JP3333869B2 - Liposomal immobilization method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a technique for immobilizing a liposome on a support, and the support on which the liposome is immobilized is used, for example, as a carrier used for chromatography or as a reactant for a bioreactor. .

[0002]

2. Description of the Related Art Conventionally, for fixing liposomes to a gel-like porous material, a so-called freeze-thaw method has been used. In the freeze-thaw method, fine liposomes are formed at a high concentration and mixed with the porous body to introduce a plurality of liposomes into the porous structure of the porous body.
By repeatedly freezing and thawing the mixture,
The liposomes are fixed to the porous body by causing fusion of the liposomes in the porous structure to form large liposomes that do not escape from the porous structure (see FIG. 7). In this method, when the mixture of fat and oil is frozen and then thawed, the fat and oil first begin to melt,
Then, since the water begins to melt, a high-concentration fat / oil dispersion mixture is likely to be generated temporarily, and when the high-concentration fat / oil dispersions flow and integrate with each other, the liposome is formed based on the phenomenon that large fat / oil droplets are likely to be generated. The constituent lipids are integrated into a porous structure to form a large liposome, and fixed using the physical restraining force of the porous structure.

[0003]

SUMMARY OF THE INVENTION The above-mentioned conventional freezing-
According to the melting method, in order to efficiently introduce the liposome into the porous structure, a high-concentration liposome must be used, handling is difficult, and a plurality of liposomes are introduced into one pore. If it is not firmly immobilized, the immobilization rate is low because it is not firmly immobilized.For example, when used as a chromatography carrier, a liposome that can obtain a high immobilization rate due to the fact that a very high separation performance cannot be expected. A fixing method is desired.

Accordingly, an object of the present invention is to provide a method for immobilizing liposomes having a high immobilization rate in view of the above circumstances. It is still another object of the present invention to provide a chromatography carrier, a bioreactor, and the like that can exhibit high performance by using a support on which liposomes are immobilized at a high immobilization rate.

[0005]

The characteristic means of the liposome immobilization method of the present invention for achieving this object will be described with reference to FIG. 1. Referring to FIG. 1, a support G composed of a gel-like porous material is used.
In addition, cyanogen bromide (CNBr), 4-nitrophen
Chloroformate (Cl-CO-O-C 6 H 4 -NO
₂ ), 1,1'carbonyldiimidazole (C ₃ N ₂ H ₃
-CO-C ₃ When N ₂ H ₃₎ less selected from the group consisting of
Also reacts with a support activator containing a compound,
An activation site is formed on the support G, and the activation site is
Biotin B is bound together with vidin A
It was provided liposome R, wherein said avidin A Biot
The liposome R is immobilized on the support G by binding
Is to determine .

FIG. 1 is a conceptual diagram merely used for reference, and the present invention is not limited to the drawings.

[Function and Effect] The support is made of a gel-like porous material.
From, for example, agarose gel, allyl dextran gel
Using a large specific surface area, such as avidin
Can be bonded to a large number of supports.
You. And according to this characteristic means, avidin-biotin
Utilizing binding, the support can be easily added to the liposome.
Can be fixed with a large coupling force. Toes
In immobilizing the liposome to the support, the binding sites involved in various forms of binding, such as avidin-biotin-binding lectin-sugar-binding antigen-antibody binding, should be applied.
That form a lipid bilayer structure of liposomes
It can be used if it is stronger than the quality interaction
In particular, non-covalent avidin-biotin binding
Easy to combine if used, and exhibits the greatest binding force
This is preferred. Moreover, according to this characteristic means,
Avidin, which has the higher molecular weight of
And bind biotin with the smaller molecular weight to liposomes.
To the liposome surface.
It is possible to expect a suitable separation function because it is hard to interfere
Becomes In other words, it is too large compared to the molecular weight of the liposome.
Liposome stability,
Adverse effects on the liposome and the outer surface of the liposome
It is easy to be overturned,
Adsorption is likely to be impeded, making it difficult to perform the separation function.
May cause problems such as
According to the characteristic means, biotin having a smaller molecular weight is
Such a risk arises from binding to the
hard. In addition, the surface of the support is activated as follows
To bind avidin to the activated activation site
Avidin can be easily bound to the support.
Can be. That is, a support activator is added to the support.
By reacting, the surface of the support is activated
And cyanogen bromide (see Chemical formula 1), 4-
Nitrophenyl chloroformate (see Chemical formula 2), 1,
Group consisting of 1'carbonyldiimidazole (see Chemical formula 3)
Support activity containing at least one compound selected from
When the agent reacts with the support, the support becomes hydrophilic.
Sexualizing sites such as cyano groups, ester groups, acyl groups, etc.
Avidin to biotin
It can be easily bonded to parts other than the binding site, and
Vidin can be easily bound, and
The activation site remains even if avidin does not bind,
Because it is hydrophilic like the outer surface of the liposome,
Is unlikely to cause interactions that hinder the approach of
Sounds are hard to occur. In addition, the above activation site depends on various uses.
For example, 1,1 'carbonyl di
Use of imidazole facilitates remaining activation sites
Can be converted to carbamoyl group,
It can be said that it is easy. And, as described above,
Avidin bound to the support is bound to the liposome
Liposomes on the support by binding to biotin
Can be fixed easily and with a large coupling force.
Because

