JP3303481B2 - Reactive endoplasmic reticulum and forming agent - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a reactive vesicle forming agent,
And a reactive endoplasmic reticulum containing the same as an endoplasmic reticulum-forming component. More specifically, pharmaceutical carriers, test agents, diagnostic agents, sensors, immobilized catalysts, bioreactors, bioelectronic devices, microcapsule substitutes, etc.
The present invention relates to a reactive vesicle-forming agent used for preparing vesicles such as various functional liposomes or fat emulsions, and a reactive vesicle containing the same as an vesicle-forming component.

[0002]

2. Description of the Related Art Liposomes are vesicles composed of a phospholipid bilayer membrane, and applications in various fields have been attempted. In particular, application to pharmaceutical carriers, sensors for diagnosis and detection, etc., has attracted attention.However, immobilizing a functional substance on the surface of a liposome or in a membrane to provide various functions, and reducing the blood concentration of the liposome Maintaining them is a major issue.

[0003] Conventionally, regarding immobilization of a functional substance on the surface of a liposome or in a membrane, the aminoethylcarbamylmethyl group substituted on a polysaccharide on the surface of a liposome coated with a pullulan derivative is attached to a γ-maleimidobutyloxysuccinate. A method in which an antibody fragment is bound via nimidyl (Biochem. Biophys. Acta., 898 , 323 (1987)), or glycolipids are added to liposome membrane-forming components in advance, and liposome formation is followed by periodate oxidation. And immobilization by reacting the resulting aldehyde group with the antibody (J. Biol. Chem., 255 , 10509 (1980)).

However, in these conventional methods, it is necessary to carry out a multi-step chemical reaction on the surface of the liposome membrane after the preparation of the liposome, so that the amount of the intended functional substance introduced is limited to a low level, and There is a problem that by-products and impurities are mixed in and the liposome membrane is greatly damaged.

On the other hand, it has been pointed out that when liposomes are administered into a living body, most of them are trapped by reticulo-organs such as the liver and spleen, and thus cannot provide a sufficient effect (Cancer Res., 43 , 5328 (1983)). Therefore, as a method of improving the problem of being trapped by the reticulo-organs or the problem of low stability such as disintegration and aggregation of the liposome itself, a polyethylene glycol chain is introduced into the surface of the liposome. (For example, Japanese Patent Application Laid-Open No. 1-249717, FEBS letters,
268 , 235 (1990)). In addition, it has been shown that liposomes modified with polyethylene glycol can maintain blood concentrations for a long period of time (Biochem. Biophyl.
s. Acta., 1066 , 29-36 (1991)). However, since the liposome into which the polyethylene glycol chain obtained by such a method has been introduced does not react with the functional substance, the functional substance cannot be immobilized on the liposome surface.

Further, JP-A-4-346918 discloses that a liposome having a maleimide group is first reacted with a protein having a thiol group (thiolated protein), and then a polyalkylene glycol having a thiol group added to the remaining maleimide group. It is described that by reacting a compound containing a (thiolated polyalkylene glycol) moiety, drug-containing antibody-bound liposomes with improved uptake in reticulo-organs can be obtained. But,
In this liposome, the antibody is hidden under the polyalkylene glycol layer, and the reaction with the antigen at the target site is hindered. Therefore, there is a problem that the expected effect cannot be sufficiently obtained.

[0007]

SUMMARY OF THE INVENTION An object of the present invention is to solve the above-mentioned problems by introducing a polyalkylene oxide chain into liposomes and other endoplasmic reticulum. A reactive endoplasmic reticulum-forming agent capable of efficiently immobilizing various functional substances by covalent bonds and increasing the amount of the introduced functional substance, and an endoplasmic reticulum-forming component comprising the endoplasmic reticulum-forming agent To provide a reactive endoplasmic reticulum.

[0008]

The present invention relates to the following reactive vesicle-forming agent and reactive vesicle. (1) A reactive vesicle forming agent comprising a reactive phospholipid derivative represented by the following general formula [1].

