JPH06126152A - Reactive liposome and excipient - Google Patents

Abstract

PURPOSE:To obtain reactive liposome, the functional substance content of which can be increased by constituting the liposome using a polyalkyleneglycol derivative of specific structure and bearing by covalent bond various functional substances on the surface or in the film of the liposome directly or through a spacer in a simple and efficient manner. CONSTITUTION:A reactive liposome is constituted using a polyalkylene glycol derivative expressed by formula I. In formula I, R1 is an aliphatic hydrocarbon group; X is -O-. In formula II, P is 1 or 2; R2 is H, a methyl group; n is the average additional number of moles and is 1 to 500. Consequently, it is possible to obtain the reactive liposome which can bear various functional substances (e.g. color) on the surface of or in the film of a liposome directly or through a spacer by covalent bond in a simple and efficient manner. In addition, this liposome can increase the content of the functional substance.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a reactive liposome-forming agent used as a membrane-forming component of liposomes, and a reactive liposome containing the same. More specifically, inspection, diagnosis, sensor, immobilized catalyst, bioreactor,
The present invention relates to a reactive liposome-forming agent used as a film-forming component of various functional liposomes such as a microcapsule substitute or a drug carrier, and a reactive liposome containing the same.

[0002]

2. Description of the Related Art Liposomes are phospholipid bilayer vesicles, and their applications have been tried in various fields. In particular, its application to a sensor for diagnosis and detection, a drug carrier, or the like is drawing attention. However, maintaining the blood concentration of liposomes,
Also, immobilization of functional substances on the surface of liposomes or in the membrane has become a major issue.

Regarding maintenance of blood concentration of liposomes,
It is disclosed in WO 91/05546 that a long-term blood concentration can be maintained by adding a polyethylene glycol derivative having no reactive functional group to the liposome membrane-forming component. However, this method is effective in maintaining the blood concentration of the liposome, but a liposome containing a polyethylene glycol derivative having no reactive functional group as a film-forming component cannot support a functional substance on the surface. It is impossible.

Further, as a method for immobilizing a functional substance on the surface of a liposome or in a membrane, γ-maleimidobutyl is substituted for an aminoethylcarbamylmethyl group substituted on a polysaccharide on the surface of a liposome coated with a pullulan derivative. Method for conjugating antibody fragments via oxysuccinimidyl (Biochem. Biophys. Acta.,
898, 323 (1987)) or a glycolipid is previously added to the liposome membrane-forming component, and after the formation of the liposome, periodic acid oxidation is carried out, and the resulting aldehyde group is reacted with the antibody to immobilize it. (J. Biol. C
hem. , 255, 10509 (1980)).

However, in these conventional methods, it is necessary to carry out a multi-step chemical reaction on the surface of the membrane after liposome preparation, which limits the amount of the target functional substance to be introduced to a low level, and also causes a side effect by the reaction. There is a problem in that products and impurities are mixed and damage to the film is large.

On the other hand, a polyethylene glycol derivative obtained by reacting polyethylene glycol with carbonyldiimidazole reacts with a primary or secondary amine compound or a primary, secondary or phenolic hydroxyl group to form a urethane bond. Alternatively, it is known to generate a carbonate bond. However, it is not known to use such a polyethylene glycol derivative as a membrane-forming component of liposomes.

[0007]

SUMMARY OF THE INVENTION In order to solve the above problems, the object of the present invention is to easily and efficiently provide various functional substances directly or via spacers on the surface or in the membrane of liposomes. An object of the present invention is to provide a reactive liposome-forming agent which can be supported by a covalent bond and can increase the amount of introduced functional substance, and a reactive liposome containing the same.

[0008]

The present invention relates to the following reactive liposome-forming agent and a reactive liposome containing the same. (1) A reactive liposome-forming agent comprising a polyalkylene glycol derivative represented by the following general formula [1].

[Chemical 3] (2) A reactive liposome comprising one or more reactive liposome-forming agents composed of the polyalkylene glycol derivative represented by the general formula [1] as a film-forming component.

The aliphatic hydrocarbon group represented by R ¹ in the above general formula [1] is a saturated, unsaturated or polymerized fatty acid having 3 or more carbon atoms, preferably 7 to 21 which constitutes a saturated, unsaturated or polymerizable fatty acid. It is preferably an aliphatic hydrocarbon group.

