JPS6332330B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION BACKGROUND OF THE INVENTION Technical Field The present invention relates to a novel medical carrier. Specifically, the present invention relates to a medical carrier made of a polymer liposome with extremely high stability. Prior Art Currently, various attempts are being made to provide pharmaceuticals by encapsulating medicinal substances, enzymes, etc. in microcapsules, and microcapsules encapsulating hemoglobin serve as artificial red blood cells. The early methods of such microencapsulation include encapsulation of a polymer compound by an emulsification method and encapsulation accompanied by the production of a polymer (polyamide) by an interfacial polycondensation reaction. However, with these methods, there are problems such as the significant toxicity of the polymer used as the encapsulation material, the toxicity due to the organic solvent required in the synthesis process remaining in the capsule, and the large particle size of the capsule (several μm to 1000 μm). To,
There was a fatal problem with its use as a medicine, as it was said to be more likely to cause disorders such as blood clots. By the way, the main purpose of encapsulating pharmaceutical substances, enzymes, hemoglobin, etc. in microcapsules is to retain the activity of pharmaceutical substances, enzymes, hemoglobin, etc., which are unstable in the body, for a long time, and to maintain their effects for a long time. . Therefore, the conditions required for microcapsule materials that are intended to be applied to living organisms or used as pharmaceuticals are that they have low toxicity to living organisms, that the capsule particle size can be miniaturized, and that capsules do not form in vivo. It must be sufficiently stable. As a method that satisfies these conditions to a considerable extent, the use of so-called liposomes, which are minute spherical aggregates formed in water of various phospholipids, which are the main components of biological membranes, as microcapsules has attracted attention. ing. However, liposomes that use natural phospholipids as they are have a short lifespan and are particularly unstable when interacting with living cells, so they cannot be used as models for cell recognition or cell-cell interactions. , drug delivery (drug delivery) in which drugs are held in liposomes and used as carriers.
In the field of liposome delivery, much research has been conducted to find stable liposomes. the current,
The most effective means is polymerization of liposomes. Therefore, the purpose of polymerizing liposomes is to stabilize the bilayer membrane and, ultimately, the endoplasmic reticulum structure by covalently bonding lipid molecules. The method is
The mainstream method is to prepare a liposome as a monomer by incorporating a functional group with polymerization ability into a lipid molecule, and then polymerize the lipid in the liposome membrane. A typical example is J.Am.
As described in Chem.Soc., 106 , 1627-1633 (1984), etc., unsaturated fatty acids are first synthesized, and this can be polymerized into phospholipids through an esterification reaction with a phospholipid hydrolyzate. This method introduces a reactive group. However, according to this method, the synthesis of unsaturated fatty acids is very labor-intensive, the purification and separation after synthesis is complicated, and the yield of the final product, polymerizable phospholipid, is low. According to the literature value, the yield is several percent, and in the results of additional tests by the present inventors, the yield was only about one-tenth of the literature value. In addition, attempts have been made to use liposomes as a material for artificial red blood cells that microencapsulate hemoglobin. It is expected that this can be effectively suppressed by using polymeric liposomes containing phospholipids. However, there are several problems in applying polymer liposomes as materials for artificial blood. First of all, polymerizable phospholipids are synthesized using pure organic chemistry through a large number of reaction steps based on extremely detailed molecular design, which not only makes it difficult to synthesize them in large quantities from a practical standpoint. This is extremely expensive. The second is a polymerization method for obtaining polymeric liposomes. That is, the polymerization reaction of polymerizable phospholipids is generally carried out using a radical polymerization initiator.