Embedded image [Wherein, R ¹ C (= 0) and R ² C (= 0) have 3 carbon atoms.
Represents an acyl residue of up to 30 fatty acids, which may be the same or different. R ³ is a hydrogen atom or a methyl group, AO an oxyalkylene group having 2 to 4 carbon atoms, n represents an average addition mole number of the oxyalkylene group, the positive number of 1 to 1,000. When n is 2 or more, the oxyalkylene groups may be the same or different, and may be added randomly or in blocks. Y represents the following organic group.

Embedded image (Here, p represents 2 or 3, q represents an integer of 0 to 3.) M represents a hydrogen atom or an alkali metal atom. (2) A reactive vesicle comprising at least one reactive vesicle-forming agent according to the above (1) as a vesicle-forming component.

In the present invention, R ¹ C of the general formula [1]
(= 0), the acyl residue of the fatty acid represented by R ² C (= 0) has 3 to 3 carbon atoms (including carbon of a carbonyl group).
0, preferably 8 to 20 acyl residues. Specific examples of such an acyl residue include propionic acid,
Butyric acid, caproic acid, caprylic acid, pelargonic acid, capric acid, undecanoic acid, lauric acid, myristic acid, palmitic acid, stearic acid, arachinic acid, behenic acid, lignoceric acid, sorotic acid, montanic acid, melicic acid, 2
Acyl residues of saturated fatty acids such as ethylhexanoic acid; oleic acid, linoleic acid, linolenic acid, erucic acid, 2,4-
Acyl residue of an unsaturated fatty acid such as octadecadienoic acid; acyl residue of branched fatty acids such as isostearic acid; ricinoleate over <br/> Le acids, fatty acids having a hydroxyl group in the alkyl group, such as 12-hydroxystearic acid Acyl residue and the like.

[0010] Among these, myristic acid,
Acyl residues of palmitic acid, stearic acid, oleic acid and 2,4-octadecadienoic acid are preferred, and acyl residues of palmitic acid, stearic acid and oleic acid are particularly preferred. R ¹ C (= 0) and R ² C (= 0) may be the same or different.

The oxyalkylene group represented by AO in the general formula [1] is an oxyalkylene group having 2 to 4 carbon atoms, such as an oxyethylene group, an oxypropylene group, an oxytrimethylene group, and an oxy-1-ethylethylene group. Oxy-1,2-dimethylethylene group, oxytetramethylene group and the like. These oxyalkylene groups are
Ethylene oxide, propylene oxide, oxetane,
It is a group obtained by addition polymerization of an alkylene oxide such as 1-butene oxide, 2-butene oxide, and tetrahydrofuran. N in the general formula [1] is a positive number of 1 to 1000, preferably 10 to 300, and more preferably 20 to 120.

When n is 2 or more, the types of oxyalkylene groups may be the same or different. In the latter case, they may be added randomly or in blocks. When imparting hydrophilicity, AO is preferably one to which ethylene oxide is independently added, and in this case, n is preferably 10 or more. When different types of alkylene oxides are added, it is desirable that ethylene oxide is added in an amount of 20 mol% or more, preferably 50 mol% or more. When imparting lipophilicity to the polyalkylene oxide chain, the number of moles added other than ethylene oxide is increased.

Y in the general formula [1] is an organic group represented by the structural formula [2]. R ^{3 in the} general formula [1] is a hydrogen atom or a methyl group. M in the general formula [1] is a hydrogen atom or an alkali metal atom such as sodium or potassium.

In order to produce the reactive phospholipid derivative represented by the general formula [1], for example, a polyalkylene glycol derivative having a carboxyl group at one end and a hydroxyl group at the other end, and dicyclohexylcarbodiimide (D
CC) to form an active form, then react with phosphatidylethanolamines, and then easily react with N, N'-carbonyldiimidazole (CDI) with the obtained compound. be able to.
Α-hydro-ω as a polyalkylene glycol derivative
A reaction formula in the case of using dipalmitoyl phosphatidylethanolamine (DPPEA) or a salt thereof as carboxyl -polyoxyethylene and phosphatidylethanolamines is shown below. In the formula, M and n represent the same as described above.

