JPH07107535B2 - Immobilization method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial field of application] The present invention relates to drug delivery, clinical testing,
The present invention relates to a method for immobilizing physiologically active substances, immunologically active substances and the like on liposomes, which are used in fields such as immunology.

[Conventional technology and its problems]

As methods for immobilizing antigens, antibodies, other proteins, etc. on liposomes, the following methods (i) to (iv) are known so far.

(I) A method in which a hydrophobic group is introduced into a protein so that the protein has an affinity for the liposome and is incorporated into a separately prepared liposome [Biochimica et Biophysica Act
a, 812 , 116 (1985), etc. ].

(Ii) A substance having a group capable of chemical modification, such as a glycolipid, is mixed in advance at the time of liposome preparation, and the aldehyde is generated by oxidizing the sugar with an oxidizing agent,
A method for binding a liposome to a protein by reacting this with an amino group of the protein to form a Schiff base [Biochimica et Biophysica Acta. 640 , 66 (1981)
etc. ].

(Iii) As in (ii), a lipid having a functional group capable of reacting with the SH group is mixed at the time of liposome preparation, and this is separately added with SH.
A method of reacting and incorporating a protein modified with an agent [Jour
nal of Immunorogical Method, 75 , 351 (1984); Biochem
ical and Biophysical Research Communications, 117 , 3
99 (1983); JP-A-60-117159. ].

(Iv) A method in which the functional groups of the liposomes and proteins are bound to each other using a cross-linking agent or a condensing agent, etc. [Biochemical and Biophysical Research Communicati
ons, 89 , 1114 (1979); Liposome Technology, 155, (198
3), CRC Press, etc. ].

However, all of these methods are methods of directly reacting and immobilizing near the surface of the liposome by using a cross-linking agent and the like, so that the membrane structure of the liposome is highly likely to be damaged during the reaction, and the liposome matrix Due to steric hindrance, the protein binding rate is not always sufficient and the efficiency is poor.

[Object of the Invention]

The present invention has been made in view of the above circumstances, and it is an immunologically active substance that does not cause any damage to liposomes containing a useful substance (without causing the inclusion substance to flow out), has a sufficient binding rate, and is efficient. Another object of the present invention is to provide a method for immobilizing a physiologically active substance or the like on a liposome.

[Outline of Invention]

 In order to achieve the above object, the present invention has the following constitution.

“To immobilize immunologically active substances such as antigens and antibodies and physiologically active substances such as haptens and drugs on liposomes, this should be done through amphipathic compounds with a molecular weight of about 5,000 to 30,000 previously introduced into liposomes. A method for immobilizing an immunologically active substance or a physiologically active substance on a liposome, which is characterized by the following: "That is, the present inventors did not damage the liposome at all and had a good binding rate to the immunologically active substance, the physiologically active substance, etc. As a result of extensive studies on a method for immobilizing liposomes on liposomes, the molecular weight of 5,000 to 30,00 was previously prepared at the time of liposome preparation.
If an amphipathic compound of about 0 is incorporated and an immunologically active substance or a physiologically active substance is bound via this, it will inevitably be immobilized at a position appropriately separated from the liposome surface. The present invention has been completed by finding that there is little disturbance and that it can be immobilized stably and efficiently.

Examples of the amphipathic compound having a molecular weight of about 5,000 to 30,000 to be incorporated in the liposome include, for example, a polysaccharide such as lipopolysaccharide (hereinafter abbreviated as LPS), a natural polypeptide having a hydrophobic group, or a natural group introduced with a hydrophobic group. (For example, one in which a hydrophobic group is introduced into the amino group or carboxyl group of insulin), a synthetic polypeptide having a hydrophobic group, a hydrophilic polymer having a hydrophobic terminal, and the like.

As a method for immobilizing an immunologically active substance, a physiologically active substance or the like on a liposome through these amphipathic compounds, a method of activating and reacting a functional group of an amphipathic compound, a monovalent crosslinking agent, a divalent All known methods such as a method of binding using a crosslinking agent such as a crosslinking agent and a method of using a binding agent can be mentioned.

