JPH07173079A - Amphiphatic polyethylene glycol derivative and its use - Google Patents

Abstract

PURPOSE:To obtain the subject compound to be used as a lipidic microcell modifier, capable of suppressing adsorption of proteins to lipidic microcells and agglomeration of these microcells due to such adsorption, also suppressing leaking out from lipidic microcell membranes when used as a membrane constituent for lipidic microcells. CONSTITUTION:This lipidic microcell modifier consists of an amphiphatic polyethylene glycol derivative made up of hydrophilic segment having polyethylene glycol chain of the formula R<1>O-(CH2CH2O)n-(CH2)m (R<1> is a 1-5C alkyl or H; (n) is an integer of 5-1000; (m) is an integer of 0-4) and polymerizable hydrophobic segment.

Description

Detailed Description of the Invention

[0001]

The present invention relates to an amphipathic polyethylene glycol (hereinafter sometimes referred to as PEG) derivative, an amphipathic agent comprising the PEG derivative, a lipid vesicle modifier, and a membrane constituent component of the modifier. The present invention relates to lipid vesicles (liposomes) containing as. More specifically, an amphipathic PEG derivative having a hydrophilic portion having a polyethylene glycol chain and a polymerizable hydrophobic portion, this P
An amphipathic agent consisting of an EG derivative, a drug carrier (drug delivery system, DDS), a carrier for a diagnostic agent, a lipid vesicle modifier used as a membrane component of a lipid vesicle, which can be used in fields such as artificial red blood cells, and The present invention relates to lipid vesicles containing this modifier.

[0002]

2. Description of the Related Art Lipid vesicles are bilayer membrane vesicles of lipid vesicle-forming components such as phospholipids, drug carriers,
Applications have been attempted in fields such as carriers for diagnostic agents and artificial red blood cells. However, in the presence of water-soluble polymers such as plasma proteins, lipid vesicles undergo structural changes such as aggregation and fusion of lipid vesicles through the adsorption of proteins on the surface of lipid vesicles, and Since it is recognized and phagocytosed by system tissues (RES), there is a problem that the residence time (half-life) in the body is short.

In order to solve such problems, a PEG derivative comprising PEG and hydrogenated egg yolk phosphatidylethanolamine is used as a modifier for lipid vesicles (JP-A-3-218309, JP-A-4-5242, JP Kaihei 3
No. 181415) or a PEG ether derivative comprising PEG and an alcohol such as a long-chain aliphatic alcohol (JP-A-2-149512). In lipid vesicles containing these modifiers as membrane constituents, the presence of hydrophilic PEG chains with a very large volume on the surface of the lipid vesicles causes adsorption of proteins and the like to the lipid vesicles.
Also, structural changes such as aggregation of lipid vesicles through such adsorption can be prevented.

However, although these conventional PEG derivatives can prevent aggregation of lipid vesicles with each other through adsorption of proteins and the like on the surface of the lipid vesicles, the PEG derivative and other lipid vesicle membrane constituents Since there is only a weak intermolecular interaction between them, external stimulation may cause leakage of the PEG derivative bound in the membrane due to the weak intermolecular interaction from the membrane. Particularly when the hydrophobic part of the PEG derivative is a single chain, for example, in the case of an ether or ester derivative consisting of PEG and a long chain fatty alcohol or a long chain fatty acid, the stability of the obtained lipid vesicle is low and the PEG derivative is Leakage easily occurs from the lipid endoplasmic reticulum membrane [Biochim.
Biophy. Acta. , 1066, 29-36 (1
991)].

It is known that PEG acts as a nonionic surfactant, disturbs the structure of the cell membrane, for example, the erythrocyte membrane, and hemolyzes erythrocytes. Therefore, when a conventional PEG derivative is used as a membrane constituent component of a lipid vesicle, there is a concern that the structure of a cell membrane such as an erythrocyte membrane may be disturbed by the PEG derivative leaking from the lipid vesicle and scattered in the solution. There is.

[0006]

It is an object of the present invention to provide new and useful amphipathic PEG derivatives. Another object of the present invention is to provide an amphipathic agent comprising the above PEG derivative. Still another object of the present invention is that when it is used as a membrane constituent component of a lipid vesicle, adsorption of a protein or the like to the lipid vesicle and the aggregation of the lipid vesicle mediated by such adsorption can be suppressed, and further, from the membrane. It is an object of the present invention to provide a lipid endoplasmic reticulum modifying agent that causes less leakage, and a lipid endoplasmic reticulum containing the modifying agent as a membrane constituent.

