JPS61155392A - Polymerizable liposome-forming lipid and production thereof - Google Patents

Abstract

NEW MATERIAL:The compound of formula [R is -(CH2)2N<+>(CH3)3, -(CH2)2N<+>H3, or -CH2CH(N<+>H3)CO<->2]. EXAMPLE:Eleostearic acid phosphatidylcholine. USE:A polymeric liposome-forming lipid for drug encapsulation. It has excellent stability, and can be polymerized easily e.g. by ultraviolet radiation. PREPARATION:The compound of formula I can be produced, e.g. by reacting 100pts.(wt.) of hydrolyzed phospholipid (preferably a complex with e.g. cadmium, etc.) with preferably 300-370pts. of tung oil fatty acid anhydride (preferably containing >=80% eleostearic acid) in the presence of preferably 68-84pts. of a catalyst (e.g. dimethylaminopyridine) in a solvent such as chloroform, in an inert gas atmosphere such as argon, preferably at 18-22 deg.C for 40-72hr, and purifying the reaction product e.g. by silica-gel column chromatography.

Description

DETAILED DESCRIPTION OF THE INVENTION 10. Background Technical Field of the Invention The present invention relates to a novel polymerizable ribosome-forming lipid and a method for producing the same. More specifically, the present invention relates to a polymerizable ribosome-forming lipid capable of forming polymeric ribosomes with excellent stability and a method for producing the same.

PRIOR ART At present, various attempts have been made to encapsulate medicinal substances, enzymes, etc. in microcapsules and provide them as pharmaceuticals, and microcapsules encapsulating hemoglobin I can serve as artificial red blood cells.

The early methods of such microencapsulation are encapsulation of a polymer compound by emulsification method and encapsulation accompanied by production of a polymer (polyamide) by interfacial polycondensation reaction. However, with these methods, there are problems such as the significant toxicity of the polymer used as the encapsulation material and the toxicity due to the organic solvent required in the synthesis process remaining in the capsule, and the particle size of the capsule is large (several μml to 1.000 μl).
), there were fatal problems with its use as a medicine, such as the tendency 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 compatibility with living organisms, that the capsule particle size can be miniaturized, and that the capsules have sufficient capacity in vivo. It must be stable, etc.

The use of so-called ribosomes, which are minute spherical aggregates formed in water by various phospholipids, which are the main components of biological membranes, as microcapsules has attracted attention as a method that satisfies these conditions to a considerable extent. are collecting.

However, ribosomes that use natural phosphorus molecules as they are have a limited lifespan and are unstable, especially when interacting with living cells, so they are used as models for cell recognition and cell interaction. or drug delivery by holding the drug in ribosomes and using it as a τ carrier.
In the field of drug delivery, many studies have been conducted to find stable ribosomes. Currently, the most effective means for achieving this goal is to polymerize ribosomes.

Therefore, the purpose of polymerizing ribosomes is to stabilize the bilayer membrane and, ultimately, the endoplasmic reticulum structure by covalently bonding lipid molecules. The mainstream method is to incorporate a functional group with polymerization ability into a lipid molecule, introduce a ribosome as a monomer, and then polymerize the lipid in the membrane of the ribosome. Representative examples include J, Am, Chew, Soc, 106.
1927-1633 (1984), etc., an unsaturated fatty acid is first synthesized, and a polymerizable reactive group is introduced into the phospholipid through an esterification reaction between this and a hydrolyzed W product of the phospholipid. It is something. However, according to this method, the synthesis of unsaturated fatty acids requires a lot of computational effort, and the purification and separation after synthesis is also difficult. ! Moreover, the yield of the polymerizable phospholipid, which is the final product, is several percent according to the literature value, and additional test results by the present inventors show that the yield is only about one-tenth of the literature value. I couldn't.

In addition, attempts have been made to use ribosomes as a material for artificial red blood cells that microencapsulate hemoglobin. It is expected that this can be effectively suppressed by using polymeric ribosomes using phospholipids.

