JP2714972B2 - Modifier for phospholipid aggregates, antiaggregation agent for phospholipid vesicles, fusion inhibitor for phospholipid vesicles, and surface immobilizing agent for phospholipid membranes - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a method for utilizing oligosaccharide fatty acid esters having a well-defined structure as phospholipids.

Phospholipids self-assemble to form monolayers, bilayers, multilayers or vesicles composed of these. These are used for the modification of ultra thin films, solid surfaces, hydrogels, carriers and the like. By introducing the oligosaccharide fatty acid ester used in the present invention, various functions can be imparted. For example, if this oligosaccharide fatty acid ester is introduced into a monolayer of phospholipids whose orientation is fixed, not only control of the surface hydration state and hydrophilic gel region, but also control of interaction with biomolecules, cells, etc. are expected. It can be applied to biosensors, cell culture bed modifiers, etc.

[Problems to be solved by conventional technology and invention]

There are many methods for immobilizing a saccharide structure on the surface of a solid phase, but for effective operation in a small amount, a method of immobilizing the sugar structure on the surface of a solid phase by a covalent bond is known. However, this method has problems such as complicated processing and limitation of the surface chemical structure.

On the other hand, in connection with genetic engineering and cell engineering, culture of many cells and cells including Escherichia coli has become important.
Conventionally, various types of culture beds have been developed, such as glass linings, polymer linings, and ceramic linings. However, culture effects have not produced the desired results. The reason for this is mainly due to the lack of hydrophilicity between the surface of the culture bed or culture tank and the cell surface.

In addition, vesicles (liposomes), which are an example of phospholipid aggregates, have been attracting attention as cell models or capsule materials. However, since they are actually unstable, phospholipids alone are suitable for use as microcapsules. Absent.
Therefore, there is an attempt to embed cholesterol and protein to improve the strength of the membrane. In these embedding examples, although the membrane strength is improved, it is necessary to introduce about 20% or more of corsterol. However, when cholesterol is introduced in an amount of 20% or more, characteristics inherent to phospholipid vesicles (eg, gel-liquid crystal phase transition, change in substance permeability to a specific stimulus, etc.) disappear, and further, the preparation method is restricted (eg, This method has been used only for limited use because the number of small-diameter phospholipid vesicles cannot be prepared and organic solvents cannot be used. In addition, these phospholipid vesicles are liable to undergo morphological changes such as fusion and aggregation after preparation, and have problems in stability over time when commercialized and dispersion stability in blood when administered by intravenous injection. As a method of solving these problems, there is a method of adding a charged substance such as phosphatidic acid to the endoplasmic reticulum, but it has not been sufficient. On the other hand, a water-soluble polymer is also used as an additive stabilizer. However, when phospholipid vesicles are used for in vivo administration, many of these substances are dissolved with a high degree of freedom, so that the efficiency of addition for stabilization is poor. Even polymers with adjusted solubility have problems in biocompatibility and metabolism.

[Means for solving the problem]

The present invention seeks to solve the problems found in the prior art described above. That is, the present invention provides the following inventions.

A modifier for phospholipid aggregates comprising an oligosaccharide fatty acid ester having a degree of sugar polymerization represented by an integer of 2 to 30 and a degree of substitution of an acyl group in a range of 1 to 5.

2. The phospholipid according to claim 1, wherein the oligosaccharide fatty acid ester comprises a saturated aliphatic chain or an unsaturated aliphatic chain containing 1 to 4 unsaturated bonds, and an acyl group having 12 to 22 carbon atoms. Modifier for aggregate.

An anticoagulant for phospholipid vesicles comprising an oligosaccharide fatty acid ester having a degree of sugar polymerization represented by an integer of 2 to 3 and a degree of substitution of an acyl group in a range of 1 to 5.

4. The anti-aggregating agent for phospholipid vesicles according to claim 3, wherein the oligosaccharide fatty acid ester has an acyl group comprising a saturated aliphatic chain and having 12 to 22 carbon atoms.