[0007]

Means for Solving the Problems The present inventors have conducted extensive studies to achieve the above object, and as a result, synthesized an amphipathic compound having a hydrophilic portion and a polymerizable hydrophobic portion, The obtained amphipathic compound is used as an amphipathic agent, or is introduced into a membrane as a modifier of lipid vesicles, and the modifiers are polymerized with each other or with a modifier and another membrane-forming component,
The inventors have found that the modifier can be prevented from leaking from the lipid vesicle membrane, and that the adsorption of proteins to the lipid vesicles and the aggregation of the lipid vesicles mediated by such adsorption can be effectively suppressed. .

That is, the present invention provides the following amphipathic PEG derivative, an amphipathic agent comprising the same, a lipid vesicle modifying agent, and a lipid vesicle containing the modifying agent as a membrane constituent. (1) General formula

Embedded image R ¹ O— (CH ₂ CH ₂ O) n— (CH ₂ ) m— [1] (In the formula, R ¹ represents an alkyl group having 1 to 5 carbon atoms or a hydrogen atom. N is 5 An integer of ˜1000 and m is an integer of 0 to 4), and an amphipathic polyethylene glycol derivative having a hydrophilic part having a polyethylene glycol chain and a polymerizable hydrophobic part. (2) An amphipathic agent comprising the amphipathic polyethylene glycol derivative according to (1) above. (3) A lipid vesicle modifying agent comprising the amphipathic polyethylene glycol derivative according to (1) above. (4) A lipid vesicle comprising the lipid vesicle modifier and the lipid vesicle-forming component according to (3) above as a membrane constituent component.

The PEG derivative of the present invention is an amphipathic polyethylene glycol derivative having a hydrophilic portion represented by the above general formula [1] and a polymerizable hydrophobic portion. The hydrophilic part and the hydrophobic part may be directly bonded by a bond such as an ester bond, an amide bond or an ether bond, or may be bonded via a spacer.

Examples of the alkyl group represented by R ¹ in the above general formula [1] include alkyl groups having 1 to 5 carbon atoms such as methyl group, ethyl group and propyl group. In the general formula [1], n is 5-1000, preferably 25-
250 and m are 0 to 4, preferably 2 to 3. When the PEG derivative of the present invention is used as a lipid vesicle-modifying agent, the degree of polymerization of polyethylene glycol represented by n is within this range. The lipid vesicles are excellent in suppressing aggregation of the vesicles and also excellent in stability.

In the PEG derivative of the present invention, the hydrophobic portion is a polymerizable hydrophobic group, for example, a hydrocarbon group having a polymerizable unsaturated bond and having 8 to 26 carbon atoms, preferably 12 to 22 carbon atoms. To be

In the present invention, the term "polymerizable" means that the PEG derivatives of the present invention or the PE of the present invention are prepared by a usual polymerization method.
This means that the G derivative and the other film-forming component can be easily bonded by a covalent bond. Examples of such a polymerizable hydrophobic group include a conjugated double bond, a hydrophobic group having a conjugated unsaturated bond such as a conjugated triple bond, a hydrophobic group having an α, β-unsaturated bond, and a vinylbenzoyl group. And a hydrophobic group having an acryloyl group. Of these, a hydrophobic group having a conjugated unsaturated bond is preferable.

Therefore, even if the group has an unsaturated bond,
Ordinary groups derived from unsaturated fatty acids are not polymerizable groups, and for example, oleyl group, linoleic acid group, linolenic acid group, etc. are not included in the polymerizable hydrophobic group of the present invention.

Specific examples of the polymerizable hydrophobic moiety in the PEG derivative of the present invention include groups represented by the following general formula [2] or [3].

CH ₃ (CH ₂ ) x- (CH = CH) y- (CH ₂ ) z- [2] (In the formula, x is an integer of 0 to 19, y is an integer of 2 to 4, and z is It is an integer of 0 to 19 and satisfies x + z = 7 to 19.)

CH ₃ (CH ₂ ) p- (C≡C) _2- (CH ₂ ) r- [3] (In the formula, p is an integer of 0 to 21, r is an integer of 0 to 21 and , P + r = 7 to 21 is satisfied.)

Specific examples of the polymerizable hydrophobic group represented by the above general formula [2] or [3] include those shown below.

