JPS6396560A - Immobilizing method - Google Patents

Abstract

PURPOSE:To immobilize an immunonogically active material such as antigen or antibody or physiologically active material such as hapten or drug to liposome by preliminarily incorporating an amphiphilic compd. into the liposome. CONSTITUTION:The amphiphilic compd. of about 50,000-30,000mol.wt. is preliminarily incorporated into the liposome at the time of preparing the liposome and the immunonogically active material, physiologically active material, etc., are bonded to the liposome via said compd. These materials are then immobilized in the position adequately apart from the liposome surface and, therefore, the damaging of the film surface of the liposome by the immobilization is prevented. In addition, the stable and efficient immobilization is exedutable. Polysaccharides such as lipopolysaccharide and natural polypeptide having a hydrophobic property, etc., are used as the amphiphilic compd.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention is applicable to r-lag delivery of pharmaceuticals, clinical testing,
This invention relates to a method for immobilizing physiologically active substances, immunologically active substances, etc. in liposomes 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 (1) to (1) are known.

(1) By introducing a hydrophobic group into the protein, it has affinity for liposomes, and this is separately prepared for IJ.
, a method for incorporating into dsomes (Bioch imi
ca et Biophysica Acta, 812
．． 116 (1985) etc. ].

(i+) At the time of lipome preparation, a substance having a group that can be chemically modified, such as a glycolipid, is mixed in advance, and the sugar is oxidized with an oxidizing agent to generate a dialdehyde group. Method 1 of binding siliposomes and proteins by reacting with amino groups to form thick bases: Btochimicaet Biophysi
ca Acta, 640.66 (1981), etc. ]
.

(iii) Similar to (ti), SH during liposome preparation
A method in which a lipid having a functional group that can react with a group is mixed, and a protein separately modified with an SH-forming agent is reacted and incorporated [Journal of Immunologic
al Method, 75.351 (1984);
Biochemical and Biophysics
calResearchCommunication
s, 117.399 (1983): Japanese Patent Publication No. 1983-1
Publication No. 17159, etc. ].

(1■) A method of bonding the functional groups of each liposome and protein with each other using a crosslinking agent or condensing agent (Biochemical and Biophys
ical ResearchCommunication
ns, 89.1114 (1979); Lipo
someTechnology, 155, (19
83), CRC Press et al. ].

However, all of these methods directly react and immobilize near the surface of liposomes using a crosslinking agent, so there is a strong possibility that the liposome membrane structure will be damaged during the reaction, and the liposome matrix Due to steric hindrance, the protein binding rate is not always sufficient and efficiency is low.

[Purpose of the invention]

The present invention has been developed in view of the above-mentioned circumstances, and is capable of producing immune activation without damaging the liposomes containing useful substances (without causing the contents to flow out), and with sufficient binding efficiency and efficiency. The object of the present invention is to provide a method for immobilizing substances, physiologically active substances, etc. onto liposomes.

[Summary of the invention]

In order to achieve the above object, the present invention consists of the following configuration.

"When immobilizing immunologically active substances such as antigens and antibodies, or physiologically active substances such as haptens and drugs, into liposomes, it is necessary to A method for immobilizing an immunologically active substance or a physiologically active substance onto a liposome, which is characterized in that the present inventors immobilize an immunoactive substance or a physiologically active substance in a liposome without causing any damage to the liposome and with a high binding rate. As a result of extensive research into methods for immobilizing physiologically active substances, etc., on liposomes, we found that the molecular weight of 5,000
If ~30,000 amphiphilic compounds are incorporated and an immunologically active substance, physiologically active substance, etc. is bound to the liposome through this, it will inevitably be immobilized at an appropriate distance from the liposome surface. The present invention was completed based on the discovery that stable and efficient immobilization can be achieved with little disturbance on the membrane surface.

Molecular weight incorporated into liposomes: 5,000-30,000
Examples of amphipathic compounds include polysaccharides such as lipopolysaccharide (hereinafter abbreviated as LPS), natural polypeptides having hydrophobic groups, and natural polypeptides having hydrophobic groups introduced therein (for example, Examples include products in which a hydrophobic group is introduced into the amino group or carboxyl group of incyrin), synthetic polypeptides having a hydrophobic group, and hydrophilic polymers with hydrophobicized terminals.