[0015]

Embedded image

In addition to the above-mentioned production method, a compound in which the carboxyl group of the above polyalkylene glycol derivative is acid chlorided with thionyl chloride, isobutyl chloroformate or the like, or activated by a compound such as succinimide or carbonyldiimidazole. An esterified compound;
The compound can also be easily produced by reacting phosphatidylethanolamines with CDI and reacting the resulting compound with CDI.

Further, in the general formula [1], Y is q = 0.
The reactive phospholipid derivative which is an organic group is produced by reacting CDI at a ratio of 2 mol or more with respect to 1 mol of a polyalkylene oxide having a hydroxyl group at both terminals, and reacting the obtained compound with phosphatidylethanolamine. The reaction can be easily performed.

These reactions can be carried out without solvent or with water, toluene, benzene, acetonitrile, methanol,
In a solvent such as 4-dioxane, tetrahydrofuran or chloroform, -4 in the air or under an inert gas atmosphere such as nitrogen, argon, helium or carbon dioxide.
0 to 120 ° C, preferably 0 to 60 ° C, 10 minutes to 2
It is desirable to carry out by stirring for 40 hours, preferably 1 hour to 48 hours. After completion of the reaction, the obtained reaction mixture is purified or isolated as it is or by distillation, recrystallization, reprecipitation, adsorbent treatment, column treatment, gel filtration, ultrafiltration, dialysis, etc. It can be used as a forming agent.

Since the reactive vesicle-forming agent of the present invention has a functional group such as an amino group, a hydroxyl group or a thiol group, particularly an oxycarbonylimidazole group having a high reactivity with a primary amino group. , Easily react with a functional substance having such a functional group to form a covalent bond. Thus, by using a reactive vesicle forming agent of the present invention as vesicle forming components, it is possible to introduce a poly A Rukire <br/>N'okishido chain to the endoplasmic reticulum, reactivity to a functional substance having the functional group Can be provided.

The reactive endoplasmic reticulum of the present invention contains the above-mentioned reactive endoplasmic reticulum-forming agent as an endoplasmic reticulum-forming component and has reactivity with the functional substance having the functional group.
The reactive vesicle forming agent may be contained alone or in combination of two or more. The vesicle-forming component may contain other vesicle-forming components capable of forming vesicles in addition to the reactive vesicle-forming agent of the present invention.

In the present invention, the term “vesicle” means a particle having a structure in which a hydrophilic group of a reactive vesicle-forming agent or a reactive vesicle-forming agent and another vesicle-forming component is oriented toward an aqueous phase at an interface. I do. Specific examples thereof include liposomes, which are closed vesicles composed of a bilayer membrane, fat emulsions in which a mixture of vegetable oil, phospholipid, and the like are emulsified, micelles, and the like.

The reactive endoplasmic reticulum-forming agent of the present invention comprises a reactive phospholipid derivative represented by the general formula [1] alone or in combination of two or more, or other endoplasmic reticulum capable of forming an endoplasmic reticulum. forming components, such as soybean lecithin, egg yolk lecithin, other phospholipids, cholesterol and Into <br/> Raripiddo (Otsuka Pharmaceutical Co., trademark), it is used as a mixture of soybean oil, safflower oil, etc. and Thus, reactive vesicles such as reactive liposomes, reactive fat emulsions, and reactive micelles can be formed. In this case, the endoplasmic reticulum can be produced by a known method. The reactive endoplasmic reticulum obtained using the reactive endoplasmic reticulum-forming agent of the present invention can utilize various functional substances by utilizing the oxycarbonylimidazole group in the compound represented by the general formula [1] as a functional group. It can be introduced by a covalent bond.

Next, each reactive vesicle will be described in detail. Reactive liposomes, which are typical reactive vesicles, contain a reactive vesicle-forming agent as a membrane-forming component (vesicle-forming component). The content of the reactive endoplasmic reticulum forming agent is 0.01 to 50 mol%, preferably 0.5 to 30 mol%, based on the total amount of the reactive endoplasmic reticulum forming agent and other film forming components. desirable. If the amount is less than 0.01 mol%, the expected effect is small, and if it exceeds 50 mol%, the stability of the liposome is reduced, so that it is not generally used. The reactive phospholipid derivative represented by the general formula [1] may contain one kind or two or more kinds in combination.

