JP3220241B2 - Stabilizer for phospholipid endoplasmic reticulum - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a stabilizer for vesicles comprising a newly synthesized disaccharide derivative having a well-defined structure, which is useful for imparting stability to phospholipid vesicles (hereinafter referred to as "vesicles"). It is about. The endoplasmic reticulum modified with the stabilizer for the endoplasmic reticulum of the present invention can be used as a drug delivery system or a sustained-release material by incorporating a physiologically active substance such as adriamycin or prostaglandin in its internal aqueous phase, Alternatively, artificial hemoglobin may be included to produce artificial red blood cells.

[0002]

2. Description of the Related Art The endoplasmic reticulum is agglomerated or fused, or
It is normal to have instability such as a change in form and leakage of inclusions due to the above phenomenon. Many proposals have been made as means for solving such instability. First, a solution by polymerization of an aggregate of a phospholipid having an unsaturated group has been attempted (for example, a review: H. Ringsdorf).
t al. Angew. Chem. , Int. Eng
l. , 27, 113 (1988)), the properties of lipids, such as gel-liquid crystal phase transition, phase separation and fluidity of the membrane, are lost due to the membrane polymerization, and the functions related to these are lost. Loss occurs.

On the other hand, a cholesterol derivative having β-aminogalactose has been used for controlling liposome aggregation (PS Wu et al., Proc. N).
atl Acal. Sic. , USA, 78, 6211.
(1981)), however, the effect of the derivative is not as great as expected, suggesting that the degree of polymerization of the sugar is strongly involved in the effect. In addition, a hyaluronic acid derivative having a liposome emulsifying action and an emulsifying system stabilizing action has been disclosed (JP-A-3-47801). However, in the hyaluronic acid derivative, since the acyl group is non-selectively ester-bonded, the stabilizing effect cannot be effectively obtained, and the effect largely depends on the introduction position and the number of the acyl groups. . In addition, the viscosity sometimes becomes high and may not be practical.

[0004] The inventors of the present invention introduced an oligosaccharide fatty acid ester developed by the present inventors into the endoplasmic reticulum bilayer membrane, thereby suppressing the aggregation and fusion of the endoplasmic reticulum,
That is, it discloses that it is extremely effective for stabilizing the dispersion of vesicles (Japanese Patent Application Laid-Open No. 1-294701). In that case, the ester derivative of oligosaccharide having a longer sugar chain shows a more excellent effect, and when a disaccharide is used as a base, a sufficient effect for suppressing the aggregation of vesicles is not obtained.

[0005]

SUMMARY OF THE INVENTION The above-mentioned Japanese Patent Laid-Open No. 1-294
Although the invention disclosed in Japanese Patent No. 701 provides many solutions to the stability of the endoplasmic reticulum, introduction of a long-chain fatty acid group into an oligosaccharide having a large number of reactive groups (hydroxyl groups) is non-selective. It is performed, and the number of introductions and introduction sites vary. Therefore, separation and purification are complicated and the yield is low.
The problem is that the effect of stabilizing the dispersion state of the endoplasmic reticulum and the difficulty of immobilizing the oligosaccharide fatty acid ester on the endoplasmic reticulum membrane surface differ depending on the isomer of the oligosaccharide. Further, the relationship between the structure of the isomer and the effect is unknown. Therefore, in the present invention, in order to solve the above-mentioned problems, it is an object of the present invention to develop a disaccharide derivative having a clearly defined structure in which a hydrophobic group is selectively introduced into a specific group of an easily available disaccharide.

[0006]

SUMMARY OF THE INVENTION In order to solve the above-mentioned problems, the present invention provides a disaccharide having a structure and effect clearly obtained by selectively introducing a hydrophobic group at the anomeric position of a disaccharide. It is concerned with providing derivatives. That is, the present invention relates to a disaccharide derivative in which a hydrophobic group is introduced at an anomeric position of a reducing terminal group of a disaccharide by an ether bond.

Hereinafter, the present invention will be described in detail. The disaccharide, which is the base of the disaccharide derivative used in the stabilizer for the endoplasmic reticulum of the present invention, is a disaccharide whose reducing terminal group is composed of aldose or ketose, and includes a sugar alcohol of the disaccharide. .