A fusion inhibitor for phospholipid vesicles comprising an oligosaccharide fatty acid ester having a degree of sugar polymerization represented by an integer of 2 to 30 and a degree of substitution of an acyl group in a range of 1 to 5.

6. The fusion inhibitor for phospholipid vesicles according to claim 5, wherein the oligosaccharide fatty acid ester has an acyl group comprising a saturated aliphatic chain and having 12 to 22 carbon atoms.

A surface immobilizing agent for a phospholipid membrane comprising an oligosaccharide fatty acid ester having a sugar polymerization degree represented by an integer of 2 to 30 and having an acyl group substitution degree in a range of 1 to 5. The surface immobilizing agent for a phospholipid membrane according to claim 7, wherein the oligosaccharide fatty acid ester has an acyl group comprising a saturated aliphatic chain and having 12 to 22 carbon atoms.

 The details will be described below.

The method for producing the oligosaccharide fatty acid ester used in the present invention is as follows.

Separation by type and degree of polymerization of oligosaccharides whose sugar polymerization degree is represented by an integer of 2 to 30 is performed by gel filtration with the column kept warm, and by using a differential refractometer for the detector, the purity is improved. Can be done higher.

To confirm the qualitativeness and purity of the fractionated oligosaccharides, use a silica gel thin plate to perform multiple development with a mixed solvent (1-butanol / pyridine / water) and develop sulfuric acid,
Alternatively, it can be carried out by subjecting to a liquid chromatography using an amino column as a stationary phase and a mixed solvent (acetonitrile / water) as a mobile phase.

The oligosaccharide used in the present invention may be a commercially available pure product as long as the structure is clear.

Means for obtaining oligosaccharide fatty acid esters include (1). A method of adding a fatty acid chloride or fatty acid anhydride to the oligosaccharide obtained as described above in the presence of an organic base catalyst of pyridine sugar and a basic polymer catalyst such as vinylpyridine / styrene copolymer,
(2). A method in which an oligosaccharide is dissolved in N, N-dimethylformamide, dimethylsulfoxide or the like, and transesterified with a fatty acid methyl or the like; and (3). Although there is a synthetic method using lipase, any method may be adopted.

After the reaction of (1), (2) or (3) is completed, the solvent is distilled off under reduced pressure, and diethyl ether,
The impurities are dissolved in a non-polar solvent such as hexane, filtered, and the residue is dried to obtain a crude product.

The oligosaccharide fatty acid ester used in the present invention mainly has the polarity, solubility in various solvents, and affinity for various column carriers of the oligosaccharide fatty acid ester, mainly due to the type of the fatty acid, the degree of substitution and the degree of sugar polymerization. Since they differ in gender, they must be separated and sorted to have a single character.

That is, the crude oligosaccharide fatty acid ester is subjected to liquid chromatography using a reversed phase column as a stationary phase and a polar solvent such as water, alcohol, or tetrahydrofuran as a mobile phase. According to this method, the crude product elutes in ascending order of the degree of substitution, so that fractions of each peak can be collected. Fractions were confirmed by Fourier transform nuclear magnetic resonance measurement (hereinafter FT-
Abbreviated as NMR. ).

The combination of the polar solvent and the type of the reversed-phase column differs depending on the type of oligosaccharide fatty acid ester to be separated and fractionated.

Thus, by using a reversed-phase preparative chromatograph, a large amount of oligosaccharide fatty acid ester having a clear structure and high purity can be obtained.

Separation and fractionation of the oligosaccharide fatty acid ester used in the present invention is not limited to the above method, for example, using silica gel as a stationary phase, normal phase liquid chromatography,
A method such as supercritical chromatography or solvent extraction can also be selected.

The qualitative and purity confirmation of the oligosaccharide fatty acid ester obtained as described above is performed by subjecting it to reverse-phase analytical affinity chromatography or by subjecting it to thin-layer chromatography under the same conditions as liquid chromatography. Can be.