Embedded image _{CH 3 (CH 2) 8 -CH} = CH-CH = CH- CH 3 (CH 2) 12 -CH = CH-CH = CH- CH 3 (CH 2) 13 -CH = CH-CH = _{CH- CH 3 (CH 2) 3} -CH = CH-CH = CH-CH = CH
_{- (CH 2) 7 - CH} 3 (CH 2) 4 -CH = CH-CH = CH-CH = CH
_{- (CH 2) 7 - CH} 3 (CH 2) 9 -C≡C-C≡C- (CH 2) 8 - CH 3 (CH 2) 9 -C≡C-C≡C- (CH 2) 9 _{- CH 3 (CH 2) 11} -C≡C-C≡C- (CH 2) 8 - CH 3 (CH 2) 12 -C≡C-C≡C- (CH 2) 8 -

In addition to the above, specific examples of the polymerizable hydrophobic portion include groups having an α, β-unsaturated bond shown below.

Embedded image _{CH 2 = CH- (CH 2)} 8 - CH 2 = CH- (CH 2) 9 -

In the PEG derivative of the present invention, those represented by the following general formulas [4] to [13], etc., in which the hydrophilic part and the hydrophobic part are directly bonded by an ester bond, an amide bond or an ether bond, etc. can give.

[Chemical 7] (In the formula, R ¹ , m, n, x, y, z, p and r are the same as above.)

Examples of the hydrophilic portion and the hydrophobic portion bonded via a spacer include those represented by the following general formulas [14] to [17].

[Chemical 8] (In the formula, R ¹ , n, x, y, z, p, and r are the same as above.)

The PEG derivative of the present invention can be produced by the following method. First, in the above-mentioned PEG derivative, the one in which the hydrophilic part and the hydrophobic part are directly bonded by an ester bond, an amide bond or an ether bond has the general formula

Embedded image ^{_{R 1 O- (CH 2 CH 2}} O) n- (CH 2) m-R 2 (18) (wherein, .R ¹ showing R ² is a hydrogen atom, a hydroxyl group, an amino group or a carboxyl group , M, and n are the same as above.) And a polyethylene glycol represented by the general formula

CH ₃ (CH ₂ ) x- (CH = CH) y- (CH ₂ ) z-X ¹ [19] (In the formula, X ¹ represents a hydroxyl group, an amino group or a carboxyl group. X, y , Z are the same as above.) Or the general formula

Embedded image CH ₃ (CH ₂ ) p- (C≡C) ₂ — (CH ₂ ) r—X ² [20] (In the formula, X ² represents a hydroxyl group, an amino group or a carboxyl group. P, r Is the same as described above.) A polymerizable long-chain alcohol, a polymerizable long-chain amine or a polymerizable long-chain fatty acid represented by it can.

As the starting material represented by the general formula [18], polyethylene glycol, monomethoxypolyethylene glycol, R ¹ O- (CH ₂ CH ₂ O) n-CH ₂ CH ₂ COOH, R ¹ O- (CH _{2 CH 2 O) n-CH} 2 CH 2 CH 2 NH
₂ (R ¹ and n are the same as above) and the like.

The starting materials represented by the above general formula [19] or [20] include n-octadeca-2,4-dienol, n-octadeca-9,11,13-trienol and 2,4-octadecadiene. Acid, 9, 11, 13-
Examples include octadecatrienoic acid.

As a concrete production method, for example, an amphipathic PEG linked with an ester bond by reacting monomethoxypolyethylene glycol with 2,4-octadecadienoyl chloride in the presence of a basic catalyst.
Derivatives can be synthesized.

In the PEG derivative of the present invention, the hydrophilic part and the hydrophobic part are bound via a spacer,
A polyethylene glycol represented by the general formula [18] or a derivative thereof, and a general formula

[Chemical 12] , Or a hydrogen atom. x, y and z are the same as above. ) Or general formula

[Chemical 13] , Or a hydrogen atom. p and r are the same as above. ) Can be produced by a known method using a phosphatidylethanolamine or a glycerol ester having a polymerizable fatty acid as a starting material.

Specific examples of the general formula [21] or [22] include 1,2-di (2,4-octadecadienoyl) -3-phosphatidylethanolamine (DODPE), 1, 2-di (9,11,13-octadecatrienoyl) -3-phosphatidylethanolamine (DOTPE), 1,2-di (2,4-octadecadienoyl) glycerol, 1,2-di (9, 1
1,13-octadecatrienoyl) glycerol and the like can be mentioned.