In the disaccharide derivative of the present invention, since it is necessary to pay attention to the adjustment of the hydrophilic-hydrophobic balance, the hydrophobic group having a hydroxyl group used herein is, for example, an alkyl chain,
When the number of carbon atoms is 12 to 22 and the compound has an unsaturated bond, the number of unsaturated compounds is preferably 1 to 4.

Examples of the hydroxyl group-containing hydrophobic group used in the present invention include primary and secondary alcohols having 12 to 22 alkyl carbon atoms. That is, examples of the linear primary saturated alcohol include lauryl alcohol, cetyl alcohol, stearyl alcohol, behenyl alcohol, and the like. In addition, 1.1 dodecenol-1,
Linear primary unsaturated alcohols such as oleyl alcohol and linolenyl alcohol, branched primary saturated alcohols, branched primary unsaturated alcohols, secondary saturated alcohols and secondary unsaturated alcohols can also be used. Further, dialkyl glycerol in which a saturated long chain or unsaturated long chain is bonded to the 1,3-position or 1,2-position of glycerin by an ether bond can be used. In addition, substances such as sterols, for example, cholesterol, cholestanol, sitosterol and ergosterol are also included in the scope of the hydrophobic group having a hydroxyl group.

[0010] The introduction of a hydrophobic group to the terminal anomeric position of a sugar chain by an ether bond is carried out by a method in which a dialkyl glycerol is ether-bonded to the terminal anomeric position of a disaccharide such as Ogawa (T. Ogawa et al., Carbohydrr.
ate Res. 93, C6 (1981); Oga
wa et al. , Agric. Biol. Che
m. , 45 (10), 2392 (1981); O
gawa et al. , Agric. Biol. Ch
em. , 46 (1), 255 (1982)).

First, protection of the hydroxyl group of the disaccharide with an acetyl group is performed by adding distilled pyridine to the disaccharide and then dropwise adding acetic anhydride under ice-cooling. This was left at 4 ° C. overnight, and recrystallized from isopropyl alcohol to obtain an acetyl form. Introduction of the hydrophobic group having a hydroxyl group to the sugar terminal anomeric position by ether bond is carried out by diluting the above-mentioned hydrophobic compound having a hydroxyl group in dichloroethane in the presence of a molecular sieve 4A powder using trimethylsilyltrifluoromethanesulfonic acid as a catalyst. The reaction is carried out by reacting with an acetylated saccharide.

Thereafter, the desired disaccharide derivative is obtained by removing the protecting group, but the purification operation of the acetylated disaccharide derivative must be sufficiently performed before deprotection. That is, the obtained acetylated product is subjected to liquid chromatography using silica gel as a stationary phase and a mixed solvent of toluene and ethyl acetate as a mobile phase. Confirmation of the fraction is performed by Fourier transform magnetic resonance measurement (hereinafter, referred to as
It is called NMR. ) Or infrared spectroscopy (hereinafter referred to as IR). The method of separating and purifying the acetylated disaccharide derivative is not limited to the above-mentioned method, and a method such as supercritical chromatography, purification using a reversed-phase column, or solvent extraction can also be selected.

Finally, the acetyl group is removed by adding a basic substance such as ammonia water or triethylamine to the acetylated disaccharide derivative in a mixed solvent of methanol and water and refluxing for 12 hours or more. Thereafter, recrystallization is performed twice or more with a polar solvent such as methanol to obtain a final product of the disaccharide derivative used as the stabilizer for the endoplasmic reticulum of the present invention. The identification of the disaccharide derivative is performed by proton-NMR, carbon-N
It can be performed by MR or IR.

Confirmation of the purity of the disaccharide derivative by the ether bond obtained as described above is performed by thin layer chromatography based on silica gel or high performance liquid chromatography.

The stability of the vesicles to which the vesicle stabilizing agent of the present invention has been applied can be confirmed as follows. Phospholipids, or cholesterol of phospholipids and membrane stabilizers, sterols such as cholestanol, or phospholipids, mixed lipids of negatively charged lipids such as stearic acid and dicetyl phosphate, the stabilizer for the endoplasmic reticulum of the present invention Is added by 5 mol% or more, preferably 10 mol% or more to prepare a hydrate. A vesicle dispersion of 50 to 200 nm is prepared from the hydrate by a known method such as an ultrasonic stirrer, a microfluidizer, or an extruder. The particle size of the vesicle dispersion liquid can be confirmed by a particle size distribution measuring device or a transmission electron microscope negatively stained.

