JP2001181213A - Vesicle having target-directing property and method for preparing the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a vesicle having a target-directing property by binding a specific lectin having an ability for selectively recognizing a high mannose type sugar chain to a vesicle. SOLUTION: This vesicle having the target-directing property, characterized by immobilizing one lectin originating from a plant belonging to the Eucheuma, selected from the group consisting of a lectin originating from Eucheuma J. Agandh, a lectin originating from Eucheuma cottoni and a lectin originating from Eucheuma amakusaensis on the surface of a vesicle. The vesicle is produced from a mixture system of Span 80 with Tween 80. The obtained lectin- immobilized vesicle preferably has a particle diameter of 1 to 1,000 nm.

Description

DETAILED DESCRIPTION OF THE INVENTION

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to the effective use of artificial cells (vesicles) having a lipid bilayer membrane structure, and more particularly to a vesicle having target directivity and a method for preparing the same.

[Prior art]

Hitherto, studies have been made using various phospholipids, sterols, and surfactants to produce highly stable vesicles. It has been studied that these vesicles contain various active substances therein and are used for external preparations for skin, drug transport into and out of the body, gene transfer to specific cells, and the like. Various drugs such as anticancer drugs and various genes for gene transfer can be considered as drugs to be put into the vesicle.

However, vesicles are merely lipid bilayer membranes, and as they are, they have very weak binding properties, such as binding due to a difference in electric charge, and randomly adhere to other substances or cells. Even if it contains the active ingredient of
It is impossible to collect vesicles at a specific part such as an injured part or a cancer cell, only by expecting that the drug effect is prolonged by encapsulation.

[0003]

Therefore, in order to impart vesicles with target directivity, studies have been made on incorporating a recognition substance component of a specific substance such as an antibody into the membrane surface.

[0004] However, depending on the recognition substance component, it is necessary to control the posture on the film surface so that the recognition site of the recognition substance component is directed to the outside of the film. I have.

In view of the above problems, the present inventors have conducted intensive studies, and as a result, obtained a finding that a specific lectin has the ability to selectively recognize a high-mannose-type sugar chain very strictly. The present invention has been completed.

[0006]

Thus, according to the invention of claim 1 of the present application, there is provided "a vesicle having target directivity in which a lectin derived from the genus Kirinsai is immobilized on the surface of a vesicle". .

[0007] In the present invention, the lectins derived from the genus Kirinsai include, as described in claim 2 of the present application, "lectins derived from Togakirinsai (ESA), lectins derived from Eucuma-Cottney (ECA), and lectins derived from Akumasakirinsai. (EAA)].

[0008] Of these, as shown in claim 3 of the present application, the lectin (ESA) derived from Rhododendron is most preferred in that it has an excellent ability to selectively recognize high-mannose-type sugar chains. . Therefore, ES in the vesicle
By immobilizing A, vesicles can be given specific adsorption ability to some cancer cells or the like in which the content of high-mannose-type sugar chains increases.

The lectin used in the present invention is prepared by extracting seaweed as a raw material with an extraction solvent selected from a buffer solution and a solvent-water mixture, and separating and purifying the extract by solvent precipitation and gel filtration. And ion exchange chromatography. For details, refer to the description of the embodiment described later.

The vesicle to which the lectin is immobilized can be synthesized by using component materials and methods known in the art.
using a mixed system of an80 and Tween80]
It is preferable because lectin can be efficiently fixed to the lipid membrane surface of the vesicle.

The particle size of the vesicle in the present invention is slightly different depending on the preparation method. For example, when a two-stage emulsification method is used, a relatively large particle having a particle size of 1 to 10 μm as described in claim 5 of the present application is obtained. If you use the law, this application "Claim 6"
Are obtained having a small particle size of 1 to 1000 nm.
From the viewpoint of the binding of vesicles to cells, the latter small particle size is preferable as described in claim 6 of the present application, but is not particularly limited thereto.