Examples of the constituent material of the liposome according to the present invention include natural lecithin (eg, yolk lecithin, soybean lecithin, etc.), dipalmitoylphosphatidylcholine (DPPC), dimyristoylphosphatidylcholine (DMPC), distearoylphosphatidylcholine. (DSPC), Dioleoylphosphatidylcholine (DOPC), Dimyristoylphosphatidylethanolamine (DMPE), Dipalmitoylphosphatidylglycerol (DPPG), Dimyristoylphosphatidic acid (DMPA) One or more, or a mixed system of these and cholesterols,
All the constituent materials usually used in the existing liposome production methods can be mentioned as usable ones. Examples of compounds that can be encapsulated by the liposome according to the present invention include, for example, enzymes, genes, nucleic acids, polynucleotides, hormones, immunoglobulins, various drugs and antibiotics, and further dyes, fluorescent substances, and luminescence. All the substances that can be encapsulated in existing liposomes are listed up to the compound.

Examples of the method for preparing the liposome according to the present invention include well-known vortexing method, ultrasonic method, surfactant method, reverse phase evaporation method (REV method), ethanol injection method, ether injection method, pre-preparation method. Pre-Vesicle method, French Press Extru
sion method, Ca ²⁺ fusion method, Annealing
Method, freeze-thaw fusion method, W / O / W emulsion method, etc., and recently SMGruner et al. [Biochemistry, 24 , 2833 (198
5)] Reported by Stable Plurilamellar vesicle
Methods (SPLV method) and the like, as well as methods of preparing liposomes known per se, such as methods of preparing liposomes called "giant liposomes" having a large encapsulation amount, can be mentioned.

Examples of the immunologically active substance or physiologically active substance that can be immobilized by the method of the present invention include α-fetoprotein, carcinoembryonic antigen (CEA), and basic fetoprotein (BF
P), tumor markers such as fetal pancreatic cancer embryonic antigen (POA), antigen proteins such as streptolysin O (SLO), C-reactive protein (CRP), immunoglobulins such as IgA, IgE, IgG, IgM, etc. Antigens or antibodies against these antigens, further insulin, hormones such as human ciliated gonadotropin (hCG) and various drugs, hapten antigens or immunologically active substances that can be immobilized on existing liposomes such as these antibodies, physiologically active substances All can be mentioned.

The outline of the method of the present invention will be described below by taking LPS as an amphipathic compound and immobilizing IgG as an example.

That is, first, LPS-liposomes incorporating LPS are prepared according to the method described in Japanese Patent Application No. 61-115405, in which liposomes are prepared by a method known per se in the presence of LPS.

Next, this suspension of LPS-liposomes [usually used by dispersing in a buffer such as Good's buffer (HEPES, PIPES, MES, etc.), Tris buffer, carbonate buffer, etc.] ] To a buffer solution of an oxidizing agent (for example, potassium periodate, sodium metaperiodate, etc.), and stirring reaction is performed at room temperature for 1 to several hours.
The sugar chain portion of LPS is oxidized to generate an aldehyde group.
The reaction solution is centrifuged to remove the supernatant, resuspended in an appropriate buffer solution, and then a buffer solution of IgG is added thereto and the mixture is stirred at room temperature for several hours to form a Schiff base. Then, a reducing agent (for example, sodium borohydride,
A buffer solution of lithium aluminum hydride or the like) is added, the mixture is stirred at room temperature for 1 to several hours, and then centrifuged to remove the supernatant, whereby IgG-bound liposomes can be obtained in high yield.

Examples will be shown below, but the present invention is not limited to these examples.