After removing the stabilizer not introduced into the endoplasmic reticulum from the dispersion of the endoplasmic reticulum by gel filtration or ultracentrifugation, the sugar of each fraction is removed by the phenol-sulfuric acid method. By quantifying, the rate of introduction of the stabilizer for the endoplasmic reticulum into the endoplasmic reticulum can be determined.
When the stabilizer for vesicles of the present invention is added in an amount of 2 mol% to 10 mol%, about 90% or more of the added amount is introduced into the vesicles. The dispersion stability of the endoplasmic reticulum can be evaluated and confirmed by measuring the change over time in absorbance (turbidity) at 500 to 800 nm with a visible spectrophotometer at room temperature for the dispersion filtered by gel filtration.

Other methods for evaluating the dispersion stability of vesicles include a method of measuring the viscosity of a dispersion of vesicles using a rotational viscometer, and a microscopic method using an electron microscope or the like.

[0018]

The stabilizer for the endoplasmic reticulum of the present invention is suitable as a modifier of the endoplasmic reticulum because of its clear structure and excellent affinity for phospholipids. Further, the vesicle stabilizing agent introduced into the vesicles can maintain the sugar structure exposed to the aqueous phase and can be uniformly dispersed on the vesicle membrane.

The stabilizer for the endoplasmic reticulum of the present invention can suppress the leakage of the inclusions in the endoplasmic reticulum, and further includes, for example, parenting, inhibition of aggregation of the endoplasmic reticulum, prevention of membrane fusion, labeling for recognition with a specific antibody, biomarker, It can be used properly according to the purpose of use such as suppression of phagocytosis in the body. In addition, the stabilizer for the endoplasmic reticulum of the present invention,
The modified vesicle dispersion has almost no increase in viscosity.

In general, when a carrier containing a functional molecule is administered to a living body in the inner aqueous phase of an endoplasmic reticulum, the longer the residence time in the blood, the higher the effectiveness. However, the endoplasmic reticulum is highly soluble in the aqueous solution present in plasma. It is usually aggregated by the interaction with molecules, calcium ions, and the like, and is usually removed from the blood in a short period of time by incorporation into the reticuloendothelial system and phagocytes. However, the residence time in the blood of the endoplasmic reticulum can be controlled by surface modification of the endoplasmic reticulum with the stabilizer for the endoplasmic reticulum of the present invention.

The stabilizer for vesicles of the present invention can be applied to all vesicle modifications. That is, the present invention can be applied not only to modification of vesicles composed of a known phospholipid having a saturated bond, but also to modification of vesicles composed of a polymerizable phospholipid copolymerized. Furthermore, the stabilizer for vesicles of the present invention is copolymerized with this in combination with vesicles comprising polymerizable phospholipids,
The fruits of stabilization can be obtained. Further, in combination with the stabilizer for the endoplasmic reticulum of the present invention and a polymerizable phospholipid, for example,
It is ideal for applying these to the culture vessel surface and copolymerizing them to convert the vessel surface to a hydrophilic and cell-philic surface. Can also be given.

[0022]

The present invention will be described below in more detail with reference to examples. Embodiment 1 FIG. 15 ml of pyridine was added to 2.00 g of maltose, and 15 ml of acetic anhydride was added dropwise while stirring at 4 ° C. This was allowed to stand at 4 ° C. for 12 hours, and then concentrated and dried.
Further, the product was recrystallized with 2-propanol, separated by filtration, and dried under vacuum to obtain acetylated maltose. The preparation per result of measuring an IR spectrum, since the peak of C = O stretching vibration based on acetyl group near 1750 cm ^-1 appeared, a peak based on O-H stretching vibration in the vicinity of 3400 cm ^-1 had disappeared, It was confirmed that all the hydroxyl groups in the sugar skeleton were acetylated. The acetylated maltose obtained here was 3.70 g, and the yield was 93.3.
%Met.