The present invention can also provide a method for preparing a vesicle having a target directivity, as described in the present application "claim 7" or "claim 8."

According to claim 7 of the present application, "Lectin derived from Kirinsai genus labeled with a fluorescent probe and Tween
A vesicle having a target directivity characterized by obtaining a vesicle having the lectin immobilized on its surface by mixing an En80 solution, adding Span80 thereto, and irradiating the resulting mixed system with ultrasonic waves. A method for the preparation of

In the above-mentioned preparation method, the step of removing the solvent required for the preparation of vesicles by the conventional ultrasonic method with an evaporator becomes unnecessary.

According to claim 8 of the present application, "Span
80 solution was first emulsified, and a lectin derived from Kirinsai genus labeled with a fluorescent probe and Tween
A method for preparing a vesicle having target directivity, characterized in that a vesicle having the lectin immobilized on the surface thereof is obtained by adding and mixing a mixed solution with an en80 solution to perform secondary emulsification.

Although the above-mentioned preparation method utilizes what is called a two-stage emulsification method, ultrasonic waves may be applied in the above-mentioned secondary emulsification stage.

In both of the above two preparation methods, the lectin is labeled with a fluorescent probe prior to immobilization on the vesicle, but this treatment increases the surface hydrophobicity of the lectin, thereby increasing the lipid membrane of the vesicle. This is preferable in that the rate of immobilization to the substrate can be increased.

[0018]

According to the invention of claim 1 of the present application, a lectin derived from the genus Kirinsai is fixed on the lipid membrane surface of the vesicle, and the vesicle does not react with a substance to which this lectin can specifically adsorb. Binding, resulting in imparting target directivity to the vesicles.

According to the invention of claim 2 or claim 9 of the present application, the lectin is lectin (ESA) derived from Togekirin rhinoceros, lectin derived from Eucuma cotney (ECA), and lectin derived from Akumasakirinsai ( EAA), the vesicles bind to each substance to which each of these lectins can be specifically adsorbed.

According to the invention of claim 3 or claim 10 of the present application, since the lectin is a lectin (ESA) derived from Rhododendron, this ESA has a very strict sugar chain recognition ability. Since the high-mannose-type sugar chain is selectively recognized, the vesicle has a selective binding ability to a substance having a high-mannose-type sugar chain depending on the presence or absence and the content of the sugar chain. In addition, since this lectin has two or more sugar chain binding sites in one molecule, there is no particular need for posture control when immobilized on the vesicle membrane surface, and it has high mannose type sugar chains. Very specific for the substance.

According to the invention of claim 4 of the present application, since the vesicle is synthesized from a mixed system of Span80 and Tween80, the water-soluble Tween80 is replaced with the oil-based Speen.
It functions well as an intermediary between an80 and the lectin, and the lectin can be immobilized on the obtained vesicles well.

According to the invention of claim 5 of the present application, the vesicles have a relatively large particle size of about 1 to 10 μm, and are easy to handle, and the immobilization ratio is also increased. Further, according to the invention of claim 6 of the present application, the vesicles have a small particle diameter of about 1 to 1000 nm, and the ability of the vesicles to bind to the target substance is enhanced.

According to the invention of claim 7 of the present application, lectin is labeled with a fluorescent probe to increase hydrophobicity,
This is dispersed and dissolved in a Tween 80 solution, and ultrasonic waves are applied to the Tween 80 solution with oily Span 80 added. Thus, the lectin is immobilized on the vesicle lipid membrane simultaneously with the production of small-sized vesicles. Will be performed.

According to the invention of claim 8 of the present application, the lectin is labeled with a fluorescent probe to increase the surface hydrophobicity. In this state, the lectin is dispersed and dissolved in Tween 80, and oily Span 80 is added and mixed. Then, the lectin is immobilized on the surface of the lipid membrane at the same time as the formation of vesicles having a relatively large particle size.

[0025]

EXAMPLES The present invention will be described below in detail with reference to examples, but the present invention is not limited by these examples.