As other membrane-forming components to be used by mixing with the reactive vesicle-forming agent, those conventionally used as liposome membrane-forming components can be used without limitation.
Specifically, diphosphatidylglycerol, cardiolipin, phosphatidylinositol, phosphatidylserine, phosphatidylethanolamine, soybean lecithin, egg yolk lecithin, phosphatidylcholine , phosphatidylcholine
Polymerizable phospholipid having a phospholipid and a fatty acid portion in unsaturated groups such as Gilles glycerol; sulphoxide ribosyl diglyceride, digalactosyldiglyceride, glycolipids such as lactosyl diglyceride; non-polar lipids, such as cholesterol; Others include non Ionic surfactant, phosphatidyl polyethylene glycol, "Biochem. Biophys. Act
a., 1066 , 29-36 (1991) ", a reaction product of phosphatidylethanolamine with polyethylene glycol, a mixture thereof and the like.

The reactive liposomes reactive vesicle forming agent and lecithin, other phospholipids, other film-forming components of the cholesterol, etc. <br/>, dissolved in a suitable solvent such as an organic solvent, Aix Trusion method, vortex mixer method,
Ultrasonic method, surfactant removal method, reverse layer evaporation method, ethanol injection method, prevesicle method, French press method, W / O /
W emulsion method, annealing method, freeze-thaw method, etc.
It can be produced by making liposomes by various known methods. In addition, reactive liposomes having various sizes and shapes such as multilamellar liposomes, small unilamellar liposomes, and large unilamellar liposomes can be produced by selecting these production methods.

In the reactive liposome thus obtained, an oxycarbonylimidazole group is bonded to the inner and outer surfaces of the liposome membrane via a spacer made of polyalkylene oxide, so that the reactive liposome has an amino group, a hydroxyl group, a thiol group and the like. A functional substance having a functional group, particularly a primary amino group, can be efficiently and easily chemically bonded to a liposome bilayer membrane by a urethane bond, a carbonate bond, or a thiocarbonate bond via a spacer made of polyalkylene oxide. Can be immobilized.

Examples of the functional substance that can be immobilized on the reactive liposome include pigments, dyes, radiolabeled compounds,
Fluorescent compounds, chemiluminescent compounds, labeled substances, such as the electrode sensitive compound; photoresponsive compounds, pH responsive compound, an external stimulus responsive compound such thermally responsive compound; enzymes, antibodies, other proteins, sugars, lipids And physiologically active substances such as glycoproteins, glycolipids and hormones;

The immobilization reaction of the functional substance on the reactive liposome is carried out by using an aqueous solvent such as various buffers, or these aqueous solvents and acetone, acetonitrile, tetrahydrofuran, 1,4-dioxane, dimethylformamide, or dimethylformamide. Cetamide, dimethyl sulfoxide, in a mixed solvent with an organic solvent such as pyrrolidone, the reactive liposome and the functional substance at -10 to 120 ° C, preferably 0 to 60 ° C, more preferably in the case of reaction with an amino group. Is 0-40
C., in the case of a reaction with a hydroxyl group or a thiol group, the reaction can be easily carried out in one step, for example, by reacting at 40 to 120 ° C. with stirring for 5 minutes to 1000 hours, preferably 30 minutes to 72 hours. it can. Outside these conditions, the stability of the liposome deteriorates, which is not preferable.

The reaction of immobilizing a functional substance having an amino group on the liposome surface is schematically shown as follows. In the formula, R ³ , AO, and n represent the same as described above.

