive liposome is provided by modifying of the structure of phospholipid, which is a component of the liposome. CONSTITUTION: Phosphatidylethanolamine derivative selected from dipalmitoylphosphatidylethanolamine, distearoyl phosphatidylethanolamine or dioleoylphosphatidylethanolamine, and method polyethylene glycol(PEG) derivative are connected by a cross linker selected from propionate, succinate or carbonate to provide phosphatidylethanolamine compound which is a component of PEG-liposome. The PEG-liposome made of PEG-phospholipid is more affinitive to human body than the existing liposome, stays longer in human body, reduces the antigenicity and also reduces the absorption and elimination of the liposome in the liver. The PEG-liposome is very useful as a carrier for hemoglobin, anti-cancer agent, antibiotics, physiological activating agent etc.

Description

Method of preparation and composition of biocompatible liposomes

The present invention relates to a human-friendly liposome component and a PEG-liposomal system using the same, although liposome systems are widely used as carriers for highly toxic drugs, the toxicity of liposomes themselves, short residence time, antigenic expression, allergy There are disadvantages such as symptom induction, and it relates to a composition and a method for preparing a new liposome that overcomes these problems and has a function as a more human-friendly injection.

In general, liposomes form a membrane composed of several types of phospholipids, and in aqueous solution, are spherical as shown in FIG. 1, and a delivery material (B) is formed in a liposomal membrane (A) constituting a sphere. It is implied. The liposome membrane (A) shows that the phospholipid molecule composed of the polar head and the hydrocarbon tail has a double membrane structure in which the polar head is disposed in the direction of the water-soluble compartment inside and the aqueous solution outside. By using this property, the liposome can be used as a medium for delivering a substance in the human body. The material to be delivered includes medicines, cosmetics, diagnostic reagents, and bioactive substances. In particular, most anticancer drugs have strong toxicity and side effects, and the liposome formulation may be used to overcome them.

Phospholipids, which are the major constituents of liposomes, include phosphatidylcholine (PC), phosphatidylserine (PS), phosphatidylethanolamine (PE), phosphatidyl inositol (PI), and phosphatidylglycerol (PG). Other additives include cholesterol and tocopherol. . More specifically, as phosphatidylcholine, dioleoyl phosphatidylcholine (DOPC), distearoyl phosphatidylcholine (PSPC), dipamitoyl phosphatidylcholine (DPPC) and the like are used. As phosphatidylserine, dioleoyl phosphatidylserine (DOPS), distearoyl phosphatidylserine (DSPS), dipamitoyl phosphatidylserine (DPPS), etc. are used. Dioleoyl phosphatidylethanolamine (DOPE), distearoyl phosphatidylethanolamine (DSPE), dipamitoyl phosphatidylethanolamine (DPPE), etc. are used also as phosphatidyl ethanolamine. Dioleoyl phosphatidyl inositol (DOPI), distearoyl phosphatidyl inositol (DSPI), dipamitoyl phosphatidyl inositol (DPPI) and the like are also used as phosphatidyl inositol. Dioleoyl phosphatidylglycerol (DOPG), distearoyl phosphatidylglycerol (DSPG), dipamitoyl phosphatidylglycerol (DPPG) and the like are also used as phosphatidylglycerol.

As a constituent of the liposome component, phosphatidylcholine, cholesterol, etc. occupy a high ratio, and phosphatidylethanolamine, phosphatidylserine, tocopherol, etc. usually occupy a low ratio. Most of the phospholipids can be extracted from soy or egg yolks.

The method for preparing liposomes begins by mixing an insoluble organic solution phase and a water soluble aqueous phase. First, a mixture of phospholipids that constitute the liposome membrane is dissolved in an organic solution. At this time, the organic solution used may include diethyl ether, methylene chloride, fluorinated hydrocarbons, methanol, ethanol and the like or mixtures thereof.

An aqueous solution is added to the organic solution prepared as described above, wherein the dissolved salt, the buffer salt, and the delivery material may be dissolved if the delivery material is water-soluble. When the organic solution and the aqueous solution prepared in this way are mixed and stirred in a mixer, liposomes are formed, and the aqueous solution in which the transport material is dissolved is placed in the liposome. However, if the material to be delivered is insoluble in water, the transport material is first dissolved in an organic solution and then stirred with an aqueous solution to form liposomes. The liposome solution thus obtained is produced by evaporating and removing the organic solution. Suitable evaporation methods for removing the organic solvent include spraying nitrogen gas over the mixed liquid, heating the mixed liquid, maintaining a reduced pressure state using a vacuum, etc., and spraying nitrogen gas into the mixed liquid under heating. .