The method for preparing the phospholipid vesicle and the phospholipid monolayer in the present invention is a general method which is usually performed, and is not special at all.

Oligosaccharides used in the present invention, glucan,
Fructan, xylan, galactan, mannan, chitin, chitosan series of other sugars in addition to the different saccharides, the degree of polymerization is a polysaccharide represented by an integer of 2 to 30, including a sugar alcohol. I have.

Further, if the fatty acids used in the present invention are exemplified, as straight-chain saturated fatty acids, lauric acid, tridecanoic acid, myristic acid, pentadecanoic acid, palmitic acid, margaric acid,
Examples thereof include stearic acid, nonadecanoic acid, arachinic acid, and behenic acid, and other examples include linear unsaturated fatty acids, branched saturated fatty acids, and branched unsaturated fatty acids known as oleic acid, linoleic acid, and linolenic acid. As long as the oligosaccharide fatty acid ester of the present invention has 12 to 22 carbon atoms, any fatty acid can be used.

The structure of a typical oligosaccharide fatty acid ester produced as a sample in the present invention is The results are summarized in Tables 1 to 8 according to the saccharide structure.

(Operation)

The oligosaccharide fatty acid ester used in the present invention has a very good affinity for phospholipids and is therefore suitable as a phospholipid modifier. Furthermore, the oligosaccharide fatty acid ester fixed to the phospholipid can maintain the sugar structure exposed to the aqueous phase and can be uniformly dispersed on the membrane.

The oligosaccharide fatty acid ester as a modifier of the phospholipid aggregate of the present invention is a compound having an adjusted hydrophilic-hydrophobic balance, and therefore is used for purposes (eg, parentalization, prevention of phospholipid vesicle aggregation, prevention of membrane fusion, (Eg, labeling for recognition by a specific antibody). In any case, the addition of a small amount is sufficiently effective, and does not change the basic physical properties of the phospholipid aggregate.

In addition, the oligosaccharide fatty acid ester of the present invention is used in combination with a polymerizable phospholipid, for example, by coating and polymerizing these on the surface of a culture vessel to convert the vessel surface to a hydrophilic, cell-philic surface. It is optimal for use, and provides a surface on which dramatic cultivation effects not seen before can be observed.

[Preparation of oligosaccharide fatty acid ester]

"Production of oligosaccharide fatty acid ester" To 1 mol of maltopentaose having a degree of polymerization of 5, 7.5 times mole of special grade pyridine and 100 times mole of special grade N, N-dimethylformamide were added and dissolved. To this was added dropwise 2.5 times mol of special grade palmitic chloride (acyl chain length 16) to start the reaction. This reaction was carried out in an anhydrous system and reacted at 50 ° C. for 24 hours with stirring. Pyridine and N, N-dimethylformamide were distilled off from the reaction mixture at 50 ° C. under reduced pressure. Next, washing with hexane was repeated to elute and remove impurities such as palmitic acid, and then collected by filtration. The obtained residue was dried under reduced pressure at 50 ° C to obtain a crude purified product.

This crude product was fractionated by a column (manufactured by Merck, Hiber Column Licrosolve RP-2). As a mobile phase, a 9: 1 mixed solvent of methanol and water was used. A differential refractometer was used for detection. Each peak separated by the column was separated, and the structure was confirmed by FT-NMR.

As a result, as shown in the chromatogram showing the degree of substitution of maltopentaose palmitate in FIG. 1, maltopentaose (degree of substitution 0 (1)), maltopentaose monopalmitate (degree of substitution 1 (2)) ), Maltopentaose dipalmitate (degree of substitution 2 (3)), maltopentaose tripalmitate (degree of substitution 3 (4)) and then maltopentaose palmitate having a degree of substitution of 4 or more (5). It became clear that taking was possible. Utilizing this knowledge, maltopentaose monopalmitate, maltopentaose dipalmitate, and maltopentaose tripalmitate can be separated from the reaction mixture with high accuracy. Separation and fractionation conditions are not limited to those described above.Waters Micro Bonder Sphere C8 and Shiseido Capsule Pack C18 are used as columns, and the mobile phase is tetrahydrofuran, 1-propanol,
Separation can also be performed using a single solvent or a mixed solvent of two or more of 2-propanol, methanol and water.