Embedded image

Various substances can be encapsulated in the reactive liposome by a known method as in the case of general liposomes. Examples of the substance to be encapsulated include labeling substances such as dyes, dyes, radiation labeling compounds, fluorescent compounds, and chemiluminescent compounds; response to external stimuli such as photoresponsive compounds, pH-responsive compounds, heat-responsive compounds, and electrode-sensitive compounds. Sex compounds; enzymes, antibodies, other proteins, sugars,
Physiologically active substances such as lipids, glycoproteins, glycolipids, and hormones; pharmaceuticals; and water-soluble polymers such as polyacrylamide, polyvinyl alcohol, polyethylene glycol, and hyaluronic acid. After completion of the immobilization reaction or the encapsulation operation, if necessary, purification can be performed by a method such as gel filtration, ultrafiltration, dialysis, centrifugation, and sedimentation by standing.

[0031] Liposomes were immobilized functional material is a polyalkylene oxide tip functional material strand is immobilized Tei Runode, the action of the functional substance without being disturbed by the polyalkylene oxide chain is fully exhibited Is done. Also,
Since the polyalkylene oxide chain has been introduced, the effects of conventional polyalkylene oxide chain introduction, such as maintaining blood concentration for a long period of time, non-immunogenicity, and preventing leakage of substances encapsulated inside liposomes are also exhibited. Can be expected. Therefore, the reactive liposome can be used as various functional liposomes, such as a pharmaceutical carrier, a test agent, a diagnostic agent, a sensor, an immobilized catalyst, a bioreactor, a bioelectronic element, and a microcapsule substitute.

In the production of the reactive liposome, a polymerizable reactive liposome can be prepared by blending a polymerizable phospholipid as another film-forming component. As the polymerizable phospholipid, known polymerizable phospholipids can be used. For example, in addition to 1,2-di (2,4-octadecadienoyl) -3-phosphatidylcholine,
Edited by Shojima Nojima, Junzo Sunamoto and Keizo Inoue, liposomes published by Nankodo in 1988, pp. 313-315, and the like. Among these, 1,2-di (2,4
-Octadecadienoyl) -3-phosphatidylcholine is preferred.

After preparing the liposome, the polymerizable liposome can be treated with UV, γ in the presence or absence of a photopolymerization initiator.
Polymerization can be easily carried out by irradiation with light such as a beam or an electron beam, or by heating with a redox initiator system or in the presence of an azo-based initiator or an organic peroxide. The thus obtained liposome after polymerization has excellent stability, and thus can be used stably while being dispersed in an aqueous solution or prepared in a powder form by freeze-drying or the like. .

Other reactive vesicles as reactive vesicles include the reactive vesicle-forming agent of the present invention, vegetable oils such as soybean oil and safflower oil, and phospholipids such as soybean lecithin and egg yolk lecithin. Fats and oils containing other additives added as necessary, for example, Intralipid (trademark, manufactured by Otsuka Pharmaceutical Co., Ltd.), an emulsifying aid, a stabilizer, a tonicity agent, a fat-soluble drug, a fat-soluble bioactive substance, and the like. The mixture is emulsified. The content of the reactive vesicle-forming agent in the fat mixture is
0.01 to 50 mol%, preferably 0.5 to 30 mol%
It is desirable that

The reactive fat emulsion can be produced by a known method. For example, a reactive endoplasmic reticulum-forming agent, a vegetable oil, a phospholipid, and an optional blending additive are mixed,
It can be produced by heating, adding water, coarsely emulsifying with a homomixer or the like, and then homogenizing with a Manton-Gaulin type pressurized injection homogenizer or the like.

The same functional substance can be easily immobilized on the thus obtained reactive fat emulsion in the same manner as in the case of the reactive liposome. Therefore, the reactive fat emulsion can be used as a pharmaceutical carrier, a test agent, a diagnostic agent, a sensor, an immobilized catalyst, and the like.

Reactive micelles, which are reactive vesicles other than those described above, may contain lecithin, other phospholipids, cholesterol, etc., even if they consist only of the reactive vesicle-forming agent of the present invention.
It may contain other components such as a metal.
The reactive micelle can immobilize the functional substance in the same manner as the reactive fat emulsion, and can be used for the same purpose.

[0038]

Since the reactive vesicle-forming agent of the present invention has a structure represented by the general formula [1], a polyalkylene oxide chain can be introduced into vesicles, and Various functional substances can be easily and efficiently immobilized on the tips by covalent bonds, and the amount of functional substances introduced can be increased.