Methods for immobilizing immunologically active substances, physiologically active substances, etc. to liposomes via these amphipathic compounds include methods of activating and reacting the functional groups of amphipathic compounds, monovalent cross-linking agents, divalent cross-linking agents, etc. All methods known per se such as the S method of bonding using a cross-linking agent such as a cross-linking agent, and the method of using a binder can be mentioned.

Constituent materials for the liposome according to the present invention include natural lecithin (e.g., egg yolk lecithin, soybean lecithin, etc.), dipalmitoylphosphatidyl coli 7 (DPPC),
Similistoylphosphatidylcholine (DMPC)
, distearoylphosphatidylcholine (DSPC)
), dioleoylphosphatidylcholine (DOPC)
), one or more phospholipids such as norimyristoylphosphatidylethanolamine (DMPE), dinoglumitoylphosphatidylglycerol (DPPG), and dimyristoylphosphatidic acid (DMPA), or these and cholesterol. All constituent materials that are commonly used in existing liposome production methods, such as mixtures with other liposomes, can be used. In addition, examples of compounds that can be encapsulated in the liposome according to the present invention include:
Substances that can be encapsulated in existing liposomes range from enzymes, genes, nucleic acids, polynucleotides, hormones, immunoglobulins (7mA), etc., to various drugs and antibiotics, as well as dyes, fluorescent substances, luminescent compounds, etc. All can be mentioned.

Methods for preparing liposomes according to the present invention include the conventionally well-known portic swing method, ultrasound method, surfactant method, reversed phase evaporation method (REV method), ethanol injection method,
Ether injection method, pre-vesicle (Pre-Vesjc)
le) method, French press extra-'/'yon (
French Press Extrusion method, Ca2+ fusion method, Annealing
) method, freeze-thaw fusion method, W10/W emulsion method, and recently, S., M., Gruner et al. [Bio
chemistry, 24.2833 (1985)
5table Pluril reported by ':1
Methods such as the amellar vesicle method (5PLV method), and even one giant liposome (G
All methods for preparing liposomes that are known per se, such as a method for preparing liposomes known as "Iant Liposoma", can be mentioned.

Examples of immunologically active substances and physiologically active substances that can be immobilized by the method of the present invention include α-fetoprotein, carcinoembryonic antigen (CEA), and basic fetoprotein (
RFP), pancreatic carcinoembryonic antigen (POA), tumor markers such as streptolysin O (SLO).

Antigenic proteins such as C-reactive protein (CRP), IgA,
IgE.

Antigens such as immunoglobulins such as IgG and IgM, or antibodies against these antigens, as well as hormones such as insulin and human ciliated gonal tropin (ICG), various drugs, hapten antigens, and existing liposomes such as these antibodies. Examples include all immunoactive substances and physiologically active substances that can be immobilized on.

The outline of the method of the present invention will be explained by using LPS as an amphipathic compound and immobilizing IgG as an example.
It will look like below.

That is, first, LPS-IJ bosomes incorporating LPS are prepared in the presence of LPS according to the method described in Japanese Patent Application No. 115405/1983, in which liposomes are prepared by the method known per se.

Next, an oxidizing agent (e.g., , potassium periodate, sodium metaperiodate, etc.) and incubate at room temperature for 1 hour.
When the reaction is stirred and reacted for several hours, the sugar chain moiety of LPS is oxidized and aldehyde P groups are generated. The reaction solution was centrifuged to remove the supernatant, resuspended in an appropriate buffer, and then added to
A ship base is formed by adding a buffer solution of gG and reacting with stirring at room temperature for several hours. Next, a buffer solution of a reducing agent (e.g., sodium borohydride, lithium aluminum hydride, etc.) is added to this, the reaction is stirred at room temperature for one to several hours, and then centrifuged to remove the supernatant. IgG
Conjugated liposomes are obtained in high yield.

Examples are shown below, but the present invention is not limited to these Examples in any way.

〔Example〕

Example 1. Production of IgG-binding liposomes (1) L
Preparation of PS-liposomes 20 mM DPPC-liooform solution 325 fi
20mM cholesterol-chloroform solution 325μl, 7.6mM DPPG-liooform/methanol (9515) solution 185μl6. and LPS
A solution of 2"f (molecular weight approximately 20,000) suspended in 1 ml of chloroform/methanol (1:1) was mixed in a test tube, and the solvent was distilled off using a rotary evaporator. In a desiccator for 3 hours. After drying, add 0.5 TLl each of chloroform and diethyl ether,
Alkaline phosphatase (hereinafter abbreviated as AP).