The liposomes thus obtained are usually distributed in various sizes ranging from 50 nm to 5000 nm, and the ideal size is narrow and relatively uniform. In order to make the liposomes relatively uniform in size, a liposome solution is passed through a filter having an appropriate pore size. Using this method, liposomes smaller than the pore size of the filter pass through, whereas large liposomes pass through the hole and tend to be as small as pore size. This can be repeated several times through the filter to control the size of the liposomes. As another method, there is also a method of forming a certain size by passing the liposome liquid through the tube of the extruder.

However, currently widely used formulations of liposomes are rapidly removed by the reticuloendothelial system of the liver after injection into the human body, resulting in pathological abnormalities of the liver tissues, and at the same time, significantly reducing the residence time. there was.

The present invention is to solve the above problems, by modifying the structure of the phospholipid used as a constituent of the liposomes raw materials that can be used in the preparation of liposomes that can have a more human-friendly properties than conventional liposomes A first object is to provide a and a second object is to provide a liposome system constructed using this raw material.

The present invention for achieving the above-described first object provides a phosphatidylethanolamine compound in which a phosphatidylethanolamine (PE) derivative and a methoxy polyethylene glycol derivative are linked by a binder.

The phosphatidyl ethanolamine derivative may be selected from the group consisting of dipamitoyl phosphatidyl ethanolamine (DPPE), distearoyl phosphatidyl ethanolamine (DSPE), dioleoyl phosphatidyl ethanolamine (DOPE) and the like, as the binder May be selected from the group consisting of propionate, succinate, carbonate, and the like.

The present invention for achieving the second object provides a PEG-liposome formed by using a phosphatidylethanolamine compound in which a phosphatidylethanolamine (PE) derivative and a methoxy polyethylene glycol derivative are linked by a conjugate.

The phosphatidylethanolamine derivative may be selected from the group consisting of dipamitoyl phosphatidylethanolamine (DPPE), distearoyl phosphatidylethanolamine (DSPE), dioleoyl phosphatidylethanolamine (DOPE), and the like. Propionate, succinate, carbonate, and the like may be selected from the group consisting of.

PEG-liposomes made from PEG-phospholipids in the form of a combination of polyethylene glycol (PEG) as described above have more human-friendly properties than conventional liposomes, thus increasing the remaining time in the body and antigenic expression. This is reduced and absorption and elimination in the liver is reduced.

1 is a block diagram of a conventional liposome system,

2 is a block diagram of a PEG-liposomal membrane according to the present invention,

3 is a schematic diagram of a PEG-liposomal system according to the present invention.

* Description of the symbols for the main parts of the drawings *

10 liposome membrane 12 PEG

Hereinafter, the configuration and operation of the present invention will be described in detail with reference to the accompanying drawings.

The phosphatidylethanolamine compound of the present invention as described above is DOPE-mPEG-propionate, DSPE-mPEG-propionate, DPPE-mPEG-propionate, DOPE-mPEG-succinate, DSPE-mPEG-succinate, DPPE-mPEG-succinate, DOPE- mPEG-carbonate, DSPE-mPEG-carbonate, DPPE-mPEG-carbonate, and the like. The compounds are shown in the following Chemical Formulas 1 to 9, respectively.

Hereinafter, the Example which manufactures each said compound is described.

Example 1 Preparation of DOPE-mPEG-propionate

The DOPE is dissolved in an organic solvent such as methanol, methylene chloride, and then SP-PEG powder is added. At this time, the molecular ratio (molar ratio) of DOPE and SP-PEG can be adjusted to 1: 5 to 1:20. The mixed solution is stirred in a reactor provided with a stirrer for 10 to 120 minutes to obtain DOPE-mPEG-propionate in which the amine group of DOPE and the propionyl group of SP-PEG are combined. Then, the organic solvent is removed using a heating method or a distillation method, and the DOPE-mPEG-propionate of Chemical Formula 1 is obtained in a solid state or a powder state.

Example 2 preparation method of DSPE-mPEG-propionate

DSPE is dissolved in organic solvents such as methanol, methylene chloride, and then SP-PEG powder is added. The molecular ratio of DSPE and SP-PEG is controlled at 1: 5 to 1:20. The mixed solution is stirred in a reactor equipped with a stirrer for 10 to 120 minutes to obtain DSPE-mPEG-propionate in which the amine group of DSPE and the propionyl group of SP-PEG are combined. Then, the organic solvent is removed using a heating method or a distillation method to obtain DSPE-mPEG-propionate of Chemical Formula 2 in a solid state or a powder state.