Alternatively, this can be done by irradiating ultraviolet light. However, methods using initiators usually require heating, and since these initiators remain in the liposomes, they are not preferred for use in living organisms. On the other hand, in the ultraviolet irradiation method, since conventional polymerizable phospholipids have low polymerizability, it is necessary to apply a large amount of irradiation, and in this case, there is a problem that internal hemoglobin is likely to deteriorate. OBJECTS OF THE INVENTION It is therefore an object of the present invention to provide a novel medical carrier. Another object of the present invention is to provide a medical carrier made of a highly stable polymeric liposome-forming lipid. Still another object of the present invention is to provide a medical carrier with little leakage of supported substances. Another object of the present invention is to provide a medical carrier useful for microencapsulating hemoglobin and the like. These objects are achieved by a medical carrier prepared by irradiating a liposome containing a liposome-forming lipid represented by the following general formula with ultraviolet rays or radiation. (However, R is ( _-CH2 ) _-2N ( _CH3 ) ₃ , ( _-CH2 )-
_{2NH3 or -CH2} -CH( _NH3 )-COO. ) The present invention also provides a medical carrier in which R is ( _-CH2 ) _-2N ( _CH3 ) ₃ . Furthermore, the present invention is a medical carrier in which the irradiation treatment is performed with ultraviolet rays. Further, the present invention is a medical carrier for supporting hemoglobin. Specific Structure of the Invention The liposome-forming lipid constituting the medical carrier according to the present invention is obtained by esterifying tung oil fatty acid containing eleostearic acid with a phospholipid hydrolyzate. The phospholipid used in the present invention is one of the complex lipids.
It is a biological component in which fatty acids and phosphoric acids of alcohols are bound, and its chemical structure consists of two parts: a nonpolar part consisting of a relatively long aliphatic hydrocarbon, and a polar part such as phosphoric acid or a base. It consists of When this phospholipid is dispersed in water, a small vesicle (vesicle) having a bilayer membrane structure, that is, a liposome is formed. Examples of such phospholipids include egg yolk lecithin (phosphatidylcholine), kephalin, and phosphatidylserine, with egg yolk lecithin being particularly preferred. Therefore, phosphatidylcholine is represented by the following general formula. Liposomes formed from such phospholipids have a short lifespan and are problematic in practical terms. In the present invention, fatty acid moieties (R ₁
and R ₂ ) with eleostearic acid, which is a natural unsaturated fatty acid, and introduces a polymerizable reactive group to synthesize a polymerizable phospholipid. Eleostearic acid used in the present invention is an unsaturated fatty acid having conjugated double bonds at the 9, 11, and 13 positions represented by the following chemical formula, and is present in tung oil as a glyceride. It accounts for 95% by weight. The tung oil fatty acid obtained by hydrolyzing this tung oil contains 60% by weight or more, preferably 80% by weight or more of eleostearic acid, and the remainder contains saturated acids, oleic acid, linoleic acid, etc. . This tung oil fatty acid may be used as it is as a natural unsaturated fatty acid, or if necessary, it may be purified by column chromatography or the like to extract only eleostearic acid and used. CH ₃ (CH ₂ ) ₃ CH=CHCH=CHCH =CH (CH ₂ ) ₇ COOH () Therefore, this tung oil fatty acid is usually subjected to esterification in the form of an acid anhydride. The amount of tung oil fatty acid used is 200 to 400 parts by weight, preferably 300 to 370 parts by weight, per 100 parts by weight of phospholipid. On the other hand, the phospholipids are used as hydrolysates, in particular as their metal complexes, for example complexes of metals such as cadmium. The esterification reaction is carried out as follows. Phospholipid hydrolyzate or its metal complex is added to a medium such as chloroform, carbon tetrachloride, methylene chloride, etc. and suspended under stirring. The above-mentioned tung oil fatty acid anhydride and catalyst are added to this suspension, and the reaction is carried out. After purging the system with an inert gas such as argon, nitrogen, or helium, the reaction is carried out in the dark at a temperature of 15 to 25°C, preferably 18 to 22°C, for 24 to 72 hours, preferably 40 to 72 hours. At this time, white insoluble matter precipitates, so it is filtered off, and the medium is distilled off under reduced pressure at room temperature, followed by a mixed solvent of chloroform, methanol, and water (volume ratio = 4/
5/1) and brought into contact with an ion exchange resin, then washed off. After distilling off the solvent under reduced pressure, the residue is dissolved in a small amount of chloroform and purified using a silica gel column or the like using a mixed solvent of chloroform and methanol. Examples of the catalyst include 4-dimethylaminopyridine, which is used in an amount of 45 to 90 parts by weight, preferably 68 to 84 parts by weight, per 100 parts by weight of the phospholipid hydrolyzate. The resulting liposome-forming lipid differs depending on the phospholipid used. For example, when egg yolk lecithin is used, eleostearate phosphatidylcholine shown by the chemical formula (), kephalin, phosphatidylhyline, etc. When these are used, liposome-forming lipids corresponding to these can be obtained. The liposome-forming lipid obtained in this way is dissolved in a solvent such as chloroform, methylene chloride, ether, methanol, etc., and then the resulting lipid solution is placed in a round-bottomed container, and the solvent is completely evaporated to form a liquid on the bottom surface. Forms a lipid membrane. Add phosphate buffer or Hepes buffer to this.