[Example 1] (1) Preparation of lectin (ESA) derived from Rhododendron

First, the collected bark beetle was thoroughly washed with seawater, freeze-dried, and pulverized with a pulverizer to obtain a seaweed powder.

Next, 1000 ml of a 20% aqueous ethanol solution was added to 100 g of the above seaweed powder, and the mixture was extracted with stirring overnight in a refrigerator to obtain a seaweed extract. The supernatant and the extraction residue were separated by centrifugation, and the supernatant was collected. The same amount of a 20% aqueous ethanol solution was again added to the extraction residue to perform secondary extraction, and the supernatant was recovered in the same manner. As a result, 840 ml of the primary extract and 900 ml of the secondary extract were obtained.

Cold ethanol is added to the above extract at a final concentration of 83%.
The lectin-containing protein was precipitated as a precipitate. The precipitate was collected by centrifugation, suspended in a 50 mM phosphate buffer (pH 7.2), and dialyzed against the buffer to remove ethanol to obtain a crude lectin solution.

The crude lectin solution was subjected to gel filtration chromatography (Pharmacia Superdex 75p as a column).
g: packed in an inner diameter of 26 mm, filled length of 60 cm, and buffered with a 50 mM phosphate buffer (pH 7.2) containing 0.1 M NaCl). Using a UV monitor, measure the liquid passing through the gel filtration column at 280 nm.
The protein was measured at 1 minute intervals while measuring the protein amount, and the agglutinating activity of each fraction was monitored using sheep erythrocytes. As a result, the largest peak near the retention time of 60 minutes coincided with the peak of the aggregation activity.

As a result of this gel filtration chromatography, 1
About 800 mg of the primary extract and about 160 mg of the lectin (ESA) derived from Rhododendron were obtained from the secondary extract.

(2) Preparation of Lectin-Immobilized Vesicle The vesicle in this example was prepared by a two-stage emulsification method. That is, 0.52 g of about 4% Span80 solution and 5.2 mg of lecithin are weighed in a 20 ml beaker, hexane is added to such an extent that the stirring section of the mini-mixer is hidden, and the mixture is stirred and dissolved at low rotation to dissolve the phosphate buffered saline ( (PBS) 2.5 ml was primary-emulsified with a homomixer at about 7000 rpm for 5 minutes while dripping with a micropipette.

On the other hand, the ESA prepared above was labeled with a fluorescent probe XRITC, and then placed in a 20 ml beaker.
% Tween 80 solution and 0.21 g of the above labeled E
2 mg of SA was weighed and 17.5 ml of PBS was added to prepare a secondary emulsifier solution.

The hexane was removed from the primary emulsion by a rotary evaporator, and then the above secondary emulsifier solution was added thereto, followed by secondary emulsification at about 2,000 rpm for 5 minutes using a homomixer. Thus, a vesicle suspension was obtained.

The vesicle suspension obtained above was subjected to gel filtration to remove the surfactant in the external solution, thereby obtaining 4 ml of vesicles. Simultaneously with the generation of the vesicles, the ESA was fixed to the lipid membrane surface of the vesicles with high efficiency. E at this time
The immobilization ratio of SA was about 30% of the charged amount. The immobilization rate was measured by the Lowry method after separating ESA not immobilized on the vesicles by gel filtration, breaking the vesicles by SDS, and removing turbidity.

The particle size of the obtained ESA-immobilized vesicles was changed by changing the rotation speed of a homomixer. The particle size was relatively large in the range of about 1 to 10 μm. Was. Hereinafter, the ESA-immobilized vesicle obtained by this method is referred to as a large particle size vesicle (GLV).

The ESA-immobilized vesicle (G
LV) was added to the medium at a concentration of 10% to react with various animal cells cultured in a 10% fetal calf serum (FCS) ERDF medium on a plastic dish and reacted at 37 ° C. for 0.1 hour. After the reaction, the cells were observed using a laser fluorescence microscope (MERIDIAN).