Since the reactive endoplasmic reticulum of the present invention contains the above-mentioned endoplasmic reticulum-forming agent as an endoplasmic reticulum- forming component, a polyalkylene oxide chain is introduced and the endoplasmic reticulum is inserted into the endoplasmic reticulum via a spacer made of polyalkylene oxide. Thus, various functional substances can be easily and efficiently immobilized by covalent bonds, and the amount of functional substances introduced can be increased.

[0040]

The present invention will be described in more detail with reference to the following Examples, but it should not be construed that the invention is limited thereto. Synthesis Example 1 α-hydro-ω-carboxyl -polyoxyethylene
( Mw: about 3000, polymerization degree: about 65) 1.0 g (0.
3 mmol) and 68 mg (0.3 mmol) of dicyclohexylcarbodiimide were dissolved in 10 ml of ethyl acetate and stirred at 5 ° C. for 1 hour. Next, 217 mg of dipalmitoyl phosphatidylethanolamine (0.3 mm
ol) was dissolved in 10 ml of an ethyl acetate solution, and the mixture was further stirred for 6 hours, allowed to stand at 0 ° C. overnight, and then the precipitate was removed by filtration. Next, carbonyldiimidazole 4 was added to the filtrate.
9 mg (0.3 mmol) was added, the mixture was stirred at room temperature for 1 hour, the obtained reaction mixture was poured into 100 ml of hexane, and the precipitate was collected by filtration to obtain the desired reactive phospholipid derivative in the form of a white powder. (Yield 88%). The progress of the reaction was determined by IR spectrum (KBr method). As an intermediate, disappearance of amino group (N ⁺ H ₂ stretch, 3000 cm ⁻¹ ) in phosphatidylethanolamine and amide bond (C = O
Stretch, 1647 cm ^-1 ), and the target product was the disappearance of hydroxyl groups (OH stretch, 3428 cm ^-1 ) at the terminal of the polyethylene glycol derivative and oxycarbonyl ether.
Formation of midazole bond (carbonyl bond) (1760c
It was confirmed by C = O extension contraction); m ^-1.

Embedded image

Example 1-1 Egg yolk phosphatidylcholine 20 mg (26 μmol),
And 3.9 mg (10 μmol) of cholesterol,
The reactive phospholipid derivative obtained in Synthesis Example 1 was put into a 10 wt% (2.4 mg, 0.7 μmol) eggplant type flask with respect to the two, dissolved in 2 ml of benzene, and then dissolved.
Lyophilization was performed. Add 1 ml of physiological saline to this,
Multilamellar liposomes were obtained by bath-type ultrasonic irradiation and vortex mixer. Further, the mixture was sequentially passed through a polycarbonate membrane of 3.0 → 1.0 → 0.2 μm by an extruder to obtain a large monolayer reactive liposome. The particle size of the obtained reactive liposome was measured with a laser scattering particle size distribution analyzer (NICOMP, NICOMP).
As a result of measurement using 370 HPL (trademark), the average particle size was 255 nm (CV value: 18%).

Example 1-2 Reactive liposomes were obtained in the same manner as in Example 1-1 except that the following compound was used as a reactive phospholipid derivative in an amount of 5 wt% (1.0 μmol) (average particle size: 278 nm). ,
CV value 23%).

Embedded image

Example 1-3 In the same manner as in Example 1-1, except that the following compound was used as a reactive phospholipid derivative in an amount of 30 wt% (3.6 μmol).
To obtain a reactive liposome (average particle size: 248 n).
m, CV value 25%).

Embedded image

Example 1-4 Reactive liposomes were obtained in the same manner as in Example 1-1, except that 1 wt% (0.1 μmol) of the following compound was used as a reactive phospholipid derivative (average particle size: 250 nm). ,
CV value 21%).

Embedded image

Example 1-5 Reactive liposomes were obtained in the same manner as in Example 1-1, except that the following compound was used as a reactive phospholipid derivative in an amount of 5 wt% (0.6 μmol) (average particle size: 250 nm). ,
CV value 21%).