However, there are several problems in applying polymeric ribosomes as materials for artificial blood.
In addition, polymerizable phospholipids are synthesized through a large number of reaction steps using 1/4 organic chemistry based on extremely detailed molecular design, which not only makes large-scale synthesis difficult from a practical standpoint.
It is extremely expensive. The second is a polymerization method for obtaining polymeric ribosomes. That is, the polymerization reaction of polymerizable phospholipids is generally carried out using a radical polymerization initiator or by irradiating with infrared light. However, methods using initiators usually require heating, and these initiators remain in ribosomes, making them unfavorable for use in living organisms.On the other hand, ultraviolet irradiation methods are Polymerizable phospholipids have low polymerizability, so many mw
In this case, there was a problem in that the internal hemoglobin was likely to deteriorate.

11 OBJECTS OF THE INVENTION Therefore, an object of the present invention is to provide a novel polymerizable ribosome-forming lipid and a method for producing the same.
The purpose of the koji of the present invention is to provide a polymeric ribosome-forming lipid with excellent stability and a method for producing the same.A further purpose of the present invention is to provide a highly stable polymeric ribosome-forming lipid and a method for producing the same. An object of the present invention is to provide a polymeric ribosome-forming lipid that can be easily polymerized under the following conditions and a method for producing the same.

These objectives are achieved by polymerizable ribosome-forming lipids of general formula I.

+CHz ”rx tPHs *riu -CH2-CH
(N'H) Also, the present invention provides R1+CHt+-t N (CH3
) is a polymerizable ribosome-forming lipid.

The purpose of these developments is to obtain a polymerizable ribosomal type represented by the general formula I, which is characterized in that tung oil fatty acid containing 60% by weight or more of eleostearic acid is esterified with a hydrolyzed phospholipid. This can also be achieved by a method for producing synthetic lipids.

Furthermore, the present invention provides that tung oil fatty acid is present in an acid anhydride form in an amount of 200 to 400 per 100,111 parts of a phospholipid hydrolyzate.
1! This is a method for producing a polymerizable ribosome-forming lipid using a certain amount. In addition, in the present invention, esterification is from 15 to
This is a method for producing polymerizable ribosome-forming lipids carried out at a temperature of 25°C.

Further, the present invention is a method for producing a polymerizable ribosome-forming lipid, wherein the phospholipid hydrolyzate is an egg yolk reticin hydrolyzate. Furthermore, the present invention is a method for producing a polymerizable ribosome-forming lipid in which the content of eleostearic acid in the tung oil fatty acid is 60% by weight or more.

10 Specific Structure of the Invention The phospholipid used in the present invention is a type of complex lipid, and is a biological component in which fatty acids and phosphoric acid are bonded to alcohols. It consists of two parts: a polar part consisting of phosphoric acid or a base, and a polar part such as phosphoric acid or a base. When this phosphorus-11 is fractionated in water, small vesicles (vesicles) having a bimolecular membrane structure, that is, ribosomes are formed. Examples of such phospholipids include egg yolk reticin (phosphatidylcholine),
Examples include cephalin and phosphatidylserine, with egg yolk reticin being particularly preferred.

Therefore, phosphatidylcholine is represented by the following general formula (■).

Ribosomes formed from such phospholipids have a short lifespan and are problematic in practical terms.
IIR, of the parts, fat I! Partial R1 and R2)
is replaced with eleostearic acid, a natural unsaturated fatty acid, and a polymerizable reactive group is introduced 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 80-9511%. The tung oil fatty acid obtained by hydrolyzing this tung oil contains 6011% or more of eleostearic acid, preferably 8011% or more.
% or more, and the rest includes saturated acids, oleic acid, linoleic acid, etc. This tung oil fatty acid may be used as it is in the form of a natural unsaturated fat layer, or if necessary, it may be purified using a columnar chromatogram or the like to extract only eleostearic acid and used.

CH3 (CH2) 30! ""CHCH"CHCH""C
1(C)I2), Cool ((Ieeee)) 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 200 parts per 1100 parts by weight of phosphoric acid. 40011 copies, rainbow or 300-370 copies I
This is part I.