〔Example〕

Example 1. Production of IgG-bound liposomes (1) Preparation of LPS-liposomes 20 mM DPPC-chloroform solution 325 µ, 20 mM cholesterol-chloroform solution 325 µ, 7.6 mM DPPG-chloroform / methanol (95/5) solution 85 µ, and LPS (molecular weight Approximately 20,000) 2 mg chloroform / methanol (1: 1)
The suspension was suspended in 1 ml, and the solution was put into a test tube and mixed, and the solvent was distilled off with a rotary evaporator. After drying in a desiccator for 3 hours, 0.5 ml each of chloroform and diethyl ether were added, and an alkaline phosphatase (hereinafter abbreviated as AP) solution [5,000 unit / manufactured by Sigma].
2.5 ml of 0.01 M, HEPES (N-2-hydroxyethylpiperazine-N'-ethanesulfonic acid) buffer solution pH 7.4] 80
μ was added and shaken vigorously with a vortex mixer. This was concentrated in a water bath (43-48 ° C) with a rotary evaporator to remove the organic solvent, and 0.01M HEPES buffer 1
ml was added and the mixture was stirred with a vortex mixer until uniformly dispersed. Transfer this to a centrifuge tube, and 40 at 34,000 rpm at 4 ℃.
After centrifugation was repeated 5 times for 5 minutes to remove free AP, 0.3M sodium bicarbonate buffer (pH 8.2, 72.5 mM NaCl was added.
3 ml) was added and suspended and stored at 4 ° C.

(2) Production of IgG-bound liposomes 1 ml of the liposome suspension obtained in (1) was supplemented with 0.706 mg of sodium metaperiodate in 1 ml of 0.3M sodium bicarbonate buffer (pH).
The solution dissolved in 8.2) was added, and the mixture was stirred at room temperature for 1 hour, transferred to a centrifuge tube, and centrifuged at 4 ° C. and 34,000 rpm for 40 minutes × 2 times. During that time, wash with 0.01 M potassium carbonate buffer (pH 9.5, containing 72.5 mM NaCl), and finally with 1 m of this buffer.
suspended in l. To this, add a solution prepared by dissolving 3 mg of IgG in the same buffer as above, stir at room temperature for 2 hours, and then add 0.6 mg of sodium borohydride to 100 μM of 0.01M potassium carbonate buffer.
Was dissolved in and added, and the mixture was further stirred for 1 hour. This was transferred to a centrifuge tube, and the centrifugation was repeated 3 times at 4 ° C. for 40 minutes × 3 times. During this time, wash with 0.01 M HEPES buffer,
Finally, it was suspended in 2 ml of the same buffer and stored at 4 ° C.

Example 2 Three types of IgG-bonded liposomes were produced according to the method of Example 1 by changing the amount of LPS introduced into the liposome, the amount of oxidizing agent NaIO _{4 and the} amount of IgG to be coupled.

Amount of bound IgG (measured by Lowry method) and inclusion (AP)
Table 1 shows the activity retention rate of the. IgG in the case of
The results of immunolysis of the bound liposomes in the presence of complement are shown in FIG.

Comparative Example 1. Three kinds of IgG-bonded liposomes were produced in exactly the same manner as in Example 2 except that LPS in Example 2 was replaced with a low molecular weight glycolipid ganglioside (molecular weight: about 2,000). The amount of bound IgG and the activity retention of the inclusions are also shown in Table 1. Further, FIG. 2 shows the results of Immunolysis in the presence of complement for IgG-bonded liposomes in the case of the table.

The AP activity retention rate was calculated by calculating the amount of AP included before and after IgG binding from lysis with a surfactant (Brij58).

In addition, FIGS. 1 and 2 show (a) various concentrations of complement on each IgG-bound liposome, and
(B) p-nitrophenyl phosphate, which is a substrate corresponding to the amount of AP released by damage to liposomes when 500-fold diluted anti-IgG antibody (antiserum) and various concentrations of complement are allowed to act The absorbance at 410 nm of the p-nitrophenol produced from the product changes with the concentration of complement. The vertical axis represents the absorbance at 410 nm and the horizontal axis represents the concentration of complement. Also, when the solid line-complement only is applied, Indicates the time when the antibody and the complement act on each other.

As is clear from Table 1, the liposome immobilized with IgG by the method of the present invention using LPS as a spacer is a method wherein IgG is immobilized with a ganglioside, which is a low molecular weight glycolipid, as a spacer (the method of the conventional method (ii) above). ) Compared to IgG
The binding amount was about twice as high, and the activity retention rate of the encapsulated AP before and after the coupling reaction with IgG was 30 times that of the latter.
While it is about 45%, it is much higher at 60-80%. This indicates that the liposome suffers much less damage in the coupling reaction for binding IgG in the method of the present invention than in the conventional method.