Example 3 preparation of DPPE-mPEG-propionate

The DPPE is dissolved in an organic solvent such as methanol, methylene chloride, and then SP-PEG powder is added. The molecular ratio of DPPE and SP-PEG is controlled at 1: 5 to 1:20. The mixed solution is stirred in a reactor equipped with a stirrer for 10 to 120 minutes to obtain DPPE-mPEG-propionate in which the amine group of DPPE and the propionyl group of SP-PEG are combined. Then, the organic solvent is removed by heating or distillation, and the DPPE-mPEG-propionate of Chemical Formula 3 is obtained in a solid state or a powder state.

Example 4 Preparation of DOPE-mPEG-succinate

DOPE is dissolved in an organic solvent such as methanol, methylene chloride, and then SS-PEG powder is added. The molecular ratio of DOPE and SS-PEG is controlled at 1: 5 to 1:20. The mixed solution was stirred in a reactor equipped with a stirrer for 10 to 120 minutes to obtain DOPE-mPEG-succinate in which the amine group of DOPE and the succinyl group of SS-PEG were combined. Then, the organic solvent is removed using a heating method or a distillation method to obtain DOPE-mPEG-succinate of Chemical Formula 4 in a solid state or a powder state.

Example 5 Preparation of DSPE-mPEG-succinate

DSPE is dissolved in an organic solvent such as methanol, methylene chloride, and then SS-PEG powder is added. The molecular ratio of DSPE and SS-PEG is controlled at 1: 5 to 1:20. The mixed solution was stirred for 10 to 120 minutes in a reactor equipped with a stirrer to obtain DSPE-mPEG-succinate in which the amine group of DSPE and the succinyl group of SS-PEG were combined. Then, the organic solvent is removed using a heating method or a distillation method to obtain DSPE-mPEG-succinate of Chemical Formula 5 in a solid state or a powder state.

Example 6 Preparation of DPPE-mPEG-succinate

DPPE is dissolved in an organic solvent such as methanol, methylene chloride, and then SS-PEG powder is added. Molar ratio of DPPE and SS-PEG is controlled at 1: 5 to 1:20. The mixed solution was stirred in a reactor equipped with a stirrer for 10 to 120 minutes to obtain DPPE-mPEG-succinate in which the amine group of DPPE and the succinyl group of SS-PEG were combined. Then, the organic solvent is removed by heating or distillation, and the DPPE-mPEG-succinate of Chemical Formula 6 is obtained in a solid state or a powder state.

Example 7 Preparation of DOPE-mPEG-carbonate

The DOPE is dissolved in an organic solvent such as methanol, methylene chloride, and then SC-PEG powder is added. The molecular ratio of DOPE and SC-PEG is controlled at 1: 5 to 1:20. The mixed solution is stirred in a reactor equipped with a stirrer for 10 to 120 minutes to obtain DOPE-mPEG-carbonate in which the amine group of DOPE and the carbonyl group of SC-PEG are combined. The organic solvent is then removed by heating or distillation to obtain DOPE-mPEG-carbonate in solid or powder form.

Example 8 Preparation of DSPE-mPEG-carbonate

DSPE is dissolved in organic solvents such as methanol, methylene chloride, and then SC-PEG powder is added. The molecular ratio of DSPE and SC-PEG is controlled at 1: 5 to 1:20. The mixed solution was stirred for 10 to 120 minutes in a reactor equipped with a stirrer to obtain DSPE-mPEG-carbonate in which the amine group of DSPE and the carbonyl group of SC-PEG were combined. Then, the organic solvent is removed by heating or distillation to obtain the DSPE-mPEG-carbonate of Chemical Formula 8 in a solid state or a powder state.

Example 9 Preparation of DPPE-mPEG-carbonate

DPPE is dissolved in an organic solvent such as methanol, methylene chloride, and then SC-PEG powder is added. The molecular ratio of DPPE and SC-PEG is controlled at 1: 5 to 1:20. The mixed solution is stirred in a reactor equipped with a stirrer for 10 to 120 minutes to obtain DPPE-mPEG-carbonate in which the amine group of DPPE and the carbonyl group of SC-PEG are combined. Then, the organic solvent is removed by heating or distillation to obtain DPPE-mPEG-carbonate of Chemical Formula 9 in a solid state or a powder state.

Using the phosphatidyl ethanol amine compound including the compound of Examples 1 to 9 can be configured as shown in FIG. 3 illustrates a spherical PEG-liposome, in which a delivery material is contained in the liposome membrane 10 and a PEG 12 is stretched outward like a branch.