In FIG. 1, the substitution degree 2 (3) and the substitution degree 3
A plurality of peaks are observed in the portion (4), and all of these peaks are isomers mainly due to differences in substitution positions.

With respect to the synthesis of other oligosaccharide fatty acid esters, 1 to 3 moles of a special grade fatty acid chloride and 2 to 15 moles of a special grade pyridine are added to 1 mole of the oligosaccharide, and the mixture is reacted at 70 ° C. or lower for 12 hours or more. A crude product was obtained in the same manner as described above. These were fractionated in the same manner, and it was confirmed by FT-NMR that each had a structure having the desired degree of substitution.

However, for the synthesis of unsaturated fatty acid esters, this operation was performed under a nitrogen stream. The reaction temperature is 40 ° C
The reaction was carried out under stirring for 24 hours or more, so that the esterification was sufficiently performed. The oligosaccharide unsaturated fatty acid ester obtained in this manner showed an unsaturated degradation of 3% by FT-NMR.
It was confirmed that:

"Modification of phospholipid with oligosaccharide fatty acid ester" [Examples] Example 1. Maltopentaose monopalmitate (Sample No. 1) in which one molecule of palmitic acid was introduced into maltopentaose (pure product) having a degree of polymerization of 5 (sample No. 1) And dipalmitoylphosfatidylcholine (hereinafter abbreviated as DPPC) 10 as a phospholipid.
After dissolving 0 mg in methanol and mixing, a thin film was formed on the inner wall of the test tube with an evaporator. Add 5 ml of pure water and several glass beads, and vortex 50
The mixture was stirred at ℃ to obtain a cloudy liquid. This cloudy liquid was treated with an extruder. Extruder filter pore size
By passing through 5 times each while changing sequentially to 0.4 μm, 0.2 μm, 0.1 μm and 0.05 μm, a white transparent DPPC vesicle dispersion was obtained. The average pore size of the endoplasmic reticulum in this state was 55 nm in diameter as a result of calculation from a negatively stained transmission electron microscope (hereinafter abbreviated as TEM) photograph.

After diluting the ER dispersion to 0.5% by weight, gel filtration (Sepharose CL-4B, manufactured by Pharmacia, 20 mmφ, 300 m
mH) and free-dissolved sugar fatty acid esters were removed from the system, and then the fraction containing the endoplasmic reticulum was colored with sugar by the phenol-sulfuric acid method to determine the rate of introduction of sample No. 1 into the endoplasmic reticulum. . As a result, Sample No. 1 contained about 50% of the additive.
% Was incorporated into the endoplasmic reticulum.

The morphological stability of the dispersion of vesicles passed through a 0.05 μm filter was evaluated by measuring the change in absorbance (turbidity) at 500 nm at room temperature. In the endoplasmic reticulum without oligosaccharide fatty acid ester added (control), the turbidity sharply increases, and the particle size distribution shows that the particle size of the endoplasmic reticulum increases over time. confirmed. In the system in which Sample No. 1 was added at 2 mol% to the lipid, it was revealed that the dispersion state was kept stable and there was no change with time. When the diameters of these endoplasmic reticulum were measured by a TEM photograph, the diameter increased to 74 nm in the control, but no change in the particle diameter was observed in the system into which sample No. 1 was introduced.