The cell line used was a human colon cancer cell (CO
LO201), human breast cancer cells (MCF7), human breast cells (MCF1
0: treated as normal cells) and human hybridoma (H
B4C5: obtained by electrofusion of human lymphocytes and human tumor cells). The observation of cells was performed using a fluorescence phase-contrast microscope (Olympus, BH-2) at an excitation wavelength of 490 n.
m, at an absorption wavelength of 630 nm. The results are shown in Table 1 below.

[0039]

[Table 1]

As a result, human colorectal cancer cell COLO21, which is considered to contain a large amount of high mannose-type sugar chains, emits the strongest fluorescence, and the human breast cancer cell (MCF7) is a cancer cell.
Fluorescence was also observed for human hybridoma (HB4C5). However, no fluorescence was observed for human mammary gland cells (MCFlO) as normal cells, confirming that ESA-immobilized vesicles (GLV) have extremely high specificity for cancer cells. .

In addition, it is generally difficult to immobilize ESA on the lipid membrane surface of the vesicle because its surface is hydrophilic. However, in this example, as described above, labeling by the fluorescent probe XRITC was used. Hydrophobized by
By mixing with the secondary emulsifier Tween80 used at the time of preparing the vesicle and incorporating the so-called ESA-Tween80 conjugate onto the lipid membrane surface of the vesicle and immobilizing the same, the immobilization rate could be increased as compared with the conventional case.

Example 2 First, the E prepared in Example 1
5 mg of SA was fluorescently labeled with a fluorescent probe FITC.
The method of ESA fluorescent labeling was performed using a FITC labeling kit (Fluoro TagTM FITC conjugation Kit: manufactured by Sigma) according to the manual.

ES labeled with the above fluorescent probe
A4 mg was added to and dissolved in 0.04 g of Tween 80 solution, and 0.1 g of Span 80 solution [that is, Spa
n80: Tween80 = 2: 1].
Add BS and irradiate ultrasonic wave for 3 minutes using a probe type ultrasonic homogenizer (Nippon Seiki, US-150T), and
An A-immobilized vesicle suspension was obtained. The suspension was subjected to gel filtration to remove the surfactant in the external solution, to give an ESA-immobilized vesicle.

The ESA-immobilized vesicle had a particle size of several tens to several hundreds of nm according to electron microscopic observation. Hereafter, this ES
The A-immobilized vesicle is called a small particle size vesicle (SLV).
At this time, the immobilization ratio of ESA was about 6% of the charged amount.

The obtained ESA-immobilized vesicle (SLV)
Was filtered and sterilized with a 0.22 μm filter, and added to the medium at a concentration of 1% to react with various animal cells cultured in a 10% fetal calf serum (FCS) ERDF medium on a plastic dish. For 0.1 hours. After the reaction, the cells were observed using a laser fluorescence microscope (MERIDIAN). Observation of cells was performed using a fluorescence phase contrast microscope (Olympus, BH-
2) was performed at an excitation wavelength of 490 nm and an absorption wavelength of 630 nm.
The results are shown in Table 1 above.

The cell lines used were human colon cancer cells (COLO201) and human breast cancer cells (MC
F7), human mammary gland cells (MCFlO; treated as normal cells) and human hybridomas (HB4C5: obtained by fusing human lymphocytes and human tumor cells by electrofusion).

As a result, human colorectal cancer cells (COLO201), which are considered to contain a large amount of high mannose-type sugar chains, emit the strongest fluorescence, and the fluorescence intensity is the large particle size vesicle obtained in Example 1. Stronger fluorescence intensity than when (GLV) was used was obtained.

Similarly, it was observed that human breast adenocarcinoma cells (MCF7) and human hybridomas (HB4C5), which are cancer cells, emit more intense fluorescence than large particle size vesicles (GLV). However, normal mammary gland cells (MCF
Regarding 10), no fluorescence was observed at all, confirming that SLV has extremely high specificity for cancer cells. Also, it is estimated that the small particle size vesicle (SLV) has a higher bonding force than the large particle size vesicle (GLV).