A mixture of 2.30 g of the acetylated maltose and 2.00 g of distearyl glycerol is dissolved in 40 ml of 1,2-dichloroethane, and powdered molecular sieve is added. Then, trimethylsilyltrifluoromethanesulfonate is gradually added. . After stirring for 20 hours, the molecular sieve was removed, and the mixture was separated and purified through a silica gel column (solvent, toluene: ethyl acetate = 3: 1 by volume). At that time, silica gel thin layer chromatography (solvent, toluene: ethyl acetate = 3: 1)
Two spots were recognized in the volume ratio of
The f-value was 0.10 for α-form and 0.29 for β-form. As a result of IR spectrum measurement of the purified product, peaks based on long-chain alkyl appeared at around 2850 cm ^-1 and 2900 cm ^-1 . Therefore, it was confirmed that distearyl glycerol bound to acetylated maltose. The weight of the powder obtained here was 3.60 g (α
Form: 1.30 g, β form: 2.30 g), and the yield was 88.4%.

1.00 g of α or β-acetylated maltose distearyl glycerol was dissolved in 20 ml of methanol, and 2 ml of triethylamine and 2 ml of pure water were added thereto, followed by refluxing for 12 hours. The reaction mixture was crystallized from methanol and dried using a vacuum drier. As a result of measuring the IR spectrum at this stage, 1750 cm
The peak of the C ＝ O stretching vibration based on the acetyl group near ⁻¹ disappeared, and the peak of the OH stretching vibration appeared near 3400 cm ⁻¹ , confirming that the acetyl group was completely hydrolyzed.

When the proton NMR spectrum was measured, the proton derived from the alkyl chain at the β-position or later from the ether oxygen, the sugar skeleton, glycerol and —OCH
The integral ratio of protons derived from ₂ CH ₂ is calculated as 2.
As a result, 2.51 was obtained as an experimental value for 33, confirming that distearyl glycerol was introduced at a molar ratio of 1: 1 to the sugar skeleton.

Further, when the carbon NMR spectrum was measured, the chemical shift of the carbon atom at the anomeric position shifted from 92 ppm of maltose to 103 ppm, confirming that distearyl glycerol was selectively bonded to the anomeric position. The yield of the obtained 1,2-dioctadecyl-O-α (β) -maltosyl-glycerol (hereinafter referred to as DOMG) was 0.72 g, and the yield was 95.0%.

The surface modification of the endoplasmic reticulum is carried out by the above-mentioned DOMG.
13 mg was dissolved in 1 ml of chloroform, and 5 ml of a chloroform solution in which 50 mg of dipalmitoylphosphatidylcholine (hereinafter, referred to as DPPC) was dissolved was mixed with the mixture so that DOMG was about 5 mol% with respect to DPPC. A thin film was formed on the inner wall of the pear-shaped flask using a rotary evaporator. Thereafter, 10 ml of pure water was added, glass beads were added, and the mixture was stirred with a vortex mixer to obtain a milky white DOMG-modified vesicle dispersion. Using an extruder (final transmission filter, pore size: 0.05 μm), the dispersion was converted into a vesicle dispersion having a controlled particle size of about 35 ± 15 nm.

DO in the case where the amount of DOMG added to DPPC was changed to 10 mol%, 15 mol%, and 20 mol%
An MG-modified vesicle dispersion was also prepared using the same means as described above. Thereafter, each dispersion was allowed to stand at 4 ° C., and the change over time in turbidity at 800 nm was tracked. As a result, the increase in turbidity was 5 mol%> 1 in DOMG increase rate.
0 mol%> 15 mol%> 20 mol%, and it was clarified that the turbidity increase rate was reduced and the stability of the endoplasmic reticulum was increased when the added amount of DOMG was increased. A comparison of the turbidity increase immediately after the preparation of the endoplasmic reticulum and that after leaving the endoplasmic reticulum at 4 ° C. for 24 hours showed that the turbidity increase rate of the unmodified DPPC vesicle was 1
00%, that of the DOMG-modified ER is DO
The ER stabilizing effect was low when 5 mol% of MG was added,
When 0 mol% was added, the turbidity increase rate was 11.2%, indicating an excellent stabilizing effect, and it was confirmed that DOMC modification can effectively stabilize the dispersion of vesicles.