70 μl of a phospholipid vesicle dispersion having a composition of sample number 1 / DPPC (1 mol / 50 mol) was sealed in an aluminum differential scanning calorimeter container, and the differential scanning calorimeter measurement was performed. When the gel-liquid crystal transition temperature was observed, an endothermic peak was observed at 41 ° C similar to that of the DPPC vesicles, and no broadening of the peak was observed. This confirmed that the introduction of the oligosaccharide fatty acid ester at about 2 mol% had no effect on the transfer of the membrane. In addition, the introduction of oligosaccharide fatty acid ester into DPPC endoplasmic reticulum was confirmed by confirming that 0.04% by weight of jack bean lectin (hereinafter abbreviated as Con A).
Was also confirmed from the aggregation of the endoplasmic reticulum by the addition of.

Example 2. Sample numbers 2 to 63 listed in Tables 1 to 8 were embedded in DPPC endoplasmic reticulum using the same method as in Example 1 and examined for their ability to prevent aggregation and fusion. The above sample was applied to lipids.
Table 9 shows the results obtained by introducing 5 mol% each and measuring them together with the results of Example 1.

Sample number 2,3,10-14,19-28,31,34,35,37-41,43,46,
49,52,54,56-63 could be embedded simply by adding the powder as it was to the vesicle dispersion and leaving it to stand at 50 ° C for several hours.

Sample Nos. 2, 3, 28, 31, 40, 43, 46, 49, 52, 54 and 56 have no effect of preventing the endoplasmic reticulum when introduced at 1 mol% or less, but sample Nos. 10 to 15, 14 ~ 27,60 Exhibited an inhibitory effect even at 0.2 mol%. With respect to the prevention of fusion, a smaller introduction amount is sufficient.

In the case of oligosaccharides having a low degree of polymerization, for example, oligosaccharides having a degree of polymerization of 2 to 3, when the degree of substitution was 3 or more, the rate of introduction into the endoplasmic reticulum decreased. Therefore, for those having a small degree of polymerization, oligosaccharide fatty acid esters having a degree of substitution of 1 or 2 are suitable for preventing fusion and aggregation of vesicles.

In addition, the introduction of oligosaccharide fatty acid ester into DPPC endoplasmic reticulum was confirmed by adding 0.04% by weight of Con A for sample numbers 2 to 15,28 to 30,46 to 48, and 0.04% by weight for sample numbers 56 and 57. Was added to wheat germ lectin (hereinafter abbreviated as WGA) to confirm the aggregation of endoplasmic reticulum.

Example 3 In the same manner as in Example 2 except that egg yolk lecithin (hereinafter abbreviated as EYL) was used as a phospholipid, sample numbers 1 to 5 were used.
57 was embedded in the endoplasmic reticulum and its effect was evaluated. The effects were almost the same, but differences were observed in the following points.

In Example 2, fusion and agglutination measurements were performed at room temperature, but EYL vesicles were hardly aggregated at room temperature, so measurement was performed at 4 ° C. When EYL is a product of an ethanol solution, in the preparation of the endoplasmic reticulum, if the removal of ethanol is incomplete, the color is developed by the phenol sulfate method. Therefore, as a result of calculating the introduction rate for the system adjusted with sufficient care, Table 1
Almost the same result was obtained. This indicates that the introduction rate and the performance of the oligosaccharide fatty acid ester depend on the structure of the sugar fatty acid ester regardless of the phospholipid structure. The introduction of oligosaccharide fatty acid esters into the endoplasmic reticulum was performed using sample numbers 1 to 15, 28 to 30, 46
In the case of ~ 48, it was confirmed from the aggregation of the endoplasmic reticulum by adding 0.04% by weight of Con A, and in the case of sample numbers 56 and 57, by adding 0.04% by weight of WGA.

Example 4. Sample Nos. 28, 29, and 30 were introduced into an endoplasmic reticulum composed of a polymerizable phospholipid, 1,2-bis (2,4-octadecadienoyl) glycerophosphocholine, and azobisisobutyronitrile was introduced. After the addition of such a radical initiator, polymerization was carried out by heating. The polymerized endoplasmic reticulum obtained here was a copolymer with an oligosaccharide fatty acid ester and stably supported a sugar structure. These are brand names "Triton X-100"
It retained its shape very stably against washing with a surfactant such as sodium chloride and sodium, and an organic solvent such as methanol. Since the endoplasmic reticulum was aggregated by Con A, stable coating of sugar chains was confirmed.