is added, shaken with a mixer, and then subjected to ultrasonication under an inert gas atmosphere such as argon, nitrogen, helium, etc. to obtain monomer liposomes. When the monomer liposomes obtained in this way are irradiated with radiation such as ultraviolet rays, gamma rays, or electron beams, especially ultraviolet rays, the three conjugated double bonds in the two aliphatic groups are easily polymerized to form polymeric liposomes. A medical carrier of the present invention is formed. Such irradiation treatment increases the stability of the medical carrier by polymerizing the monomeric liposome. This medical carrier can carry pharmaceutical substances, enzymes, hemoglobin, etc. When the supported substance is hydrophilic, it is encapsulated and supported within a polymeric liposome,
On the other hand, if it is hydrophobic, it is supported on the aliphatic portion of the polymer liposome. There are various supporting methods, but after mixing a polymerizable liposome-forming lipid with the substance to be supported, a suspension of monomer liposomes is formed by ultrasonication, and by centrifugation, monomer liposomes are formed. can be carried by By irradiating such supported monomer liposomes with ultraviolet rays, radiation, etc., supported polymer liposomes can be obtained. Irradiation conditions vary depending on the type of radiation source, but for example, in the case of ultraviolet rays, the monomer liposome suspension is placed in a container that can transmit ultraviolet rays, such as a quartz glass container, and the inside of the container is deaerated or filled with argon. After replacing the sample with an inert gas atmosphere such as nitrogen or helium, the sample is water-cooled or air-cooled using a light source capable of emitting ultraviolet rays, such as a mercury lamp or a xenon lamp, at an irradiation distance of 5 to 20 cm, preferably 10 to 15 cm. UV rays are irradiated for 15 minutes to 16 hours, preferably 2 to 12 hours. Next, the present invention will be explained in more detail with reference to Examples. Production example of polymerizable liposome-forming lipid Production of eleostearic anhydride Tung oil fatty acid equivalent to 80 g of eleostearic acid was dissolved in 600 ml of carbon tetrachloride immediately after dehydration distillation.
Dicyclohexylcarbodiimide 32.6 to this solution
g was added, the inside of the container was replaced with argon gas, the container was sealed, and the container was left as it was at 25° C. for 24 hours (with occasional stirring). Insoluble components were filtered off and distilled to dryness. When this was purified with silica gel using dichloromethane as a developing solvent, eleostearic anhydride was obtained in a yield of 29%. Production of egg yolk lecithin (phosphatidylcholine) hydrolyzate cadmium complex Dissolve 45 g of egg yolk lecithin (Kewpie PL-100) in 450 ml of dehydrated ether, filter out insoluble materials,
% concentration of tetrabutylammonium hydroxide in methanol was added and the mixture was shaken vigorously at a temperature of 25°C. As the reaction progresses, the solution becomes cloudy and the layers gradually separate, so let it stand.
A brown oil was allowed to settle out well and the supernatant was decanted. Dissolve the brown oil with 100ml of dehydrated ether.