Examples of such aliphatic hydrocarbon groups include butyric acid, caproic acid, caprylic acid, pelargonic acid, capric acid, undecanoic acid, lauric acid, myristic acid,
Palmitic acid, stearic acid, arachidic acid, behenic acid,
Saturated aliphatic hydrocarbon groups constituting saturated fatty acids such as lignoceric acid, cerotic acid, montanic acid and melissic acid;
Unsaturated aliphatic hydrocarbon groups constituting unsaturated fatty acids such as linoleic acid, linolenic acid and erucic acid; 2,4-octadecadienoic acid and "Adv. Polym. Sci.,"
64, 1-62 (1985) ”and polymerizable aliphatic hydrocarbon groups constituting a surfactant or a surfactant.

In the general formula [1], R ² is a hydrogen atom or a methyl group. n represents the average number of moles added, and the value is 1 to 500, preferably 4 to 250. When n is 2 or more, it may be a single adduct of ethylene oxide in which R ² is a hydrogen atom or a single adduct of propylene oxide in which R ² is a methyl group, and a random adduct or block adduct of ethylene oxide and propylene oxide. May be

The liposome-forming agent of the present invention can be easily produced by, for example, a method of reacting a polyalkylene glycol compound represented by the following general formula [2] with carbonyldiimidazole (hereinafter abbreviated as CDI) or other methods. can do.

[0013]

[Chemical 4] (In the formula, R ¹ , X, p, R ² and n are the same as described above.)

Specific examples of the polyalkylene glycol compound represented by the above general formula [2] include polyoxyethylene oleyl ether, polyoxyethylene cetyl ether, polyoxyethylene stearyl ether, polyoxyethylene lauryl ether and polyoxyethylene tridecyl. Ether, polyoxyethylene monolaurate,
Commercially available surfactants such as polyoxyethylene monostearate, polyoxyethylene monooleate, polyoxyethylene nonyl phenyl ether, polyoxyethylene octyl phenyl ether, and polyoxyethylene octadecyl amine are easily available, which is preferable.

In addition to the above, polyoxypropylene oleyl ether, polyoxyethylene polyoxypropylene oleyl ether, polyoxypropylene cetyl ether, polyoxyethylene polyoxypropylene cetyl ether, etc., such as R ¹ , X, R ² , n, p Compounds in various combinations may be mentioned.

The reactive liposome-forming agent of the present invention is preferably a polyalkylene glycol derivative obtained by reacting the above polyalkylene glycol compound with CDI.

The liposome-forming agent of the present invention contains, for example, 1000 moles or less of CDI, preferably 1.5 moles or less, without a solvent, or toluene based on 1 mole of the polyalkylene glycol compound represented by the above-mentioned general formula [2]. , Benzene, acetonitrile, methanol, 1,4-dioxane, tetrahydrofuran, chloroform, etc., in the atmosphere or in an inert gas such as nitrogen, argon, helium, carbon dioxide, etc., at -40 to 120 ° C., preferably 0. It can be easily obtained in a high yield by stirring at -60 ° C for 1 minute to 48 hours, preferably 1 minute to 1 hour. The reaction mixture thus obtained is used as it is, or is purified or isolated by a method such as distillation, recrystallization, reprecipitation, adsorbent treatment, column treatment, gel filtration, ultrafiltration, dialysis, etc. to obtain a liposome forming agent. be able to.

The reactive liposome-forming agent of the present invention has a high reactivity with various functional substances, especially those having an amino group or a hydroxyl group, and especially those having a primary amino group. . Therefore, these functional substances are easily covalently bonded by a urethane bond or a carbonate bond. The binding between the reactive liposome-forming agent and the functional substance can be performed directly or via a spacer made of polyalkylene glycol or the like.

Examples of the functional substance include labeling substances such as dyes, dyes, radiation labeling compounds, fluorescent compounds and chemiluminescent compounds; external stimulus responsiveness such as photoresponsive compounds, pH responsive compounds and thermoresponsive compounds. Compounds; enzymes, antibodies,
Other proteins, physiologically active substances such as sugars, lipids, glycoproteins, glycolipids, hormones, and the like; among drugs and the like, those having an amino group or a hydroxyl group in the molecule can be mentioned.

An example of the reaction in which the reactive liposome-forming agent and the functional substance having a primary amino group are immobilized by a urethane bond is shown in the following general formula [3].

[Chemical 5] (In the formula, R ¹ and n have the same meanings as described above.)

The reactive liposome-forming agent of the present invention is
It has a good ability to form bilayer membranes and can form liposomes alone or in combination with other membrane-forming components such as phospholipids.