Example 5. Sample Nos. 56 and 57 were prepared according to Example 4 using 1,2-bis (2,4
(Octadecadienoyl) glycerophosphocholine, and after adding a radical initiator of azobisisobutyronitrile sugar, the mixture was heated and polymerized. The polymerized endoplasmic reticulum obtained here stably supports an oligosaccharide fatty acid ester, and is washed with a surfactant such as “Triton X-100” or sodium cholate, and an organic solvent such as methanol. , The shape was maintained extremely stably. Since the endoplasmic reticulum was aggregated by WGA, stable coating of sugar chains was confirmed.

Example 6. Sample numbers 28, 29, and 30 were introduced into a phospholipid monolayer composed of 1,2-bis (2,4-octadecadienoyl) glycerophosphocholine. Furthermore, after adding a radical initiator such as azobisisobutyronitrile and heating and polymerizing, a polymerized phospholipid thin film was obtained. Since these oligosaccharide fatty acid esters are immobilized by covalent bonds, these films are prepared as films whose sugar chains are stably modified on the surface. In such a system, no sugar was eluted in the water. In addition, it was revealed by fluorescence microscopy using Con A labeled with a fluorescent probe that the membrane surface was easily recognized by lectin.

Example 7. Sample numbers 56 and 57 were introduced into a phospholipid monolayer composed of 1,2-bis (2,4-octadecadienoyl) glycerophosphocholine. Furthermore, after adding a radical initiator such as azobisisobutyronitrile and heating and polymerizing, a polymerized phospholipid thin film was obtained. Since these oligosaccharide fatty acid esters are immobilized by covalent bonds, these films are prepared as films whose sugar chains are stably modified on the surface. In such a system, no sugar elutes into the water. In addition, it was revealed by fluorescence microscopy using WGA labeled with a fluorescent probe that the membrane surface was easily recognized by lectin.

Example 8. Sample Nos. 28, 29 and 30 were introduced into vesicles composed of 1-palmitoyl 2- (2,4-octadecadienoyl) glycerophosphocholine and copolymerized with ultraviolet irradiation or a radical initiator. A phospholipid / oligosaccharide fatty acid ester copolymer having a sugar chain introduced therein was prepared. This one,
After freeze-drying, dissolve in an organic solvent such as chloroform, apply on a glass plate, and dry in a nitrogen stream to easily obtain a thin film, and redisperse in water to expose sugar chains to the aqueous phase The obtained endoplasmic reticulum was obtained. This endoplasmic reticulum structure
TEM confirmed it. Agglutination by the action of lectin was also confirmed.

Example 9. As an example of the oligosaccharide fatty acid ester according to the present invention, sample numbers 1,4,18,28,29 were prepared using polymerizable phospholipids, 1,2-bis (2,4-octadecadienoyl), respectively. Mixed with glycerophosphocholine, sugar portion is 2 mol% of total
Was prepared. Using this, the inner surface of a normal container used for cell culture was covered with a monomolecular film. For the examples of Sample Nos. 28 and 29, additional radical initiator was added. To increase the stability of the polymerized system. Next, a culture vessel was filled with a culture vessel coated with a phospholipid monolayer containing these oligosaccharide fatty acid esters, and E. coli was incubated at 37 ° C.
Cultured for 48 hours. The bacteria after culturing were collected, and the numbers of bacteria were compared by a turbidimetric method. As a result, the results shown in Table 10 were obtained. The cultivation efficiency was higher than that of the uncoated example. The culture efficiency was defined as the number of Escherichia coli cultured in the uncoated incubator being 1, and the number of Escherichia coli obtained by culturing in each incubator for this was 1.
It is shown with a multiplier of 10.