After washing twice, the mixture was heated and dissolved in 125 ml of dehydrated methanol, 1 g of a decolorizing agent was added under reflux at the boiling point, and the mixture was filtered while hot. After cooling, 250 ml of dehydrated ether was added to the filtrate, the precipitate was left behind and decanted, and the precipitate was dissolved in 40 ml of hot water. To this, add 8 g of cadmium chloride hemihydrate dissolved in 20 ml of pure water,
Further, 2.5 g of activated carbon and 2 g of a decolorizing agent were added, and the mixture was refluxed to the boiling point, and then filtered using a filter paper and a 0.25 μm Millipore filter. When 100 to 150 ml of ethanol was added to this, a colored precipitate was formed, so this was removed and only a cloudy solution was collected, and then 100 to 150 ml of ethanol was added and vigorously shaken.
White crystals began to precipitate. After standing overnight at a temperature of 0 to 5°C, the precipitated crystals were collected by filtration, washed with dehydrated methanol, dehydrated ether, and dehydrated benzene in this order, and further vacuum-dried over phosphorus pentoxide at a temperature of 80°C overnight. However, a cadmium complex of phosphatidylcholine hydrolyzate was obtained with a yield of 56%. Production of polymerizable lipids by esterification Egg yolk lecithin hydrolyzate cadmium complex 6.74g
160 ml of chloroform immediately after distillation was added to the solution and suspended under stirring. Add to this tung oil fatty acid anhydride 24.70
g and the catalyst 4-dimethylaminopyridine
After adding 5.61 g, the inside of the container was replaced with argon gas, the container was tightly stoppered, and the reaction was allowed to proceed in the dark with stirring at a temperature of 25° C. for 60 hours. At this time, a white insoluble substance was precipitated, so this was filtered off, and the solvent was distilled off under reduced pressure at room temperature. Methanol/chloroform/water = 5/4/1
Redissolve in 100ml of mixed solvent. This solution is filtered again and the filtrate is treated with ion exchange resin AG-501-X8(D).
(Bio-Rad) Column and the mixed solvent 500
Washed off with ml. After the solvent was distilled off under reduced pressure at a temperature of 25° C., the residue was redissolved in chloroform and purified using a silica gel column. Phosphatidylcholine eleostearate was obtained in a yield of 30%. Its infrared absorption spectrum was as shown in FIG. Production of liposomes from polymerizable phospholipids 200 mg of phosphazylcholine eleostearate
was dissolved in 6 ml of chloroform. The lipid solution thus obtained was placed in an eggplant-shaped flask, and the solvent was completely removed using an evaporator to form a lipid film on the bottom of the eggplant-shaped flask. After adding 10 ml of Hepes buffer (10 mM, PH8.0) to this and shaking it with a vortex mixer, it was treated with a tip-type ultrasonic irradiator (40 to 50 W) under an argon stream for 10 minutes. The treatment liquid changed from a cloudy state to a transparent dispersion, and the formation of liposomes was confirmed. Furthermore, spherical particles with a diameter of 0.2 to 0.5 μm were observed using a scanning electron microscope, confirming the formation of liposomes. Example 1 Polymerization of liposomes (manufacture of medical carrier) Irradiation distance was 12 cm using a 75 W mercury lamp as the light source.
When the sample concentration was 10 mg/ml and irradiated with ultraviolet rays in a water bath with a water temperature of 25°C under deaerated conditions, the second
As shown in the figure, the absorbance at 272 nm based on triene decreased with the passage of irradiation time, which confirmed that polymerization was progressing. Example 2 Preparation of hemoglobin-encapsulated liposomes 46 mg of phosphatidylcholine eleostearate obtained in the production example of polymerizable liposome-forming lipids
(58μmol), cholesterol 22.4mg (58μmol)
Chloroform solution containing and tung oil fatty acid
Add a chloroform solution containing 2.4 mg (8.5 μmol), put the resulting solution in a 50 ml eggplant-shaped flask, blow nitrogen to form a thin film on the bottom of the flask, and dry it in an evaporator at 25 °C for 1 hour. Thereafter, vacuum drying was performed at a temperature of 25° C. for two and a half hours. Next, the hemoglobin converted into carbon monoxide was
After adding 10 ml of a physiological saline solution containing 10% and shaking with a vortex mixer, the mixture was treated with a bath-type ultrasonic irradiator (20W) under an argon stream for 30 minutes.