As a specific production method, a starting material having a hydroxyl group as an end group and having PEG or a polymerizable hydrophobic portion is activated by previously reacting it with cyanuric chloride or succinic anhydride. , By reacting with another starting material having a carboxyl group or an amino group (starting material having a polymerizable hydrophobic portion or PEG), to bond the PEG and the polymerizable hydrophobic portion via a spacer You can

For example, monomethoxypolyethylene glycol is reacted with succinic anhydride to introduce a carboxyl group at the end of PEG, and then 1,2-di (2,4-octa) is added in the presence of a dehydrating agent such as carboximide. By reacting with decadienoyl) -3-phosphatidylethanolamine, an amphipathic PEG derivative composed of PEG and a polymerizable phospholipid can be obtained.

Since the amphipathic PEG derivative thus produced has a hydrophilic portion and a hydrophobic portion, it can be used as an amphipathic agent or a surfactant. This amphipathic agent is a drug that gathers at the interface between the hydrophilic phase and the hydrophobic phase to form micelles. Since the amphipathic agent of the present invention has a polymerizable hydrophobic portion, by polymerization, a composite film of a hydrophobic polymer layer and a large-volume hydrophilic layer composed of PEG chains extending velvety from the polymer layer is formed. It is formed. Such an amphipathic agent can be used as a modifier for lipid vesicles and other film forming agents.

The lipid vesicle modification agent of the present invention comprises the above-mentioned amphipathic PEG derivative, wherein the hydrophobic portion of the modification agent serves as a lipid vesicle bilayer forming component and the hydrophilic portion of the modification agent is the lipid vesicle. It is modified so that it extends from the surface of the to the aqueous phase. The modification may be carried out during the formation of lipid vesicles,
It may be performed after formation.

When modification is carried out during the formation of lipid vesicles, the lipid vesicle-forming components are aggregated by forming the lipid vesicle-forming component by using the lipid vesicle-forming component and the modifier as a membrane-forming component. Upon exposure, the hydrophilic portion of the modifier collects on its surface and coats the lipid vesicle membrane. By carrying out the polymerization in this state, the polymerizable group of the modifier is polymerized to stabilize the structure. At this time, if a polymerizable component is used as the vesicle-forming component, polymerization also occurs between the polymerizable group of the vesicle and the polymerizable group of the modifier, and the binding force between the vesicle and the modified membrane is increased.

When modification is carried out after the formation of lipid vesicles, when a modifying agent is added to the formed lipid vesicles, the hydrophobic portion of the modifying agent penetrates into the bilayer membrane of the lipid vesicles, and the hydrophilic portion becomes lipid. Collect on the surface of the endoplasmic reticulum. Further, the modifier can be polymerized to enhance the stability of the modified lipid vesicle.

As the lipid vesicle-forming component, known natural or synthetic lipid vesicle-forming components can be used without limitation,
For example, diphosphatidylglycerol, cardiolipin, phosphatidylinositol, phosphatidylserine, phosphatidylethanolamine, phosphatidylcholine and other phospholipids; Polymerizable phospholipid having a residue; "Ad
v. Polym. Sci. , 64, 1-62 (198
5) ”, polymerizable phospholipids and surfactants; glycolipids such as sulfoxyribosyl diglyceride, digalactosyl diglyceride and lactosyl diglyceride; nonpolar lipids such as cholesterol; and mixtures thereof. To be

Among these, it is desirable to use a polymerizable phospholipid in order to prevent leakage of the amphipathic PEG derivative from the lipid vesicle membrane. As the polymerizable phospholipid,
In particular 1,2-di (2,4-octadecadienoyl) -3
-Phosphatidylcholine (DODPC), 1,2-di (9,11,13-octadecatrienoyl) -3-phosphatidylcholine (DOTPC) and the like are preferable.

Lipid vesicles are prepared from these lipid vesicle-forming components by, for example, extrude method, vortex mixer method, ultrasonic method, surfactant removal method, reverse layer evaporation method, ethanol injection method, prevesicle method, French press. Law,
It can be produced by various methods such as a W / O / W emulsion method, an annealing method, and a freeze-thaw method.