Example 10. For sample numbers 1, 4, 10, 13, 19, 22, 61, 62 and 63, the method of Example 1 was repeated using human plasma at 25 ° C instead of pure water. The effect of preventing fusion and aggregation of the endoplasmic reticulum was evaluated. As a result, fusion of any sample compared to the non-added system,
The effect of preventing aggregation was observed.

Maltose monopalmitate, a disaccharide derivative that was ineffective in pure water (Sample No. 62), was found to have an effect of preventing the fusion and aggregation of phospholipid vesicles in plasma.

(Test example)

The membrane stability of the oligosaccharide fatty acid ester-introduced phospholipid vesicles shown in the examples was tested by releasing carboxyfluorescein (hereinafter abbreviated as CF).
That is, CF encapsulated in the endoplasmic reticulum using DPPC as a phospholipid
The membrane stability of the oligosaccharide fatty acid ester-introduced phospholipid vesicles was evaluated from the measurement of the release of phospholipids, and compared with the performance of the phospholipid vesicles without the addition of oligosaccharide fatty acid esters.

4 ml 100 mM in 1 ml 0.5 wt% phospholipid vesicle dispersion
CF (pH 8.6) was added, and CF was included in the phospholipid endoplasmic reticulum. Inclusion is as reported previously (Makromol Chem. Rapid Commun ; 8 , 215
(1987)). Next, after the unencapsulated CF was removed by gel filtration, the fluorescence was measured. That is, 3m
300 μl of sample in 1 l of Tris buffer (50 mM, pH 8.0)
After the addition of, the increase in fluorescence intensity accompanying the release of CF was measured (excitation wavelength = 330 nm, fluorescence wavelength = 520 nm). that time,
The surfactant, Triton X-100, was added to destroy the phospholipid vesicles, and the fluorescence intensity when CF was completely released was defined as the time when 100% CF was released.

As an example, phospholipid vesicles (7) to which sample numbers 10, 13 were added at a ratio of 1 mol to 50 mol of DPPC, respectively,
FIG. 2 shows the results of comparing the CF release behavior of the system (8) with the phospholipid vesicle (6) without the addition of oligosaccharide fatty acid ester.

As shown in this figure, the addition of the oligosaccharide fatty acid ester suppressed the release of CF, no disturbance in the packing structure of lipids was observed, and it became clear that more stable phospholipid vesicles were obtained. . This result was also observed in a system to which another sample was added.

As the amount of the oligosaccharide fatty acid ester increased, the CF release rate decreased and then increased again. Therefore, it was recognized that the addition ratio of the oligosaccharide fatty acid ester most effective in suppressing CF release was present. Generally, this value is 50: 1 in molar ratio, as used in this study.
Before and after.

〔The invention's effect〕

Saccharose long-chain fatty acid esters and polysaccharide long-chain fatty acid esters, which are amphiphilic polymers, are widely used in cosmetics, food additives, and even solubilizers for proteins because of their low biotoxicity. . These are considered as materials that can set a characteristic chemical environment, rather than functional molecules, and use properties to guide properties. The biochemical utilization of such polysaccharide long-chain fatty acid esters is, of course, proceeding. For example, Prof. Sunamoto of Nagasaki University and others report an attempt to introduce long-chain fatty acids into polysaccharides with a large molecular weight through ester bonds and use them as artificial cell walls as stabilizers for phospholipid vesicles. (“Polymers in Medicine”, Plemum Press (1
983) p157) shows that the oligosaccharide fatty acid ester of the present invention is sufficiently effective only by adding a small amount to phospholipid vesicles, and thus the original small saccharide vesicles are compared with the polysaccharide-coated vesicle system. It is characterized by maintaining the basic physical properties of the endoplasmic reticulum.

On the other hand, the polysaccharide fatty acid ester having a large degree of polymerization is difficult to precisely separate and purify according to the sugar chain length and the degree of substitution of the fatty acid ester. All are average values. On the other hand, the oligosaccharide fatty acid ester used in the present invention can be obtained as a pure product, and the degree of sugar polymerization, the degree of substitution of the fatty acid ester and the like can be strictly defined. By changing the properties, various properties such as the polarity and solubility of the oligosaccharide fatty acid ester can be adjusted, and the oligosaccharide fatty acid ester can be properly used depending on the purpose.