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 in chloroform,
The tung oil fatty acid anhydride and catalyst are added to a medium such as carbon tetrachloride or methylene chloride and suspended under stirring, and the reaction system is filled with an inert gas such as argon, nitrogen, or helium. After the substitution, the reaction is carried out in the dark at a temperature of 15 to 25°C, preferably 18.22°C, for 24 to 72 hours, preferably 40 to 72 hours. At this time, white insoluble matter precipitates and is removed by filtration, and the resulting filtrate is brought into contact with an ion exchange resin and then washed off. After the medium is distilled off under reduced pressure at room temperature,
Mixed solvent of chloroform, methanol, and water (disability ratio -4
15/1) and purified using a chloroform and methanol mixed solution using a silica gel column or the like. Examples of catalysts include 4-dimethylaminopyridine, and phosphorus WI
45 to 90 f per 100 parts by weight of quality hydrolyzate
Preferably 1 part of 68-84fi is used.

The ribosome-forming lipid obtained depends on the phospholipid used; for example, when egg yolk lecithin is used,
．． When phosphatidylcholine eleostearate represented by the chemical formula (IV), cephalin, phosphatidylserine, etc. are used, ribosome-forming lipids corresponding to these can be obtained.

The ribosome-forming lipids obtained in this way are 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. Forms a lipid membrane. Add this to phosphorus mts* liquid or Hebesubatsu 77 (H
After shaking with a mixer, monomeric ribosomes are obtained by ultrasonication in an inert gas atmosphere such as argon, nitrogen, helium, etc.

This monomeric ribosome can carry pharmaceutical substances, enzymes, hemoglobin, etc.

When the monomer ribosome thus obtained is irradiated with ultraviolet rays, gamma rays, electron beams, etc., especially ultraviolet rays, the three conjugated double bonds in the 21 aliphatic groups are easily polymerized, forming polymeric ribosomes. is formed. Such macromolecules increase ribosome stability. This polymeric ribosome can also carry pharmaceutical substances, enzymes, hemoglobin, etc.

If the supported substance is hydrophilic, it will be encapsulated and supported within a monomer or polymeric ribosome, whereas if it is hydrophobic, it will be supported on the aliphatic portion of the monomer or polymeric ribosome. Ru.

There are various supporting methods, but a suspension of monomeric ribosomes is formed by mixing a polymerizable ribosome-forming lipid with the substance to be supported and then subjecting it to ultrasonication.
By centrifuging this, it can be supported on the monomer ribosome.

By irradiating such supported monomer ribosomes with ultraviolet rays, radiation, etc., supported polymeric ribosomes can be obtained.

Next, the present invention will be explained in more detail with reference to Examples.

Example: A tung oil fatty acid corresponding to eleostearin 180Q of the eleostearin group was dissolved in 600 parts and 1 part of carbon tetrachloride immediately after dehydration distillation.To this solution, 32.69 parts of dicyclohexylcarbodiimide was added,
Purge the inside of the container with argon gas, seal it, and leave it for 25 minutes.
The mixture was left for 24 hours at ℃ (with occasional stirring). Insoluble components were filtered off and distilled to dryness. When this was purified with silica gel using dichloromethane as a solvent, eleostearin anhydride was obtained in a yield of 29%.

Egg yolk lecithin (Kewpie PL-100) 459 dehydrated with ether 450m! , and after filtering off insoluble matter, 10
% concentration of tetrabutylammonium hydroxide in methanol (5C) mA was added and shaken vigorously at 25°C. As the reaction progressed, the solution became cloudy and the layers gradually separated, so the solution was allowed to stand, a brown oil was sufficiently precipitated, and the supernatant was decanted. The brown oil was washed three times with 100 ii of dehydrated ether, then dissolved under heating in dehydrated methanol 125-rILIL, 1 g of decolorizer was added under reflux at the S point, and the mixture was filtered while hot. After cooling, 250 xl/dry ether was added to the filtrate, the precipitate was left behind and decanted, and the precipitate was dissolved in hot WA 40vtlt. To this, 8 g of cadmium chloride pentahydrate was added to pure 2011Lttk:WIWI, and then 2.5 g of activated carbon and 20 g of decolorizing agent were added, and after refluxing at the boiling point,
It was filtered using 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 white solution was collected. When 100 to 150 ml of ethanol was added and vigorously shaken, white crystals were precipitated. .

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 vacuum-dried over phosphorus pentoxide overnight at a temperature of 80°C. As a result, a cadmium complex of phosphatidylcholine hydrolyzate was obtained with a yield of 56%.