[0008]

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[0009]

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[0010]

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Further, the biotin-bound liposomes
The provision of the over-time, for example, the liposome is integral lipid and to be configured to advance to bind the biotin to lipid capable of constituting the liposome, are coupled the biotin <br/> combinators By forming a liposome from the lipid aggregate by associating the lipid with the lipid , a lipid bilayer is formed in a shape in which the connector is projected, and the lipid bilayer is formed into a spherical shape. Thus, a liposome can be formed. This method is particularly advantageous because, for example, an active reaction site is provided on the surface of the liposome and biotin can be bound after forming the liposome and can be introduced under extremely mild conditions. It is also advantageous in that liposomes can be formed easily.

[0012]

As the avidin, avidin derived from egg white can be used, and avidin derived from microorganisms such as streptavidin derived from Streptomyces avidinii can also be used. The liposome can be usually formed from phospholipids, but can also be formed from glyceroglycolipids or the like.

[0014]

DESCRIPTION OF THE PREFERRED EMBODIMENTS Embodiments of the present invention will be described below with reference to specific examples.

[Embodiment 1] Example 1 in which liposomes of SUVs (small unilamellar vesicles) are immobilized on support G of agarose gel <1> Preparation of support Agarose gel activated by CNBr by the following steps (Pharmacia Biotech Co., Ltd. C
NBr activated Sepharose 4B) (hereinafter simply referred to as Sepharose) egg white avidin and (Pierce Co. avidin) A engaged forming as supports G a.

First, 10 ml of 1 mM dilute hydrochloric acid (hereinafter referred to as this concentration unless otherwise noted) was added to 2 g of freeze-dried Sepharose G to swell to 7 ml of gel.
This gel is filled in a glass filter and 200
After washing with ml of diluted hydrochloric acid, the washing solution is removed by gentle suction. On the other hand, 30 mg of avidin A is dissolved in 8 ml of a coupling buffer (described later) and refrigerated at 4 ° C. The washed gel is further washed with 30 ml of the coupling buffer, dried by suction, poured into the avidin solution, and mixed and reacted. After reacting at room temperature for 2 hours, the reaction mixture is further washed with a coupling buffer, and the filtrate is recovered to determine the amount of avidin not immobilized. The washed reaction mixture is poured into a blocking buffer (described later) to inactivate a portion of Sepharose where the CNBr activation site is not bound to avidin A. It is necessary to avoid such inconvenience that the interaction works and hinders the separation. The resulting gel is again coupled with coupling buffer,
After washing twice with an acetate buffer (described later) sequentially to remove proteins and the like adsorbed on the gel, the gel is obtained by precipitation from a coupling buffer at 4 ° C. This gel has 3.5 mg of avidin bound per 1 ml, and a large number of avidins are bound to the agarose surface to form a support G having a large number of first binding sites (that is, binding sites for biotin). . This support G was prepared by removing the supernatant and replacing it with an equal volume of Tris buffer.
Store at ° C.

○ Coupling buffer: pH 8.1 Sodium hydrogen carbonate 0.1 M Sodium chloride 0.5 M ○ Blocking buffer: pH 8.0 Monoethanolamine 1.0 M ○ Acetate buffer: pH 4.0 Sodium acetate 0.1 M Sodium chloride 0.5 M Acetic acid pH adjustment ○ Tris buffer: pH 7.5 Tris 10 mM EDTA 0.1 mM Sodium azide 1 mM