The results are shown in Table 1 together with the results of Comparative Examples 1 to 5 described later. Table 1 shows 5 wt% hydrated DPPC
It shows the turbidity increase immediately after the preparation of the vesicle dispersion liquid and after standing at 4 ° C. for 24 hours. The turbidity increase rate of unmodified DPPC vesicles (referred to as increase rate in Table 1) is 100.
%, And the numerical value of the turbidity increase rate of various derivative-modified DPPC vesicles with respect to the percentage was expressed by%. In the table, the amount in parentheses indicates the amount of the modifier added, and the number indicates mol%. The value of the turbidity increase rate of the endoplasmic reticulum indicates that the smaller one has high stability, and from the table, the stabilizer for the endoplasmic reticulum of the present invention is extremely useful, and the results are as follows: This was almost comparable to the effect of the oligosaccharide derivative, which was excellent in stabilizing the dispersion of the endoplasmic reticulum described later in Comparative Examples 1 and 2. That is, it was clearly shown that the stabilizing agent for vesicles of the present invention exhibited an extremely excellent effect by increasing the amount of addition.

Comparative Example 1 DOMG according to Example 1
Under the same conditions as in the preparation of the modified endoplasmic reticulum, dioctadecylmaltopentaosylglycerol (hereinafter referred to as DOMPG) was introduced into the 5 wt% hydrated DPPC endoplasmic reticulum at a molar ratio of 5 mol%. As a result, the turbidity increase of the DOMG-modified DPPC vesicle was 12.1% with respect to the turbidity increase rate of the unmodified DPPC vesicle.

[0031]

[Table 1]

Comparative Example 2 As in Comparative Example 1, maltopentaose monopalmitate (hereinafter referred to as MPMP) was introduced into the DPPC vesicle at a concentration of 5 mol%.
As a result, the turbidity increase of the MPMP endoplasmic reticulum
The turbidity increase rate of the endoplasmic reticulum was 13.5%.

Comparative Example 3 As an example of sucrose fatty acid ester, sugar ester (P-1695: trade name, manufactured by Mitsubishi Kasei Food) was used in the same manner as in Example 1 at 5 mol%, 10 mol%, 15 mol%, and 20 mol%. , DPPC
Comparative experiments were performed as ER modifiers. as a result,
Even when the amount of the modifier added was 20 mol%, the turbidity increase rate was 32.5%, and the aggregation suppressing effect was not sufficient.

Comparative Example 4 Polyethylene glycol monocetyl ether using commercially available polyethylene glycol having a degree of polymerization of 8 (Nonion P-208: trade name, manufactured by NOF Corporation)
Was used as a modifier of DPPC vesicles, the turbidity increase rate was 30.1% when 15 mol% was added, and when added more, vesicle breakage occurred and the turbidity rapidly decreased. .

Comparative Example 5 When polyethylene glycol monostearyl ether (Nonion S-215: trade name, manufactured by NOF Corporation) using commercially available polyethylene glycol having a polymerization degree of 15 was used as a modifier for DPPC vesicles, the turbidity increase rate was increased even when 15 mol% was added. Was 29.3%. However, at 20 mol%, the endoplasmic reticulum was destroyed and measurement was impossible.

Embodiment 2 FIG. D obtained in the same manner as in Example 1.
2.4 mg of OMG was dissolved in 1 ml of chloroform, and 5 ml of chloroform in which 100 mg of DPPC was dissolved was mixed with the solution (DOMG was 2 mol% based on DPPC). The obtained mixture was made using a rotary evaporator. A thin film was formed on the inner wall of the flask. Thereafter, 10 ml of pure water was added so that the vesicle concentration became 1.0 wt%, glass beads were added, the mixture was stirred with a vortex mixer, and further, DOMG modification with a controlled particle size using an extruder. D
A PPC vesicle dispersion was prepared. At the same time, a part of the dispersion was diluted 5-fold with pure water, and the ER concentration was 0.2 wt.
% Dispersions were also prepared.

The ER concentration of 1.0 wt% and 0.2 wt%
Each of the wt% DOMG-modified ER dispersions was placed at 4 ° C. for 2 hours.
After standing for 4 hours, ultracentrifugation (300,000 g,
(60 minutes), DOMG released in the supernatant was quantified by the phenol / sulfuric acid method, and the introduction rate of DOMG into the endoplasmic reticulum was calculated. As a result, the introduction rate at an endoplasmic reticulum concentration of 1.0 wt% was 94.3%, and the introduction rate was 77.0% at 0.2 wt%.
However, these results are shown in Table 2 together with the results of Comparative Examples 6 and 7 in which the oligosaccharide derivative described below was used as a modifier of the endoplasmic reticulum. In Table 2, the introduction rate (%) represents the rate of the modifying agent introduced into the endoplasmic reticulum. Therefore, the higher the numerical value of the introduction rate, the more stably the modifier was fixed in the lipid membrane of the endoplasmic reticulum.