6.74 g of egg yolk lecithin hydrolyzate cadmium complex molded with ester,
Chloroform 160xJ right after distillation! was added and suspended under stirring. Tung oil fat a! Anhydride 24.70a and catalyst 4-dimethylaminopyridine 5.619
Add , and fill the container with argon gas! ! After replacing the mixture, the mixture was sealed and reacted in a dark place at a temperature of 25° C. with stirring. At this time, a white insoluble material was precipitated, so this was filtered off, the solvent was distilled off under reduced pressure at room temperature, and the material was redissolved in 100 ift of a 5/4/1 mixed solvent of methanol/chloroform/water. This solution was filtered again and the filtrate was collected using ion exchange resin A.
G-501-X8 (D) CBio-Red) column and washed off with the above mixed solvent 500 IF. After the solvent was distilled off under reduced pressure at a temperature of 25° C., the residue was redissolved in chloroform and subjected to rR preparation using a silica gel column, whereby phosphatidylcholine eleostearate was obtained in a yield of 30%. Its infrared absorption spectrum is
As shown in Figure 1.

200-q of phosphadylcholine eleostearate of ribosomes from Juhachi phospholipid was dissolved in 1 chloroform solution. 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. Hepes buff 777 (Hepes buff
er) (10s M, D H8゜0>10y*1
After shaking with a portex mixer, the mixture was treated with a chip-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 ribosomes was confirmed. Also, scanning electronic! Spherical particles with a diameter of 0.2 to 0.5 μL were observed using an IQ microscope, confirming the formation of ribosomes.

Polymerization example of ribosomes 1 For irradiation using a 75W mercury lamp as a light source [12Ca, sample concentration 110l1/Ll, under degassing,
Fi-fi UV rays in a water bath with a water temperature of 25 degrees Celsius! As shown in FIG. 2, the absorbance at 272 nm due to triene decreased as the irradiation time increased, indicating that polymerization was progressing.

Weight of ribosome 2 Phosphatidylcholine eleostearate obtained in El example of hemoglobin-encapsulated ribosome 461G
(58 μ■ 01), cholesterol 22.4 mg (5
A chloroform solution containing 8 μmof) and a chloroform solution containing 12,4 my of tung oil fat (8,5 μm01) were added and the resulting solution was divided into 50×1. The mixture was placed in an eggplant-shaped flask, nitrogen was blown to form a sm on the bottom of the flask, and the mixture was dried in an evaporator at 25° C. for 1 hour, followed by vacuum drying at 25° C. for 2 and a half hours. Then, -carbohydrated hemoglobin was added to 10
% raw M saline solution containing 10mj! After adding and shaking with a portex mixer, bath type ultrasonic wave 1! II
The mixture was treated with injection m (20 W) for 30 minutes under an argon stream. Phosphoric acid solution (pH 7,4) 4C) + tl was added to this, and after centrifugation (10,0 O Orp-, 10 minutes), vcl
11 parts of K, L, and II temporary solutions (pH 7,4) were added.

I got it! 3.5xl of the turbid liquid was collected, and after replacing with -carbon oxide, it was stirred at a certain point overnight, and then heated at 5゜000 r.
Centrifuge at p- for 10 minutes, add 5iui of phosphoric acid solution (
pH 7.4) was added to obtain a monomer ribosome suspension with a constant volume of 3×1. This monomer ribosome suspension 0
．． 4xl with bovine rIn serum or phosphorus lII! l! Equilibrium 3.
Added to 6xl.

In addition, 3.5 x 1 of the obtained suspension was taken out, and after replacing with carbon oxide, the mixture was stirred at a temperature of 25°C for 12 hours at a irradiation distance of 120 x using a 75 W mercury lamp as a light source, and irradiated with external radiation. This was followed by centrifugation at 5,000p for 10 minutes, followed by adding phosphorus a II solution (pH 7,4) to obtain a polymer ribosome suspension with a constant volume of 3xl. 0.4 zIt of ribosome suspension was added to 3.6 liters of bovine plasma or phosphorus solution.

Hemoglobin leakage test Monomers and polymers of hemoglobin ribosomes were left in bovine plasma and phosphorus-SaW solution for a certain period of time, and the visible spectrum of the entire suspension (peak around <400 ns based on hemoglobin) was measured by centrifugation. Separation (bovine plasma; 10.0OOru.

Rin II! 1 buffer solution: 5.0 OOrl) 110 minutes) The spectra of the shida supernatant were compared, and hemoglobin leakage from ribosomes was quantified. The results were as shown in Table 1.