<2> Preparation of liposome The terminal amino group of biotin B ( binding site for avidin)
Phosphatidylethanolamine having an amide bond (a part other than the second bonding site) (each of the two alkyl groups has 18 carbon atoms and one of them has one double bond) (B-PE) (Equivalent to conjugate-bound lipid )
And phosphatidylcholine (EPC) derived from egg white are dissolved and mixed in chloroform at a molar ratio of 1:50.
The solvent is volatilized and dried to form a film. After dissolving again in ether, the solvent is volatilized, the atmosphere is replaced with nitrogen gas, and then the film is vacuum-dried for 24 hours to obtain a film-like phospholipid. In addition, a Hepes buffer (described later)
When 5 ml is added and dissolved, a suspension of 25 mM MLVs (multilamellar vesicles) is obtained. The suspension is irradiated with an ultrasonic wave using a probe-type ultrasonic wave irradiator.
When performed for 3 minutes at 0 minutes, transparent SUVs can be obtained by centrifugation at 100,000 G for 1 hour. The diameter of these SUVs is
It was approximately 42.9 ± 12.6 nm. As a result, a large number of biotins B are bound to the surface, and a liposome R provided with the second binding site 2 is prepared.

○ Hepes buffer: pH 7.5 HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) 10 mM Sodium chloride 150 mM EDTA 0.1 mM

<3> Immobilization If the support and the liposome obtained in the above step are simply mixed (about 1 to 2 hours under stirring conditions), the first binding site of avidin A provided on the support (agarose gel) G Since the second binding site 2 of biotin B provided in the liposome R is firmly bound to the liposome 1, the liposome R is fixed to the support G through the binding between the first binding site and the second binding site. (See FIGS. 1 and 2 ). At this time, the mixing ratio of the liposome R to the support G is variously changed, and the liposome R
Examination of the immobilization rate was as shown in FIG. As a result, it was found that an immobilization rate of nearly 4 times was obtained with the same amount of liposome as the conventional one. The immobilization rate was determined from the amount of liposome R recovered after mixing the liposome R with the support G, and the amount of the reduced liposome R was determined as the ratio of the reduced amount of the liposome R to the amount of the liposome R used. .

[Embodiment 2] Example 2 in which SUVs liposomes are immobilized on a support of agarose gel Immobilization is similarly performed by replacing avidin A bound to the support G with streptavidin (Streptavidin manufactured by Pierce). was determined the rate, it was as shown in Figure 4. Again, it can be seen that a high immobilization rate was obtained.

[Embodiment 3] Example of immobilizing SUVs liposomes on support G of allyldextran gel Allyxdextran gel (Sephacryl S-100 manufactured by Pharmacia Biotech Co., Ltd.) is used instead of the above support.
0) (hereinafter simply was determined the immobilization rate of the liposomes using called Sephacryl) was as shown in FIG.
The preparation of the support is performed as follows.

<4> Preparation of Support Sephacryl G is washed with acetone. When washing, use an aqueous solution of acetone.
Using an acetone whose concentration is gradually increased, finally, washing with pure acetone is performed. 50 ml of Sephacryl G in 20 ml
Transfer to a 0 ml flask and mix with acetone solution (0.3 M) of 4-nitrophenyl chloroformate (Aldrich) as support activator. The mixture is stirred gently with 4-dimethylaminopyridine (0.3
When 24 ml of a 5M) anhydrous acetone solution is dropped and reacted, a yellow suspension is obtained by stirring for about 1 hour.
The obtained reaction mixture is added with sodium hydroxide to the washed mother liquor until acetone and acetone · 2 no longer turn yellow.
-Propanol (1: 1) mixture, 2-propanol.
Washing successively with a water (1: 1) mixture and distilled water to remove residual raw materials allows the preparation of sephacryl activated by 4-nitrophenyl chloroformate. The activated sephacryl can be treated with the same method as the above-mentioned CNBr-activated sepharose to bind avidin. As a result, per 1 ml of support,
4 mg of avidin could be bound.

[Embodiment 4] Example 1 in which liposomes R of LUVs (large unilamellar vesicles) are immobilized on a support G of an allyldextran gel In addition to preparing LUVs, the support prepared in <4> above was used. fixed, it was determined a fixed rate, was as shown in FIG. In addition, preparation of LUVs is performed as follows.

<5> Preparation of LUVs A freeze-thawing method in which a freezing step of freezing MLVs prepared in the above step <2> with liquid nitrogen and a melting step of raising the temperature of the frozen liposomes to 25 ° C. are sequentially performed. Five steps were performed, and then using a high-pressure vesicle extruder (manufactured by Lipex Biomembrane), a polycarbonate porous film having a pore size of about 100 nm was extruded and passed to enlarge the diameter of the liposome. This makes it almost 132 ±
Liposomes with a diameter of 46.1 nm were obtained. In addition, SU
The diameter of both Vs and LUVs was measured by a dynamic light scattering method (DLS-7000 manufactured by Otsuka Electronics Co., Ltd.).