Hereinafter, the Example which manufactures PEG-liposome using the phosphatidylethanolamine compound of this invention is described.

Example 11 Preparation of PEG-Liposomes

Phosphatidylcholine (PC), cholesterol, and phosphatidylethanolamine (PE) are dissolved in organic solvents such as methylene chloride, methanol, ethanol and the like. At this time, the composition ratio of the three phospholipids, PC: cholesterol = 1: 1 to 1: 0.5 is appropriate and PC: PE = 1: 0.1 to 1: 0.3 is appropriate. And it adjusts so that the concentration of total phospholipid may be 10-100 mg / mL.

The PE used at this time is DOPE-mPEG-propionate, DSPE-mPEG-propionate, DPPE-mPEG-propionate, DOPE-mPEG-succinate, DSPE-mPEG-succinate, DPPE-mPEG-succinate as described above in Examples 1 to 10. , DOPE-mPEG-carbonate, DSPE-mPEG-carbonate, and DPPE-mPEG-carbonate are used. At this time, only one type of PE may be used, or one or more types may be mixed and used.

On the other hand, when the transport material is water-soluble, the transport material is dissolved in an aqueous solution. The delivery material may be hemoglobin, an anticancer agent, an antibiotic, a physiological active agent, a hormone, a cytokine, an anti-inflammatory drug, a drug such as a gene, or various kinds of cosmetic raw materials. At this time, the aqueous phase is dissolved in 0.01 ~ 0.5M Nacl, 1 ~ 50mM Na-phosphate, pH is adjusted according to the nature of the delivery material. On the other hand, when the delivery material is hydrophobic, the delivery material is dissolved in an organic solvent in which phospholipid is dissolved. Thus prepared organic solution phase and aqueous phase are mixed and stirred in the reactor. The reactor should be equipped with temperature control, pH control and gas ejection.

The mixture is stirred in a reactor at a speed of 100-2000 rpm with a stirrer. The reaction temperature at this time is 10 ~ 40 ℃ is appropriate and adjusted according to the nature and stability of the delivery material. At this time, in order to make the mixing more appropriate, it is also beneficial to blow nitrogen gas into the mixture. Thus, once the PEG-liposomes are formed, the polycarbonate filter having a pore size of 100-200 nm is repeatedly passed about 5 to 10 times in order to uniformize the size of the PEG-liposomes to the desired size. Alternatively, the size of the PEG-liposome may be adjusted by passing through an extruder instead of using a filter. In this way, the size of the PEG-liposome is adjusted, followed by evaporation using a rotary evaporator to remove the solvent, or removal of the solvent by 99.9% using a dialysis method.

DOPE-mPEG-propionate, DSPE-mPEG-propionate, DPPE-mPEG-propionate, DOPE-mPEG-succinate, DSPE-mPEG-succinate, DOPE-mPEG-succinate, DOPE-mPEG-carbonate, DSPE-mPEG-carbonate, Phosphatidylethanolamine, including DPPE-mPEG-carbonate and the like, is a new component of PEG-liposomes, the final product of the present invention, which changes the PEG-liposomes in a human-friendly manner, and thus, such PEG-liposomes may be used for hemoglobin, anticancer agents, antibiotics, There is an effect that can be used as a delivery medium of drugs such as physiological active agents, hormones, anti-inflammatory drugs or various kinds of cosmetic raw materials.

Claims (6)

A phosphatidyl ethanol amine compound in which a phosphatidyl ethanol amine (PE) derivative and a methoxy polyethylene glycol derivative are linked by a conjugate. The method of claim 1, wherein the phosphatidyl ethanolamine derivative is selected from the group consisting of dipamitoyl phosphatidyl ethanolamine (DPPE), distearoyl phosphatidyl ethanolamine (DSPE), dioleoyl phosphatidyl ethanolamine (DOPE), etc. Phosphatidyl ethanol amine compound. The phosphatidylethanolamine compound according to claim 1, wherein the binder is selected from the group consisting of propionate, succinate, carbonate, and the like. A PEG-liposome formed by using a phosphatidylethanolamine compound in which a phosphatidylethanolamine (PE) derivative and a methoxy polyethylene glycol derivative are linked by a conjugate. The method of claim 4, wherein the phosphatidyl ethanolamine derivative is selected from the group consisting of dipamitoyl phosphatidyl ethanolamine (DPPE), distearoyl phosphatidyl ethanolamine (DSPE), dioleoyl phosphatidyl ethanolamine (DOPE), etc. PEG-liposomes. The PEG-liposome according to claim 4, wherein the conjugate is selected from the group consisting of propionate, succinate, carbonate, and the like.