Comparative Example Diene phosphatidylcholine 4 represented by the following chemical formula
5*a (60μm01), cholesterol 23.2-g
(60μ■01) and tung oil fat! 22゜4■18.
Hemoglobin-encapsulated ribosomes were prepared in the same manner as in Ribosome Polymerization Example 2 using 5 μmol).

Regarding the obtained hemoglobin ribosome monomer and polymer, hemoglobin leakage was performed in the same manner as in ribosome polymerization example 2, and the results were as shown in Table 2.

As is clear from Table 1, Hbl release from eleostearate phosphatidylcholine monomer ribosomes is
It increases over time in bovine plasma, whereas in polymer ribosomes it increases by 13! Even after testing, no hemoglobin leakage was observed, indicating that ribosome stability was significantly improved by polymerization. On the other hand, as is clear from Table 2, with dienephosphatidylcholine, hemoglobin leakage is 1! even in polymer ribosomes! The effect of polymerization is low.

■8 Specific Effects of the Invention As mentioned above, the polymerizable ribosome-forming lipid according to the present invention has three conjugated diamides derived from eleostearic acid in its hydrophobic group, as shown in the general formula (■). Because of the presence of heavy bonds, monomeric ribosomes formed from these lipids are easily polymerized by ultraviolet irradiation, and the ribosomes after polymerization have improved stability compared to ribosomes made only of natural phospholipids. . Therefore, if a polymeric ribosome formed from the polymerizable ribosome-forming lipid according to the present invention is loaded with a pharmaceutical substance, an enzyme, hemoglobin, etc., extremely excellent medicines, artificial red blood cells, etc. can be obtained.

Furthermore, since the method for producing polymerizable ribosome-forming lipids according to the present invention consists of esterification with a hydrolyzate of phospholipids such as tung oil fat layer containing 60 Ii mass % or more of eleostearic acid, natural Fatty acids obtained from fats and oils can be used, and compared to conventional methods, the synthesis process of unsaturated fatty acids is not required, the process can be significantly shortened, and the yield is 1.
Since it is improved by more than 0 times, it becomes cheaper.

[Brief explanation of the drawing]

FIG. 1 is a chart showing an example of the infrared absorption spectrum of the polymerizable ribosome-forming lipid according to the present invention, and FIG. 2 is a chart showing the degree of polymerization of ribosomes formed from the ribosome-forming lipid according to the present invention by ultraviolet irradiation. It is a chart showing an example of an absorption spectrum.

Claims (7)

[Claims] (1) A polymerizable ribosome-forming lipid represented by the following general formula I. ▲There are mathematical formulas, chemical formulas, tables, etc.▼ (However, R is -(CH_2)-_2N^■(CH_3
)_3, -(CH_)-2+2N^■H_3 or -
CH_2-CH(N^■H_3)-COO^■. ) (2) R is -(CH_2)-_2N^■(CH_3)_
3. The polymerizable ribosome-forming lipid according to claim 1, which is No. 3. (3) A method for producing a polymerizable ribosome-forming lipid represented by the following general formula I, which comprises esterifying a tung oil fatty acid containing 60% by weight or more of eleostearic acid with a phospholipid hydrolyzate. ▲There are mathematical formulas, chemical formulas, tables, etc.▼ (However, R is -(CH_2)-_2N^■(CH3_
)_3, (CH_2)-_2N^■H_3 or -CH
_2-CH(N^■H_3)-COO^■. ) (4) The polymerizable ribosome-forming lipid according to claim 3, wherein the tung oil fatty acid is used in the form of an acid anhydride in an amount of 200 to 400 parts by weight per 100 parts by weight of the phospholipid hydrolyzate. Production method. (5) The method for producing a polymerizable ribosome-forming lipid according to claim 3 or 4, wherein the esterification is carried out at a temperature of 15 to 25°C. (6) The method for producing a polymerizable ribosome-forming lipid according to any one of claims 3 to 5, wherein the phospholipid hydrolyzate is an egg yolk reticin hydrolyzate. (7) The content of eleostearic acid in tung oil fatty acid is 6
The method for producing a polymerizable ribosome-forming lipid according to any one of claims 3 to 6, wherein the content is 0% by weight or more.