In addition, as is clear from FIGS. 1 and 2, Immunolysis was clearly observed in the liposome (where LPS was used as a spacer) on which IgG was immobilized by the method of the present invention, whereas ganglioside, which is a low molecular weight glycolipid, was observed. Immunolysis was not observed at all in the liposomes on which IgG was immobilized using the as a spacer.

Example 3 Production of peptide-bonded liposomes A solution of 2.14 mg of sodium metaperiodate in 1 ml of 0.3M sodium bicarbonate buffer was added to 2 ml of a liposome (containing 2 mg of LPS) prepared by the REV method, and the mixture was allowed to stand at room temperature for 1 hour. After stirring for an hour, transfer to a centrifuge tube, and 4 minutes at 34,000 rpm for 40 minutes x 2
Centrifugation was performed. Meanwhile, washing was performed with 0.01 M potassium carbonate buffer, and finally, the suspension was suspended in 2 ml of the same buffer. In addition to this, the C-terminal peptide of the β-subunit of hCG (CTP118
~ 145) A solution of 3 mg (about 1 µmol) in 1 ml of the same buffer was added, and the mixture was stirred at room temperature for 2 hours, then 0.9 mg of sodium borohydride was added to 100 µ of the same buffer, and the mixture was further stirred for 1 hour. Transfer this to a centrifuge tube, and 40 at 34,000 rpm at 4 ℃.
The centrifugation was repeated 3 times for 3 minutes. During this time, wash 0.01
Perform with M HEPES buffer, and finally suspend in 2 ml of the same buffer,
Stored at 4 ° C.

The peptide content of the peptide liposome prepared as described above was determined by a fluorescence measurement method using fluoresamine, and it was found that 9.6 nmol of the peptide was bound. That is, about 1.5 nmol of peptide is bound to 1 μmol of lipid.

〔The invention's effect〕

As described above, the present invention provides a method for immobilizing an immunologically active substance, a physiologically active substance or the like in a liposome extremely efficiently without damaging the liposome encapsulating a useful substance. When the antigen or antibody is immobilized by the method of the present invention, the immunolysi
Since s occurs, it can be used as a stable Immunolyposome for immunoassay.

Further, when the antigen is immobilized by the method of the present invention, immunization with this Immunolyposome is also effective in antibody production.

Furthermore, when the antibody is immobilized by the method of the present invention,
It can be expected that the inclusions can be incorporated into target cells in vivo sufficiently stably.

It has a remarkable effect on the above points.

[Brief description of drawings]

FIG. 1 shows the results of Immunolysis in Example 2.
Further, FIG. 2 shows the results of Immunolysis in Comparative Example 1. In both FIG. 1 and FIG. 2, the vertical axis represents the absorbance at 410 nm, the horizontal axis represents the concentration of complement, and the solid line − represents Ig.
When only complement is allowed to act on the G-bonded liposome, Shows the case where complement and anti-IgG antibody (antiserum) were allowed to act on IgG-bonded liposomes, respectively.

Claims (3)

[Claims] 1. A method for immobilizing an immunologically active substance such as an antigen or an antibody or a physiologically active substance such as a hapten or a drug on a liposome, a molecular weight of 5,000 to 30,000 previously introduced into the liposome.
A method for immobilizing an immunologically active substance or a physiologically active substance on a liposome, which is characterized in that this is performed through a certain degree of amphipathic compound. 2. The immobilization method according to claim 1, which is carried out by activating the hydrophilic part of the amphipathic compound or by using a crosslinking agent or a condensing agent. 3. An amphipathic compound wherein lipopolysaccharide (LPS), a natural polypeptide having a hydrophobic group or a natural polypeptide having a hydrophobic group introduced therein, a synthetic polypeptide having a hydrophobic group, or a hydrophobic end group. The immobilization method according to claim 1 or 2, wherein the immobilization method is a hydrophilic polymer.