ESA is more effective than MCFlO as a normal cell.
7. Fluorescence microscopy revealed that COLO201 has a specific binding site for cancer cells. This indicates that ESA has a strong binding ability with high mannose,
It is considered that the cause is that the above-mentioned cancer cells have a larger amount of high mannose on the surface than normal cells.

Example 3 Preparation of ESA-immobilized vesicles was performed using a probe-type ultrasonic homogenizer (Nippon Seiki, US-1).
50T). Span80 and Tween80,
Mix at a ratio of 2: 1 and add PBS with ESA dissolved,
Ultrasonic irradiation was applied for 3 minutes. The obtained ESA-immobilized vesicle suspension was purified by gel filtration. ESA obtained
The particle size of the immobilized vesicle is 10 to 200 nm,
It was LV. The immobilization rate of ESA is about 6% of the charged amount.
%Met.

The ESA-immobilized vesicle was sterile-filtered with a 0.22 μm filter, and then placed on a plastic dish.
Human colon cancer cells (COLO201), human breast cancer cells (MCF7), and fibroblasts (Fibroblast) cultured in 10% fetal calf serum (FCS) ERDF medium in the same manner as in Example 1 [this is a normal cell , And cultured, and the change in cell number was measured. The results are shown in FIGS.
(The culture was performed at 37 ° C. in a CO ₂ incubator. Control was obtained by culturing only cells, vesicle was obtained by adding vesicles to cells, and ES was prepared by ES.
The A-immobilized vesicles are cells obtained by adding ESA-immobilized vesicles to cells and culturing the cells, respectively.
The number of cells on day 3 in 1 was used as a reference. )

As can be seen from these figures, the proliferation of colon cancer cells (COLO201) and mammary gland cancer cells (MCF7) was suppressed, whereas that of normal fibroblasts was suppressed. Did not inhibit proliferation.

Furthermore, both ESA-immobilized vesicles and vesicles suppress the growth of cancer cells, but the former is more frequent. This indicates that immobilization of vesicles on ESA has a greater cancer cell growth inhibitory effect. This may be due to differences in the sugar chain structure on the cell surface or in the cytoskeleton.

[0054]

According to the invention of claim 1 of the present application,
Since the lectin from the genus Kirin is fixed on the lipid membrane surface of the vesicle, the vesicle can be bound to a substance to which this lectin can specifically adsorb,
Vesicles can be provided with target directivity.

According to the invention of claim 2 or claim 9 of the present application, the lectin is a lectin derived from Togekirin rhinoceros (ESA), a lectin derived from Eucuma cotney (ECA) and a lectin derived from Akumasakirinsai ( EAA), the vesicle can be bound to each substance to which each of these lectins can be specifically adsorbed.

According to the invention of claim 3 or claim 10 of the present application, the lectin is a lectin (ESA) derived from Rhododendron oryzae, and this ESA has a very strictly high sugar chain recognition ability. Since a mannose-type sugar chain is selectively recognized, a selective binding ability to a substance having a high mannose-type sugar chain can be imparted to a vesicle depending on the presence or absence and the content of the sugar chain.

ESA has a sugar chain binding site in one molecule.
Since it has two or more, there is no particular need for posture control when immobilizing it on the vesicle membrane surface, and the specificity for a substance having a high mannose type sugar chain is extremely high.

According to the invention of claim 4 of the present application, since vesicles are synthesized from a mixed system of Span80 and Tween80, water-soluble Tween80 is converted to oily Sp
It functions well as a mediator between an80 and the lectin, and can increase the immobilization rate of the lectin to the obtained vesicles.