Furthermore, the oligosaccharide fatty acid ester used in the present invention is easy to separate and purify, so that it does not contain any impurities and has no factors that adversely affect the membrane characteristics of phospholipids. Many of these have relatively low solubility in water and are therefore stably immobilized on the lipid assembly membrane. Since some oligosaccharides have higher physiological activity than polysaccharides, if they are immobilized on the surface of phospholipid vesicles, they can be used as vesicles for recognition on the surface.

In practicing the present invention, an oligosaccharide fatty acid ester having an unsaturated bond in an acyl chain can be introduced into a polymerizable phospholipid aggregate, and copolymerized by ultraviolet irradiation, addition of an initiator, or the like. As a result, the oligosaccharide fatty acid ester can be covalently immobilized on the polymer phospholipid aggregate. Such a high molecular weight phospholipid aggregate in which the sugar chain moiety is covalently immobilized on the surface is stable against heat, an organic solvent, and a physical or chemical stimulus such as a surfactant. It can be used effectively. For example, high molecular weight phospholipid monolayers and bilayers to which oligosaccharide fatty acid esters are covalently bonded are extremely sensitive to lectin, which is an agglutinin, and thus serve as these sensors.

Therefore, according to the present invention, phospholipids can be used as high-value-added functional materials.

[Brief description of the drawings]

FIG. 1 is a chromatogram showing the separation of maltopentaose palmitate according to the degree of substitution, and FIG. 2 is a graph showing the CF release behavior of phospholipid vesicles. The reference numerals in the figure have the following meanings. 1... Substitution degree 0.2... Substitution degree 1.3... Substitution degree 2.4
... Substitution degree 3.5 ... Substitution degree 4 or more. 6. Phospholipid vesicle without oligosaccharide fatty acid ester. 7 ... Sample. 10 added phospholipid vesicles. 8 ...
Sample 13 added phospholipid vesicles.

Claims (8)

(57) [Claims] 1. A sugar polymerization degree is represented by an integer of 2 to 30, and
A modifying agent for phospholipid aggregates comprising an oligosaccharide fatty acid ester having a substitution degree of an acyl group in the range of 1 to 5. 2. An oligosaccharide fatty acid ester having an acyl group having 12 to 22 carbon atoms, which is composed of a saturated aliphatic chain or an unsaturated aliphatic chain containing 1 to 4 unsaturated bonds. Modifier for phospholipid aggregates. 3. The sugar polymerization degree is represented by an integer of 2 to 30, and
An anticoagulant for phospholipid vesicles comprising an oligosaccharide fatty acid ester having a substitution degree of an acyl group in the range of 1 to 5. 4. The anticoagulant for phospholipid vesicles according to claim 3, wherein the oligosaccharide fatty acid ester has an acyl group comprising a saturated aliphatic chain and having 12 to 22 carbon atoms. 5. The sugar polymerization degree is represented by an integer of 2 to 30, and
A fusion inhibitor for phospholipid vesicles comprising an oligosaccharide fatty acid ester having an acyl group substitution degree in the range of 1 to 5. 6. The fusion inhibitor for phospholipid vesicles according to claim 5, wherein the oligosaccharide fatty acid ester has an acyl group comprising a saturated aliphatic chain and having 12 to 22 carbon atoms. 7. The sugar polymerization degree is represented by an integer of 2 to 30, and
A surface immobilizing agent for phospholipid membranes comprising an oligosaccharide fatty acid ester having a degree of acyl group substitution in the range of 1 to 5. 8. The surface immobilizing agent for a phospholipid membrane according to claim 7, wherein the oligosaccharide fatty acid ester has an acyl group comprising a saturated aliphatic chain and having 12 to 22 carbon atoms.