After adding 40 ml of phosphate buffer (PH7.4) to this and centrifuging (10,000 rpm, 10 minutes), 11 ml of phosphate buffer (PH7.4) was added to the precipitate. 3.5 ml of the resulting suspension was taken out, replaced with carbon monoxide, stirred overnight in the dark, and then
The mixture was centrifuged at 5000 rpm for 10 minutes, and phosphate buffer (PH7.4) was added to obtain a monomer liposome suspension with a constant volume of 3 ml. 0.4 ml of this monomer liposome suspension was added to 3.6 ml of bovine plasma or phosphate buffer. In addition, 3.5 ml of the obtained suspension was collected,
After replacing the carbon monoxide, UV irradiation was performed using a 75W mercury lamp as a light source at a distance of 12cm at a temperature of 25°C for 12 hours with stirring, followed by centrifugation at 5000 rpm for 10 minutes, and a phosphate buffer (PH7.4 ) and 3
A polymer liposome suspension with a fixed volume of 1 ml was obtained.
0.4 ml of this polymer liposome suspension was added to 3.6 ml of bovine plasma or phosphate buffer. Hemoglobin leakage test Hemoglobin liposome monomers and polymers are left in bovine plasma and phosphate buffer for a certain period of time, and the visible spectrum of the entire suspension (peak around 400 nm based on hemoglobin)
The spectra of the supernatant obtained by centrifugation (bovine plasma; 10,000 rpm, phosphate buffer; 5,000 rpm, 10 minutes) were compared, and hemoglobin leakage from the liposomes was quantified. The results were as shown in Table 1. Comparative example 45 mg (60 μmol) of dienephosphatidylcholine and 23.2 mg of cholesterol shown by the following chemical formula
Hemoglobin-encapsulated liposomes were prepared in the same manner as in Liposome Polymerization Example 2 using (60 μmol) and 2.4 mg (8.5 μmol) of tung oil fatty acid. The monomer and polymer of the obtained hemoglobin liposome were subjected to a hemoglobin leakage test similar to that in Liposome Polymerization Example 2.
It was as shown in Table 2.

【table】

[Table] As is clear from Table 1, Hb leakage from eleostearate phosphatidylcholine monomer liposomes increases over time in bovine plasma, whereas with polymer liposomes, hemoglobin levels increase even after one week. No leakage was observed,
A remarkable improvement in liposome stability was observed due to polymerization. On the other hand, as is clear from Table 2, in the dienephosphatidylcholine system, leakage of hemoglobin is observed even in polymer liposomes, and the effect of polymerization is low. Specific Effects of the Invention As described above, the medical carrier according to the present invention has
As shown in the above general formula, since three conjugated double bonds derived from eleostearic acid are present in the hydrophobic group, the monomer liposome formed from the lipid easily polymerizes by irradiation with ultraviolet rays. Furthermore, the stability of the polymerized liposome carrier is improved compared to liposomes made only of natural phospholipids. Therefore, if the medical carrier, which is the polymer liposome of the present invention, supports pharmaceutical substances, enzymes, hemoglobin, etc., extremely excellent medicines, artificial red blood cells, etc. can be obtained.

[Brief explanation of the drawing]

FIG. 1 is a chart showing an example of the infrared absorption spectrum of the liposome-forming lipid, which is the main component before polymerization of the medical carrier according to the present invention.
The figure is a chart showing an example of an ultraviolet absorption spectrum showing the degree of polymerization of the medical carrier of the present invention formed from liposome-forming lipids by ultraviolet irradiation.

Claims (1)

[Scope of Claims] 1. A medical carrier obtained by irradiating a liposome containing a liposome-forming lipid represented by the following general formula with ultraviolet rays or radiation. (However, R is ( _-CH2 ) _-2N ( _CH3 ) ₃ , ( _-CH2 )-
_{2NH3 or -CH2} -CH( _NH3 )-COO. )2R is ( _-CH2 ) _-2N ( _CH3 ) ₃ , the medical carrier according to claim 1. 3. The medical carrier according to claim 1 or 2, wherein the irradiation treatment is performed with ultraviolet light. 4. The medical carrier according to any one of claims 1 to 3, which is for supporting hemoglobin.