Therefore, by using such a reactive liposome-forming agent as a membrane-forming component of a liposome, various functional substances can be easily attached to the surface of the liposome or in the membrane by a covalent bond, directly or through a spacer. It is possible to obtain a reactive liposome that can be loaded onto

The reactive liposome of the present invention is a reactive liposome containing one or more of the above-mentioned reactive liposome-forming agents as a film-forming component. In this case, it is preferable to form liposomes from one or more of the above liposome-forming agents and other film-forming components. The content of the reactive liposome forming agent is 0.001 to 60 with respect to the other film forming components.
It is desirable that the content is wt%, preferably 0.1 to 30 wt%. With such a content, stable liposomes can be obtained.

As other membrane-forming components, known liposome membrane-forming components can be used without limitation, and examples thereof include phospholipids such as diphosphatidylglycerol, cardiolipin, phosphatidylinositol, phosphatidylserine, phosphatidylethanolamine and phosphatidylcholine;
Polymerizable phospholipid having a polymerizable fatty acid residue in the fatty acid moiety such as 1,2-di (2,4-octadecadienoyl) -3-phosphatidylcholine; "Adv. Polym. Sc.
i. , 64, 1-62 (1985) ”; polymeric phospholipids and surfactants; glycolipids such as sulfoxyribosyl diglyceride, digalactosyl diglyceride, lactosyl diglyceride; and nonpolar lipids such as cholesterol. A mixture of these may be mentioned. Of these, 1,2-di (2,4-octadecadienoyl) -3-phosphatidylcholine is particularly preferable. The alkylene glycol compound represented by the general formula [2], which is a raw material for producing the reactive liposome-forming agent, is
It may remain as it is as another component.

The reactive liposome of the present invention can be prepared, for example, by the extrusion method, vortex mixer method, ultrasonic method, surfactant removal method, reverse layer evaporation method, ethanol injection method, pre-vesicle method, French press method, W / O. It can be produced by various methods such as the / W emulsion method, the annealing method, and the freeze-thaw method. In addition, reactive liposomes of various forms and various sizes such as multilamellar liposomes, small unilamellar vesicles, and large unilamellar vesicles can be prepared depending on the types of these production methods.

Since the reactive liposome of the present invention contains the above-mentioned reactive liposome-forming agent as a film-forming component,
It has a high reactivity as shown in the above reaction formula [3], and therefore, a large amount of a functional substance can be easily and efficiently added to the surface or the membrane of the liposome directly or via a spacer. It can be immobilized and supported by covalent bonding. In addition, by-product contamination due to immobilization,
Damage to the film can be reduced. Examples of the functional substance to be immobilized include the above-mentioned functional substances.

Immobilization of the functional substance on the reactive liposome is carried out by using an aqueous solvent such as various buffers or acetone, acetonitrile, tetrahydrofuran, 1,4-dioxane, dimethylformamide, dimethylacetamide, dimethylsulfoxide, pyrrolidone. In a single step by reacting in an organic solvent such as or a mixed solvent thereof at −10 to 120 ° C., preferably 0 to 40 ° C. for 5 minutes to 168 hours, preferably 30 minutes to 72 hours. Can be done. In addition, stable liposomes can be obtained by reacting under such conditions.

Various substances can be encapsulated inside the reactive liposome of the present invention. For example, dye,
Labeling substances such as dyes, radiation labeling compounds, fluorescent compounds, chemiluminescent compounds; photo-responsive compounds, pH-responsive compounds,
External stimuli-responsive compounds such as thermo-responsive compounds; enzymes, antibodies, other proteins, physiologically active substances such as sugars, lipids, glycoproteins, glycolipids, hormones; pharmaceuticals and the like can be encapsulated.

When a polymerizable component such as the above-mentioned polymerizable phospholipid is used as another membrane-forming component, a polymerizable reactive liposome can be obtained. Polymerization of the reactive liposome is carried out by irradiating with UV, radiation, electron beam or the like in the presence or absence of a photopolymerization initiator after preparation of the liposome, or by a redox initiator system, or at a Tg of 10 or less of a bilayer membrane. It can be easily carried out by heating in the presence of a polymerization initiator having a time half-life temperature.

When purification is required, gel filtration, immobilization of functional substances, encapsulation or after completion of polymerization, gel filtration,
It can be purified by ultrafiltration, dialysis, centrifugation, sedimentation and the like.

Since the reactive liposome of the present invention has excellent stability, it can be tested, diagnosed, sensored or immobilized while being dispersed in an aqueous solution or after being prepared into powder by a method such as freeze-drying. Catalyst, bioreactor,
It can be used as various functional liposomes such as a microcapsule substitute or a drug carrier.