) solution [manufactured by Sigma 5,000 units 72.5 m/
! 80 μl of a 0.01 M HEPES (N-2-hydroxyethylpidecyl-N'-nitanesulfonic acid) buffer solution, pH 7.4, was added and the mixture was vigorously shaken with a portex mixer. I took this in a water bath (43-4
8°C) and concentrated using a rotary evaporator to remove the organic solvent. 1 ml of OIMHEPES buffer was added and stirred with a portex mixer until uniformly dispersed. This was transferred to a centrifuge tube and centrifuged 5 times for 40 minutes at 4°C and 34,000 rpm to remove free AP.
, 72, containing 5mM NaC7) was added to suspend the mixture and stored at 4°C.

(2) Production of IgG-binding liposomes Add 0.706 mF of sodium metaperiodate dissolved in 1 ml of 0.3 M sodium bicarbonate buffer (pH 8,2) to 1 ml of the liposome suspension obtained in (1). After stirring at room temperature for 1 hour, transfer to a centrifuge tube and store at 4°C.
Centrifugation was performed twice for 40 minutes at 34,000 rpm. Meanwhile, washing was carried out with 0.01M potassium carbonate buffer (PH
9,5,72,5mM NaCt), and finally suspended in the same buffer lrnl. A solution of 1g03m9 dissolved in the same buffer as above was added to this, and after stirring at room temperature for 2 hours, 0.6g of sodium borohydride was added to the solution.
The mixture was dissolved in 100 μl of 1M potassium carbonate buffer, added, and further stirred for 1 hour. Transfer this to a centrifuge tube and keep at 4℃ for 34 minutes.
Centrifugation was repeated 3 times for 40 minutes at 00 Q rpm. During this time, the cleaning is 0. It was carried out using OIM HEPES buffer, and finally suspended in 2 ml of the same buffer and stored at 4°C.

Example 2゜Example 1 by changing the amount of LPS introduced into the liposome, the amount of oxidizing agent Na IO4, and the amount of IgG to be coupled.
．． Three types of IgG-binding liposomes were produced according to the method of .

Amount of bound IgG (measured by Lowry method) and inclusions (A
Table 1 shows the activity retention rate of P). In addition, for the IgG-binding liposomes in the case of 0 in the table, Immunolysis was performed in the presence of complement.
The results of the nolysis are shown in FIG.

Comparative example 1゜Example 2. Example 2 except that LPS in Example 2 was replaced with a low molecular weight glycolipid ganglioside (molecular weight approximately 2,000).
Three types of IgG-binding liposomes were produced in exactly the same manner as described above. The amount of bound IgG and the activity retention rate of the inclusions are also shown in Table 1. Further, FIG. 2 shows the results of Irrnunolysis performed on the IgG-bound liposomes in the case of ■ in the table in the presence of complement.

The AP activity retention rate was calculated by determining the AP inclusion amount before and after rgG binding from 1ysis with a surfactant (Br1j 58).

In addition, FIGS. 1 and 2 show the results when (a) various concentrations of complement were applied to each IgG-binding liposome, and
(b) The substrate p-nitrophenyl phosphate corresponds to the amount of AP released when liposomes are damaged when a 500-fold diluted anti-IgG antibody (antiserum) interacts with various concentrations of complement. 41 of p-nitrophenol produced from
It shows how the absorbance at 0 nm changes with the concentration of complement, and the vertical axis shows the absorbance at 410 nm.
Moreover, the horizontal axis represents the concentration of complement. Further, the solid line - represents the case when only complement was applied, and the broken line -1111 represents the case when antibody and complement were applied.

As is clear from Table 1, IgG can be immobilized by the method of the present invention using LPS as a spacer. Somes contain Ig using gangliosides, which are low molecular weight glycolipids, as spacers.
The method in which G is immobilized (the amount of IgG binding is about twice as high as that of the conventional method (method +D), and the activity retention rate of the encapsulated AP before and after the coupling reaction with IgG is also higher than that of the conventional method (method +D). It is about 30 to 45 inches, while 60 to 8
It is much higher at 0chi.