[0038]

[Table 2]

Comparative Example 6 Under the same conditions as in Example 2, M
PMP was introduced into the DPPC endoplasmic reticulum, and the introduction rate of MPMP was calculated. That is, 2% per 1.0 wt% hydrated DPPC vesicle
Although MPMP was introduced at a rate of mol%, the introduction rate was
DO as the stabilizer for the endoplasmic reticulum of the present invention shown in Example 2
In some cases, the introduction rate of MG into the DOMG-modified DPPC endoplasmic reticulum was poor.

Comparative Example 7 As in Comparative Example 6, 1.0
DOMP at a ratio of 2 mol% to the wt% hydrated DPPC endoplasmic reticulum
G was introduced, but the introduction rate was again the same as in Example 2.
DOM which is one of the stabilizers for the endoplasmic reticulum of the present invention shown in FIG.
G fell short of the results.

Embodiment 3 FIG. After acetylated cellobiose obtained from 5.0 g of cellobiose was mixed with cholesterol in which tetraethylene glycol was ether-bonded, that is, equimolar amount of tetraethylene glycosyl cholesterol was mixed by the same acetylation method as in Example 1, and then mixed. According to the method of Example 1, cellobiosyltetraethyleneglycosyl cholesterol (hereinafter, referred to as STEC) was synthesized to obtain 5.8 g of the same (total yield: 44.8).
%). The IR spectrum of STEC obtained here contains 27
A peak based on tetraethylene glycol was observed at 00 to 2900 cm ^−1, and the measurement of carbon NMR spectrum showed that the chemical shift of the anomeric carbon atom moved from 92 ppm of cellobiose to 104 ppm,
It was confirmed that tetraethylene glycosyl cholesterol was selectively bound to the anomeric position of cellobiose.

The mixed lipid powder containing the above-mentioned STEC (DPP)
C: cholesterol: stearic acid: STEC = 7:
(7: 2: 2 molar ratio) was dispersed in 100 ml of physiological saline and stirred at 4 ° C for 24 hours.
The microfluidizer treatment was performed under the conditions of 0 psi and 15 minutes. The dispersion is then centrifuged (1000
(00 g, 30 minutes) to obtain an STEC-modified ER layer in the lower layer. A 2.5 wt% solution of dextran (molecular weight: 40000) was added to the obtained endoplasmic reticulum layer, and 800
The turbidity in nm was measured to evaluate the stability of the STEC-modified ER. As a result, the turbidity increase rate was unmodified ER (DPPC: cholesterol: stearic acid = 7: 7:
(Molar ratio of 2) was 58% of the increase in turbidity, and it was confirmed that STEC, one of the stabilizers for the endoplasmic reticulum of the present invention, had an inhibitory effect on the dextran aggregation action of the endoplasmic reticulum.

Embodiment 4 FIG. Example 1 After mixing acetylated lactose obtained from 5.0 g of lactose with equimolar dioctadecylglycerol having a polymerizable group, Example 1
7. Synthesis of dioctadecyllactosylglycerol (hereinafter referred to as DOLG) according to the method of 8.
9 g were obtained (total yield 64.5%). DOLG obtained here
Was measured by carbon NMR, the chemical shift of the anomeric carbon atom was 103 ppm from lactose 92%.
The shift to ppm confirmed that dioctadecylglycerol selectively bound to the anomeric position of lactose.

The mixed lipid powder containing DOLG (DPP)
C: cholesterol: stearic acid: DOLG = 7:
(A molar ratio of 7: 2: 2) was dispersed in 100 ml of physiological saline, glass beads were added, and the mixture was stirred with a vortex mixer to obtain a DOLG-modified vesicle dispersion, and the particle size was adjusted using an extruder. It was adjusted. 5 mM calcium chloride was added to the DOLG-modified vesicle dispersion liquid whose particle diameter was adjusted, and the stability to calcium ions was evaluated based on the change in viscosity. That is, when the viscosity at 37 ° C. was measured using a rotational viscometer (Vismetron VS-AK), no increase in the viscosity was observed, and the shear rate was 1.1.
It was 5 cp at 5 sec ^-1 .