According to the invention of claim 5 of the present application, the vesicles have a relatively large particle size of about 1 to 10 μm, and can be easily handled, and the immobilization rate can be increased. According to the invention of claim 6 of the present application, the particle size of the vesicle is as small as about 1 to 1000 nm, and the ability of the vesicle to bind to the target substance can be greatly enhanced.

According to the invention of claim 7 or claim 8 of the present application, by labeling lectin with a fluorescent probe, it is possible to impart hydrophobicity to the originally hydrophilic lectin surface. With water-soluble Tween8
Lectin and Tween 8
After formation of a conjugate with 0, the oil-based Span80
The lectin can be smoothly immobilized on the vesicle lipid membrane at the same time as the vesicles are formed by adding vesicles.

Further, according to the invention of claim 7 of the present application, vesicles having a small particle diameter can be generated by irradiating a mixed system to which Span80 is added with ultrasonic waves.

[Brief description of the drawings]

FIG. 1 is a graph showing the growth inhibitory effect of ESA-immobilized vesicles of the present invention on colon cancer cells, together with comparative examples.

FIG. 2 is a graph showing the growth inhibitory effect of ESA-immobilized vesicles of the present invention on mammary adenocarcinoma cells together with comparative examples.

FIG. 3 is a graph showing the effect of ESA-immobilized vesicles of the present invention on human mammary gland cells together with comparative examples.

 ──────────────────────────────────────────────────の Continued on the front page (72) Inventor Takuya Sugawara 3-5-7 Tarumi, Matsuyama-shi, Ehime Pref., Faculty of Agriculture, Ehime University (72) Inventor Akihiro Kawakubo 1698-1, Yoneminato, Iyo City, Ehime Pref. 72) Inventor Hizuo Namioka 728 Mori, Iyo City, Ehime Prefecture F-term in Marine Greens Co., Ltd. (reference) DA80 EA20 FA71 GA05 GA22

Claims (10)

[Claims] 1. A vesicle having target directivity in which a lectin derived from the genus Kirinsai is immobilized on the surface of the vesicle. 2. A lectin derived from Kirinsai sp.
The vesicle having target directivity according to claim 1, which is one selected from the group consisting of a lectin derived from Rhododendron rhinoceros (ESA), a lectin derived from Eucuma-Cottney (ECA), and a lectin derived from Akumasakirin rhinoceros (EAA). 3. The lectin derived from the genus Kirinsai is a lectin (ESA) derived from Rhododendron.
A vesicle having the described targeting. 4. The vesicles are Span80 and Tw.
2. A mixed system of een80.
4. The vesicle having target directivity according to any one of to 3 above. 5. The vesicle has a particle size of 1 to 10 μm.
The vesicle having target directivity according to any one of claims 1 to 4. 6. The vesicle has a particle size of 1 to 1000.
The vesicle having target directivity according to claim 1, wherein the vesicle has a nm. 7. A mixture of a lectin derived from the genus Kirinsai and a Tween80 solution labeled with a fluorescent probe, Span80 is added thereto, and the resulting mixed system is irradiated with ultrasonic waves, whereby the lectin is fixed to the surface. A method for preparing a vesicle having target directivity, characterized in that a vesicle is obtained. 8. The Span80 solution is first emulsified, and a mixed solution of a lectin of the genus Kirinsai and a Tween80 solution, which is labeled with a fluorescent probe, is added and mixed, and the mixture is secondarily emulsified. A method for preparing a vesicle having target directivity, comprising obtaining a vesicle fixed to a vesicle. 9. A lectin derived from Kirinsai sp.
The target directivity according to claim 7 or 8, which is one selected from the group consisting of a lectin derived from Rhododendron rhinoceros (ESA), a lectin derived from Eucuma cotney (ECA), and a lectin derived from Akumasakirin rhinoceros (EAA). A method for preparing vesicles. 10. A lectin derived from Kirinsai sp.
The method for preparing a vesicle having targeting property according to claim 7 or 8, which is a lectin (ESA) derived from Rhododendron rhinoceros.