Embedded image

Example 1-6 A reactive liposome was obtained in the same manner as in Example 1-1, except that the following compound was used as a reactive phospholipid derivative in an amount of 5 wt% (0.1 μmol) (average particle size: 291 nm). ,
CV value 24%).

Embedded image

Example 1-7 In the same manner as in Example 1-1, except that the following compound was used as a reactive phospholipid derivative in an amount of 20 wt% (4.0 μmol).
To obtain a reactive liposome (average particle size: 221 n).
m, CV value 18%).

Embedded image

Example 1-8 An average was obtained in the same manner as in Example 1-1 except that 1,2-di (2,4-octadecadienoyl) -3-phosphatidylcholine (DODP) was used instead of egg yolk phosphatidylcholine. A large unilamellar reactive liposome having a particle size of 254 nm (CV value: 23%) and having polymerizability was obtained. The liposome was irradiated with 0.75 Mrad of γ-ray to polymerize DODP. The resulting polymerized liposomes are Sephad
After gel filtration using ex G-50, a powder sample was obtained by freeze-drying. This liposome powder could be regenerated by swelling with physiological saline.

Example 2 0.5 ml of the reactive liposome solution (solid content: 2.5 wt%) obtained in Example 1-1 and 1 mg / ml of horseradish peroxidase (hereinafter abbreviated as HRP) / 0.
The 1M phosphate buffer (pH 7.5) was stirred at 4 ° C. for 24 hours to immobilize HRP on the reactive liposome. This was subjected to gel filtration using Sephadex G-50,
The liposome-containing fraction was collected. To this was added a solution of HRP substrate 1,2-phenylenediamine (10 mmol /
l) 0.1 ml was added, the mixture was incubated at 30 ° C. for 10 minutes, and 10 μl of 0.1N sulfuric acid was further added, whereby brown coloration was observed. From this result, the reactive liposomes of Example 1 -1 was confirmed to be able to immobilize HRP simply stirring with HRP.

Comparative Example 1 Egg yolk phosphatidylcholine 20 mg (26 μmol),
By using only 3.9 mg (10 μmol) of cholesterol and cholesterol, a large unilamellar liposome having a solid content of 2.5 wt% was obtained in the same manner as in Example 1-1. HRP was allowed to act on the liposome solution in the same manner as in Example 2, purified by gel filtration, and 0.1 ml of a 1,2-phenylenediamine solution (10 mmol / l) was added.
For 10 minutes, and then add 0.1N sulfuric acid 10
μl was added, but no color development was observed. This result indicates that HRP could not be immobilized in the liposome of Comparative Example 1 containing no reactive phospholipid derivative as a film-forming component.

Continuation of front page (56) Reference European Patent 557199 (EP, B1) See Blume et al, Biochimica et Biophysica Acta, July 7, 1993, 1149, 180-184. Blume et al, Biochimica et Biophysica Acta, May 27, 1993, 157-168 (58) Fields investigated (Int. Cl. ⁷ , DB names) A61K 47/18 A61K 9/127 C07F 9/10 CA (STN) REGISTRY (STN)

Claims (2)

(57) [Claims] 1. A reactive vesicle forming agent comprising a reactive phospholipid derivative represented by the following general formula [1]. Embedded image [Wherein, R ¹ C (= 0) and R ² C (= 0) have 3 carbon atoms.
Represents an acyl residue of up to 30 fatty acids, which may be the same or different. R ³ is a hydrogen atom or a methyl group, AO an oxyalkylene group having 2 to 4 carbon atoms, n represents an average addition mole number of the oxyalkylene group, 1 to 10
Represents a positive number of 00. When n is 2 or more, the oxyalkylene groups may be the same or different, and may be added randomly or in blocks.
Y represents the following organic group. Embedded image (Here, p represents 2 or 3, q represents an integer of 0 to 3.) M represents a hydrogen atom or an alkali metal atom. ] 2. A reactive vesicle comprising at least one reactive vesicle-forming agent according to claim 1 as a vesicle-forming component.