In the present invention, the modifying agent as a membrane-forming component of lipid vesicles, that is, the amphipathic PEG derivative is added in an amount of 0.2 to 25% by weight, particularly 1 to 8% by weight of the lipid vesicle-forming component. Is preferred. If the amount of the amphipathic PEG derivative added is smaller than that, the effect as a modifier is not so remarkable, and if it is larger than that, the stability of lipid vesicles is hindered, which is not preferable.

When forming a modified membrane in forming lipid vesicles, for example, an amphipathic PEG derivative which is a modifier is dissolved in an organic solvent such as benzene together with other lipid vesicle-forming components, and the solvent is removed. , Amphipathic P
A mixed lipid containing an EG derivative is prepared. Using the obtained mixed lipid, a lipid vesicle containing a PEG derivative can be prepared by the above-mentioned known lipid vesicle granulation method.

When a modified membrane is formed after the formation of lipid vesicles, for example, an amphipathic PEG derivative as a modifier is added to a dispersion liquid of lipid vesicles prepared in advance, and the mixture is incubated. Amphiphilic PEG
The derivative can be introduced into the membrane of the lipid vesicle.

The polymerization component introduced into these lipid vesicle membranes can be polymerized by known methods such as initiator polymerization, photopolymerization and radiation polymerization. For example, in the case of polymerization using an initiator, a polymerization initiator is mixed in advance and polymerization is performed. In the case of photopolymerization, for example, polymerization can be performed by irradiating ultraviolet rays from a high pressure mercury lamp while cooling with ice water. In the case of radiation polymerization, the lipid vesicles are irradiated with γ-rays or the like to give a 0.1
Polymerization is possible at doses of 5 to 1.0 Mrad.

The lipid vesicle obtained as described above contains a lipid vesicle-forming component and the modifying agent as a membrane constituent component, and has a structure in which a modified layer is formed on the surface of the lipid vesicle. Since the modified layer is composed of PEG chains that extend in a velvety state as described above, it is possible to prevent adsorption of proteins and the like to lipid vesicles and aggregation of lipid vesicles through this adsorption. As a result, when administered into the body, it is possible to prevent the blockage in the blood vessel due to the aggregate, and the inhibition of the blood flow due to this.

Since the lipid vesicles are polymerized with an amphipathic PEG derivative as a modifier, the lipid vesicles are less likely to leak from the membrane and have good compatibility with blood components such as red blood cells. It is extremely suitable as an artificial red blood cell. Furthermore, since this lipid vesicle has very high storage stability, it can be used as a diagnostic agent or biosensor by supporting a physiologically active substance such as an antigen or an antibody as a carrier with less nonspecific adsorption of proteins to the surface. Is also available.

[0040]

EXAMPLES The present invention will be described in more detail with reference to Examples, Comparative Examples and Test Examples.

Example 1 0.15 mol of 2,4-octadecadienoic acid (ODA)
Was dissolved in 800 ml of dry benzene, and then 0.18 mol of thionyl chloride was added to this solution. The mixture was reacted with stirring at 10 ° C. for 1 hour and further at 40 ° C. for 30 minutes. After cooling the solution, the solvent was removed with a rotary evaporator under reduced pressure to obtain 2,4-octadecadienoyl chloride (ODC).
Got

After dissolving 0.15 mol of monomethoxy PEG (molecular weight 5000) in 6000 ml of pyridine,
0.15 mol of the above ODC was added, and the mixture was reacted at 60 ° C. for 10 hours with stirring. After the reaction was completed, the solvent was removed under reduced pressure, and the product obtained was used as an eluent for ethanol / water (4 / l, v /
v) and purified by column chromatography,
2,4-Octadecadienoic acid PEG ester (ODE-
PEG5000) was obtained with a yield of 75%.

The spectral data of this ODE-PEG5000 is as follows. ¹ H-NMR (270 MHz, CDCl ₃ , δ (pp
m)) 0.88 (3H, t), 1.25 (22H, m), 2.
17 (2H, m), 3.44 (3H, t), 3.6-
3.8 (455, m), 5.35 (1H, d, J = 15)
Hz), 5.87 (2H, m), 7.2 (1H, m) IR (KBr, cm ^-1 ) 1720 (-C (= O) -O-C-) 1640, 1610 (-C = C). -C = C-)

The structural formula of ODE-PEG5000 is shown below.