This shows that liposomes are significantly less damaged in the method of the present invention during the coupling reaction for binding IgG than in the conventional method.

Furthermore, as is clear from FIGS. 1 and 2, in the liposomes (using LPS as a spacer) in which fiIgG was immobilized by the method of the present invention, Immunolys t
In contrast, in liposomes in which IgG was immobilized using ganglioside, which is a low-molecular-weight glycolipid, as a substrate, Immuno 1y s t s was not observed at all.

Example 3. Production of peptide-bound liposomes Liposomes prepared by the REV method (LPS 2 my gold-containing suspension 2
ml of sodium metaperiodate 2.1411g,
Add a solution dissolved in 1 ml of 3 M bicarbonate sodium buffer and stir at room temperature for 1 hour, then transfer to a centrifuge tube.
Centrifugation was performed twice for 40 minutes at 4°C and 34,000 rpm. During that time, washing was performed with 0.01M potassium carbonate buffer and finally suspended in 2 ml of the same buffer.

This is added to the C-terminal peptide r of the β-subunit of hCG (C
Add a solution of TP 118-145) 3 (approximately 1 μmol) dissolved in 1 ml of the same buffer, stir at room temperature for 2 hours, and then dissolve IJium 0.9-9 in 100 μl of the same buffer. The mixture was added and stirred for an additional hour. Transfer this to a centrifuge tube and incubate for 40 min at 4°C and 34,000 rpm.
Centrifugation was repeated 3 times for 3 minutes. During this time, 0 washes,
Performed with OIM HEPES buffer, and finally with the same buffer 2.
ml and stored at 4°C.

The peptide content of the peptide liposome prepared as described above was determined by fluorescence measurement using fluorescamine, and it was found that 9.6 nmol of peptide r was bound. That is, about 1.5 nmo per 1 μmol of lipid.
This means that the vepti r of l is bonded.

〔Effect of the invention〕

As described above, the present invention provides a method for immobilizing immunologically active substances, physiologically active substances, etc. onto liposomes in an extremely efficient manner without causing any damage to liposomes encapsulating useful substances. ■When an antigen or antibody is immobilized by the method of the present invention, it easily reacts with the antibody or antigen to cause Immunization.
lysis occurs, resulting in stable immunity
It can be used as a posome for immunoassay.

(2) Furthermore, when the antigen is immobilized by the method of the present invention, immunization with this Immunolyposome is effective in producing antibodies.

(2) Furthermore, when the antibody is immobilized by the method of the present invention, it can be expected that the inclusions can be introduced into target cells in a sufficiently stable manner in vivo.

It has remarkable effects in the following points.

[Brief explanation of the drawing]

FIG. 1 shows Example 2. Immunolys i in
The results of s are shown. In addition, FIG. 2 shows comparative example 1. The results of Immunolys is shown in FIG. In both Figures 1 and 2, the vertical axis represents the absorbance at 410 nm, and
The horizontal axis shows the concentration of complement, and the solid line - shows the case when only complement acts on the IgG-bound liposome, and the broken line -
-- indicates the case where complement and anti-IgG antibody (antiserum) were made to act on IgG-bound liposomes, respectively. Patent applicant: Wako Pure Chemical Industries, Ltd. No./I! 1 Victim & (*Explanation side prison Fig. 2 body groove L (→Navigation)

Claims (3)

[Claims] (1) When immobilizing immunoactive substances such as antigens and antibodies, or physiologically active substances such as haptens and drugs, into liposomes, the molecular weight of 5,000 to 30,0
1. A method for immobilizing an immunologically active substance or a physiologically active substance in liposomes, which is characterized in that this is carried out via an amphipathic compound of about 0.000. (2) The immobilization method according to claim 1, wherein this is carried out by activating the hydrophilic part of the amphiphilic compound or by using a crosslinking agent or a condensing agent. (3) The amphipathic compound is lipopolysaccharide (LPS), a natural polypeptide with a hydrophobic group, a natural polypeptide into which a hydrophobic group has been introduced, a synthetic polypeptide with a hydrophobic group, or a hydrophobized terminal. The immobilization method according to claim 1 or 2, wherein the immobilization method is a hydrophilic polymer.