[0032]

INDUSTRIAL APPLICABILITY As described above, according to the present invention, various functional substances can be supported on the surface of a liposome or in a membrane easily and efficiently by covalent bond directly or via a spacer. Moreover, a reactive liposome-forming agent capable of increasing the amount of introduced functional substance can be obtained.

Further, according to the present invention, since the above-mentioned reactive liposome-forming agent is contained as a membrane-forming component, various functional substances can be directly and efficiently attached onto the surface of the liposome or in the membrane. Alternatively, it is possible to obtain a reactive liposome which can be supported by a covalent bond via a spacer and which can increase the introduced amount of the functional substance.

[0034]

EXAMPLES The present invention will now be described in more detail by way of examples, which should not be construed as limiting the invention thereto.

Example 1 Polyoxyethylene oleyl ether [C ₁₈ H ₃₅ O (CH ₂ CH ₂ O) _n H, n = 6] 1 in 10 ml of chloroform
g and an equimolar amount of CDI were dissolved and mixed, and the mixture was mixed at room temperature for 3 times.
After reacting for a time, an activated polyalkylene ether derivative was obtained.

Next, 20 mg of egg yolk phosphatidylcholine
(26 μmol), cholesterol 3.9 mg (10 μm
mol) and 20 wt% of the polyalkylene glycol derivative obtained above were placed in an eggplant-shaped flask and dissolved in 2 ml of chloroform, and then the chloroform was distilled off under reduced pressure by a rotary evaporator, followed by vacuum drying for 12 hours. Let By adding 1 ml of physiological saline to this and irradiating with Votex and Bath type ultrasonic waves, multilamellar liposomes could be obtained.

Examples 2 to 13 In Example 1, the following various polyalkylene glycols were used in place of polyoxyethylene oleyl ether as a raw material for the polyalkylene glycol derivative.
Further, by changing the mixing ratio of the polyalkylene glycol derivative in the total lipids variously and performing the same operation as in Example 1, a multilamellar liposome could be obtained. The types and mixing ratios of the polyalkylene glycol derivative used in each example are shown below.

Example 2: C ₁₈ H ₃₅ O (CH ₂ CH ₂ O) _n
H n = 30 2wt% Example _{3: C 16 H 33 O (} CH 2 CH 2 O) n H n = 8 10wt% Example _{4: C 16 H 33 O (} CH 2 CH 2 O) n H n = 13 10 wt% Example 5: C ₁₂ H ₂₅ O (CH ₂ CH ₂ O) _n H n = 42 2 wt% Example 6: C ₁₁ H ₂₃ COO (CH ₂ CH ₂ O) _n H n = 42 2 wt% Example 7: C ₁₇ H ₃₅ COO (CH ₂ CH ₂ O) _n H n = 10 30 wt% Example 8: C ₁₇ H ₃₃ COO (CH ₂ CH ₂ O) _n H n = 6 10 wt% Example 9: C ₉ H ₁₉ CON [(CH ₂ CH ₂ O) _n H] ₂ n = ₂₁ 1 wt% Example 10: C ₁₈ H ₃₇ N [(CH ₂ CH ₂ O) _n H] ₂ n = 10 10 wt%

[0039]

[Chemical 6]

Example 15 1,2-di (2,4-octadecadienoyl) -3-phosphatidylcholine 20.3 mg (26 μmol, cholesterol 3.9 mg (10 μmol) and 10 wt% of them in the following formula The resulting polyalkylene glycol derivative was placed in an eggplant-shaped flask, dissolved in 2 ml of chloroform, the chloroform was distilled off under reduced pressure by a rotary evaporator, and further vacuum dried for 12 hours.C ₁₇ H ₃₅ COO (CH ₂ CH ₂ O ) _N H n = 20

To this, 1 ml of physiological saline was added, and Vot was added.
Multilayer liposomes could be obtained by irradiation with ultrasonic waves of ex and Bath types. This was irradiated with 0.75 Mrad of γ-ray to obtain a polymerized liposome. This liposome was subjected to gel filtration using Sephadex G-50,
Freeze-drying gave a powdery sample. Furthermore, it could be regenerated by swelling with physiological saline.

Example 16 0.5 ml of the liposome solution (solid content 2.5% by weight) obtained in Example 1 and 1 mg / ml horseradish peroxidase (hereinafter abbreviated as HRP) /0.1 M phosphoric acid The buffer solution (pH 7.5) was stirred at 4 ° C. for 24 hours.
This was subjected to gel filtration using Sephadex G-50 to collect a liposome-containing fraction. To this, 10 μl of 0.07% H ₂ O ₂ aqueous solution which is a substrate of HRP and 1,
2-Phenylenediamine solution (10 mmol / l) 10
When 0 μl was added and incubated at 30 ° C. for 60 minutes, yellow coloration was observed.