CH ₃ (CH) ₁₂ CH = CH-CH = CHCO
-(OCH ₂ CH ₂ ) nOCH ₃ (n = about 114)

Example 2 According to the procedure of Example 1, 0.1 mol of 2,4-octadecadienoyl chloride (ODC) was synthesized.

PEG with 0.1 mol of amine end groups
_{(CH 3 O (CH 2 CH} 2 O) nCH 2 CH 2 CH 2 NH 2, n =
45, molecular weight 2000) was dissolved in 4000 ml of dry benzene, and then 0.12 mol of ODC was added, and the mixture was added at 20 ° C.
And reacted with stirring for 12 hours. After completion of the reaction, the solvent was removed under reduced pressure to obtain a reaction product. This reaction product was purified in the same manner as in Example 1 to obtain 2,4-octadecadienoic acid PEG amide (ODA-PEG2000).

The spectral data of this ODA-PEG2000 is as follows. ¹ H-NMR (270 MHz, CDCl ₃ , δ (pp
m)) 0.88 (3H, t), 1.25 (22H, m), 1.
80 (2H, q), 2.17 (2H, m), 2.80
(2H, t), 3.44 (3H, t), 3.55-3.
90 (180H, m), 5.35 (1H, d, J = 15)
Hz), 5.87 (2H, m), 7.20 (1H, m) IR (KBr, cm ^-1 ) 1680 (-CO-NH-) 1640, 1610 (-C = C-C = C-).

The structural formula of ODA-PEG2000 is shown below.

CH ₃ (CH ₂ ) ₁₂ CH = CH-CH = CHCO
NH (CH ₂ ) ₃ (OCH ₂ CH ₂ ) nOCH ₃ (n = 45)

Example 3 ODE-PEG5000 obtained in Example 1 and a polymerizable lipid 1,2-di (2,4-octadecadienoyl) -3
A mixture of phosphatidylcholine (DODPC) and cholesterol (Chol) (DODPC / Chol /
ODE-PEG5000 = 22/10/1, weight ratio)
Was dissolved in benzene at a concentration of 5 wt% and the solvent was removed to obtain a mixed lipid. 15 wt% of the obtained mixed lipid
The lipid vesicles were prepared by the extrusion method in addition to physiological saline. The resulting lipid vesicles were cooled under ice,
The polymerizable component of the lipid vesicle membrane was polymerized by γ-ray irradiation (irradiation dose 0.75 Mrad) to obtain a polymerized lipid vesicle modified with ODE-PEG5000.

Example 4 In Example 3, the ODA-PEG2000 obtained in Example 2 was used instead of ODE-PEG5000 to obtain a polymerized lipid vesicle modified with ODA-PEG2000 in the same manner as in Example 3. .

Comparative Example 1 ODE which is an amphipathic PEG derivative in Example 3
-Unmodified polymerized lipid vesicles were obtained in the same manner as in Example 3 except that PEG5000 was not mixed.

Comparative Example 2 Ester (S-PEG5) of non-polymerizable stearic acid and monomethoxy PEG (polymerization degree 114, molecular weight 5000)
000), distearoylphosphatidylcholine (DS
PC) and cholesterol mixture (S-PEG50
00 / DSPC / Chol = 22/10/1, weight ratio)
Was dissolved in benzene at a concentration of 5 wt% and the solvent was removed to obtain a mixed lipid. Using this mixed lipid, a lipid vesicle was prepared by the extrusion method in the same manner as in Example 3,
Non-polymerized lipid vesicles modified with S-PEG5000 were obtained.

Comparative Example 3 2,4-Octadecadienoic acid and CH ₃ O (CH ₂ CH ₂ O) ₃
-H ester (ODE-PEG150)
Polymerized lipid vesicles were obtained in the same manner as in Example 3 except that E-PEG5000 was used instead of.

Comparative Example 4 An ester (OLE-PEG) consisting of oleic acid and monomethoxy PEG (molecular weight 5000) was converted into ODE-PEG5.
Lipid vesicles were obtained in the same manner as in Example 3 except that the lipid vesicles were used instead of 000.

Test Example 1 Human plasma was added to the lipid vesicles obtained in Examples 3 and 4 and Comparative Examples 1 to 4 at a concentration of 5 wt%, and the mixture was incubated at 37 ° C. for 10 minutes, and then lipids were observed by a phase contrast microscope. The aggregation behavior of the endoplasmic reticulum was investigated.