On the other hand, a mixed lipid powder containing no DOLG (DPPC: cholesterol: stearic acid = 7: 7:
When the unmodified vesicle dispersion is prepared from the above (molar ratio of 2) and calcium ions are similarly added, the vesicles are immediately aggregated and have a viscosity of 11c because the vesicle surface is negatively charged.
p. Therefore, it was confirmed that DOLG, which is one of the stabilizers for the endoplasmic reticulum of the present invention, has a strong inhibitory effect on the calcium ion aggregation action of the endoplasmic reticulum.

Embodiment 5 FIG. 200 mg of di (2,4-octadecadienoyl) phosphatidylcholine (hereinafter referred to as DODPC), which is a polymerizable phospholipid, was dissolved in 5 ml of chloroform, and maltose and 2,4-octadecady were obtained by the method of Example 1. Polymerizable 2, obtained from enol
After mixing 2.0 mg of 4-octadecadienyl-O-α (β) -maltose (hereinafter referred to as ODM) with 1 ml of a chloroform solution in which 2.0 mg was dissolved, a thin film was formed on the inner wall of a mold flask formed by a rotary evaporator. , 2
While adding 0 ml of pure water, glass beads were added and the mixture was stirred with a vortex mixer. ODM obtained here
The modified vesicle dispersion was prepared using an extruder to prepare a vesicle dispersion having a substantially uniform particle size. Irradiating the vesicle dispersion with γ-rays to convert ODM into polymerizable phospholipid DODPC
And partially dilute 5 times with pure water.
C. and left for 24 hours. Each copolymerized vesicle dispersion was centrifuged (300,000 g, 60 minutes), and the ODM in the supernatant was quantified by the phenol / sulfuric acid method to calculate the introduction rate. As a result, the introduction rate was reduced even after dilution. Without doing this, the value was as high as 94.2%.

On the other hand, in a system in which ODM is not copolymerized,
By diluting the ER dispersion five-fold, the introduction rate of ODM into the ER was reduced from 93.8% to 66.9%.

Further, the vesicle dispersion before the 5-fold dilution was added to 4
When the turbidity after standing at 24 ° C. for 24 hours was compared with the turbidity of the ODM-unmodified vesicle under the same conditions, the turbidity increase rate was 10.9%.
Is extremely low, and one of the ER stabilizers of the present invention, O
It was also confirmed that DM was extremely useful for stabilizing the endoplasmic reticulum by copolymerizing it with the endoplasmic reticulum.

[0049]

EFFECTS OF THE INVENTION The stabilizer for vesicles of the present invention, when used as a modifier for phospholipid vesicles, not only inhibits aggregation of phospholipid vesicles themselves, but also Can stabilize the dispersion, and the effect of suppressing phagocytosis in vivo can be obtained. Also,
The use of readily available bases such as disaccharides greatly contributes to the industry.

──────────────────────────────────────────────────続 き Continuing on the front page (72) Inventor Hiromi Sakai 1-6-9, Sekichominami, Nerima-ku, Tokyo (72) Inventor Hidetoshi Tsuchida 1-10-10, Sekichominami, Nerima-ku, Tokyo (56) Reference Document JP-A-5-294983 (JP, A) JP-A-1-294701 (JP, A) (58) Fields investigated (Int. Cl. ⁷ , DB name) B01F 17/00-17/56

Claims (3)

(57) [Claims] 1. A stabilizing agent for phospholipid vesicles comprising a disaccharide derivative having a hydrophobic group introduced at the anomeric position of the reducing terminal group of the disaccharide by an ether bond. 2. Hydrophobic group having hydroxyl group having 12 to 2 carbon atoms.
2. A stabilizer for phospholipid vesicles comprising the disaccharide derivative according to claim 1 , which has an alkyl chain of 2. 3. The alkenyl chain having 12 to 22 carbon atoms, wherein the hydrophobic group has a hydroxyl group and has 1 to 4 unsaturated bonds.
A stabilizer for phospholipid vesicles comprising the disaccharide derivative according to claim 1.