[0026]

EXAMPLES Examples in which the gel in which liposomes are immobilized according to the above-described examples are used for various purposes will be described. <1> The support into which the liposome is incorporated can be used as a carrier for chromatography.
Various substances can be separated. In addition, by incorporating various enzymes, membrane proteins, and the like into the liposome, when used as a carrier for chromatography,
Since the incorporated enzyme, protein and the like show a specific interaction with the substance to be separated, it is possible to easily isolate a substance which has conventionally been difficult to separate. Specifically, for example, when a liposome incorporating an enzyme that recognizes only the D-form of sugar is immobilized on a support and used as a carrier for chromatography, the racemic sugar can be optically resolved. On this occasion,
The immobilization rate of the liposome greatly affects the separation performance, and a higher immobilization rate can form a carrier having a higher separation performance, and is useful for separating a mixture that is more difficult to separate.

<2> The support is similarly used as a bioreactor for producing a substance specifically reacted and produced by the enzyme by immobilizing the liposome with various enzymes incorporated therein. be able to. Specifically, for example, a support holding a liposome incorporating γ-glutamyl transpeptidase can be used to synthesize γ-glutamyl-p-nitroanilide from glycylglycine and p-nitroaniline. It is considered that the use of a support having a high liposome immobilization rate enables high-yield synthesis. Incidentally, the incorporation of enzymes, membrane proteins, etc. into the liposome is carried out by dispersing in an aqueous solution together with oils and fats using a surfactant at the time of liposome formation, and removing the surfactant by dialysis along with the liposome formation. It can be done easily.

[Brief description of the drawings]

FIG. 1 is a conceptual diagram showing a bond for immobilizing a liposome on a support.

FIG. 2 is a conceptual diagram of a liposome immobilized on a support according to the present invention.

FIG. 3 is a diagram showing the liposome immobilization rate of the first embodiment.

FIG. 4 is a diagram showing a liposome immobilization rate according to the second embodiment.

FIG. 5 is a diagram showing the liposome immobilization rate of the third embodiment.

FIG. 6 is a diagram showing the liposome immobilization rate of the fourth embodiment.

FIG. 7 is a conceptual diagram of a liposome fixed to a support by a conventional method.

[Explanation of symbols]

 1 first binding site 2 second binding site R liposome G support

Continuing from the front page (72) Inventor Shuichi Shukuchi 5-9-9 Tokodai, Tsukuba City, Ibaraki Prefecture Stanley Electric Co., Ltd. Tsukuba Research Laboratories (72) Inventor Hideki Toyama 5-9-9 Tokodai, Tsukuba City, Ibaraki Prefecture Stanley Electric Co., Ltd. Tsukuba Research Laboratories (72) Inventor Jun Miyake 1-1-3 Higashi, Tsukuba City, Ibaraki Pref. Institute of Biotechnology, Industrial Technology Institute (72) Inventor Masayuki Hara 1-3-1 Higashi Tsukuba City, Ibaraki Pref. Within the Institute of Biotechnology, Institute of Industrial Science and Technology (72) Inventor Yasuo Asada 1-3-1 Higashi, Tsukuba, Ibaraki Prefecture Examiner, Hiroshi Miyazawa, Institute of Biotechnology, Institute of Industrial Science and Technology (56) References 325570 (JP, A) Table 6-505913 (JP, A) (58) Fields investigated (Int. Cl. ⁷ , DB name) G01N 30/48 C12N 11/00

Claims (3)

(57) [Claims] (1) A support made of a gel-like porous material is provided on a support.
Anogen bromide (CNBr), 4-nitrophenylc
Rorohorumeto (Cl-CO-O-C 6 H 4 -NO 2),
1,1 'carbonyldiimidazole (C ₃ N ₂ H ₃ -CO
At least one member selected from the group consisting of —C ₃ N ₂ H ₃ )
Reacting a support activator containing the compound of
Forming an activation site in the body, and adding avidin to the activation site
A liposome bound and biotin-bound
And bonding the avidin and the biotin to form the support
A liposome immobilization method for immobilizing liposomes on a liposome. 2. A binder-bound fat to which biotin is bound.
The liposome is formed from lipid aggregates containing
The method for immobilizing a liposome according to claim 1, which is performed . 3. A liposome fixing method according to claim 1 or 2, wherein the avidin is streptavidin.