As a result, in the case of lipid vesicles modified with an amphipathic PEG derivative (Examples 3 and 4 and Comparative Example 2,
4), almost no aggregates of lipid vesicles were observed, whereas unmodified lipid vesicles (Comparative Example 1) or modified with a modifier consisting of PEG with a degree of polymerization of 3 (Comparative Example) 3), large aggregates were observed. In addition, it was found that the aggregates collected by the ultracentrifugation method contained plasma proteins, and the adsorption of plasma proteins on the surface of lipid vesicles was involved in the aggregation of lipid vesicles. The results are summarized in Table 1.

Test Example 2 The lipid vesicles obtained in Examples 3 and 4 and Comparative Examples 1 to 4 were incubated at 37 ° C. for 180 minutes, and then the lipid vesicles were precipitated by ultracentrifugation. In the case of polymerized lipid vesicles (Examples 1 and 2 and Comparative Example 3), no PEG derivative was detected in the supernatant, whereas in the case of unpolymerized lipid vesicles (Comparative Example 2) and non-polymerizable PEG. In the case of a lipid vesicle using a derivative (Comparative Example 4), PEG was obtained from the supernatant.
A derivative was detected. It was found that in the lipid vesicles of Comparative Examples 2 and 4, the PEG derivative leaked from the membrane. The results are summarized in Table 1.

Test Example 3 The lipid vesicles obtained in Examples 3 and 4 and Comparative Examples 1 to 4 and fresh human red blood cells were mixed in equal volumes and incubated at 37 ° C. for 60 minutes. When the hemolysis of erythrocytes was observed by controlling physiological saline, compared with the control, in the case of polymerized lipid vesicles (Examples 1 and 2 and Comparative Example 3), hemolysis of blood cells was scarcely observed. In the case of non-polymerized lipid vesicles (Comparative Example 2) and the case of lipid vesicles using a non-polymerizable PEG derivative (Comparative Example 4), hemolysis of erythrocytes was about 5%. The results are summarized in Table 1.

[0059]

[Table 1]

From the results shown in Table 1, it can be seen that the lipid vesicles containing no PEG derivative as a membrane constituent (Comparative Example 1) adsorb proteins to the surface thereof and aggregate the lipid vesicles. In the case of using a non-polymerizable PEG derivative (Comparative Example 2) and the case of lipid vesicles using a non-polymerizable PEG derivative (Comparative Example 4), the PEG derivative leaked from the membrane,
You can see that red blood cells are hemolyzed. On the other hand, when the polymerizable PEG derivative is used (Examples 3 and 4), protein adsorption does not occur and the PEG derivative does not leak out, so that red blood cell elution does not occur.

[0061]

According to the present invention, a novel and useful amphipathic PEG derivative can be obtained. Further, since the amphipathic agent of the present invention contains the above amphipathic PEG derivative as a component, the amphipathic agent gathers at the interface between the hydrophobic phase and the hydrophilic phase to form a micelle having a polymerizable hydrophobic portion and a hydrophilic portion having a large volume. Can be formed.

Since the lipid vesicle modifying agent of the present invention comprises the above-mentioned amphipathic PEG derivative as a component, the hydrophilic part of the modifying agent is aggregated on the surface of the lipid vesicle, so that the adsorption of proteins etc. can be prevented. Layers can be formed.

The lipid endoplasmic reticulum of the present invention comprises the above-mentioned amphipathic P
Since the EG derivative is used as a membrane component, the above modified layer is formed on the surface of the lipid vesicles to prevent aggregation due to adsorption of proteins, etc., and to be compatible with blood components and highly stable in storage. The endoplasmic reticulum is obtained.

Claims (4)

[Claims] 1. A compound represented by the general formula: R ¹ O- (CH ₂ CH ₂ O) n- (CH ₂ ) m- [1] (wherein R ¹ is an alkyl group having 1 to 5 carbon atoms or hydrogen). A represents an atom, n is an integer of 5 to 1000, and m is an integer of 0 to 4.) An amphipathic polyethylene glycol having a hydrophilic part having a polyethylene glycol chain and a polymerizable hydrophobic part. Derivative. 2. An amphipathic agent comprising the amphipathic polyethylene glycol derivative according to claim 1. 3. A lipid vesicle modifying agent comprising the amphipathic polyethylene glycol derivative according to claim 1. 4. A lipid vesicle comprising the lipid vesicle modifier and the lipid vesicle-forming component according to claim 3 as a membrane constituent component.