Comparative Example 1 Egg yolk phosphatidylcholine 20 mg (26 μmol),
Multilayer liposomes having a solid content of 2.5% by weight were obtained by the same operation as in Example 1 using only 3.9 mg (10 μmol) of cholesterol. This was treated with HRP in the same manner as in Example 16 and purified by gel filtration to give 0.07
% H ₂ O ₂ aqueous solution 10 μl and 1,2-phenylenediamine solution (10 mmol / l) 100 μl were added,
Incubation at 60 ° C. for 60 minutes showed no color development.

From the results of Examples and Comparative Examples, it was confirmed that the reactive liposome of the present invention can immobilize a functional substance such as HRP easily and efficiently.

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[Procedure amendment]

[Submission date] November 27, 1992

[Procedure Amendment 1]

[Document name to be amended] Statement

[Correction target item name] 0018

[Correction method] Change

[Correction content]

The reactive liposome-forming agent of the present invention is high in various functional substances, particularly those having an amino group , a hydroxyl group or a thiol group , and especially those having a primary amino group. It has reactivity. Therefore, these functional substances are easily covalently bonded by a urethane bond or a carbonate bond. The binding between the reactive liposome-forming agent and the functional substance can be performed directly or via a spacer made of polyalkylene glycol or the like.

[Procedure Amendment 2]

[Document name to be amended] Statement

[Correction target item name] 0019

[Correction method] Change

[Correction content]

Examples of the functional substance include labeling substances such as dyes, dyes, radiation labeling compounds, fluorescent compounds and chemiluminescent compounds; external stimulus responsiveness such as photoresponsive compounds, pH responsive compounds and thermoresponsive compounds. Compounds; enzymes, antibodies,
Other proteins, physiologically active substances such as sugars, lipids, glycoproteins, glycolipids, hormones, and the like; among drugs and the like, those having an amino group , a hydroxyl group or a thiol group in the molecule can be mentioned.

[Procedure 3]

[Document name to be amended] Statement

[Name of item to be corrected] 0029

[Correction method] Change

[Correction content]

When a polymerizable component such as the above-mentioned polymerizable phospholipid is used as another membrane-forming component, a polymerizable reactive liposome can be obtained. The polymerization of the reactive liposome, after liposome preparation, UV in the presence or absence of a photopolymerization initiator, radiation, light irradiation such as an electron beam, or by a redox initiator systems or organic,
It can be easily carried out by heating in the presence of a polymerization initiator such as a peroxide .

[Procedure amendment 4]

[Document name to be amended] Statement

[Correction target item name] 0035

[Correction method] Change

[Correction content]

Example 1 Polyoxyethylene oleyl ether [C ₁₈ H ₃₅ O (CH ₂ CH ₂ O) _n H, n = 6] 1 in 10 ml of chloroform
g and an equimolar amount of CDI were dissolved and mixed, and the mixture was mixed at room temperature for 3 times.
After reacting for a time, an activated polyalkylene glycol derivative was obtained.

[Procedure Amendment 5]

[Document name to be amended] Statement

[Correction target item name] 0036

[Correction method] Change

[Correction content]

Next, 20 mg of egg yolk phosphatidylcholine
(26 μmol), cholesterol 3.9 mg (10 μm
mol) and 20 wt% of the polyalkylene glycol derivative obtained above were placed in an eggplant-shaped flask and dissolved in 2 ml of chloroform, and then the chloroform was distilled off under reduced pressure by a rotary evaporator, followed by vacuum drying for 12 hours. Let By adding 1 ml of physiological saline to this and irradiating with Vortex and Bath type ultrasonic waves, multilamellar liposomes could be obtained.

[Procedure correction 6]

[Document name to be amended] Statement

[Correction target item name] 0041

[Correction method] Change

[Correction content]

1 ml of physiological saline was added to this, and Vor
Multilayer liposomes could be obtained by tex and Bath type ultrasonic irradiation. To this, 0.75Mrad
Was irradiated with γ-ray to obtain a polymerized liposome. The liposome was subjected to gel filtration using Sephadex G-50 and then freeze-dried to obtain a powdery sample. Furthermore, it could be regenerated by swelling with physiological saline.

Claims (2)

[Claims] 1. A reactive liposome forming agent comprising a polyalkylene glycol derivative represented by the following general formula [1]. [Chemical 1] 2. A reactive liposome, which comprises, as a film-forming component, at least one reactive liposome-forming agent comprising a polyalkylene glycol derivative represented by the following general formula [1]. [Chemical 2]