JPH06234626A - Polymerizable proteoliposome - Google Patents

Abstract

PURPOSE:To obtain a polymerizable proteoliposome imparted with stability by the introduction of polymerizable functional group and resistant to the deterioration of the acquired function of protein. CONSTITUTION:This polymerizable proteoliposome contains a polymerizable phospholipid of the formula [one or both of R1 and R2 are acyl of the formula R'-CH=CHCH=CHCO (R' is 5-17C alkyl) or one is the above acyl and the other is a saturated or unsaturated 10-22C fatty acid residue] and a protein capable of being supported with the liposome membrane constituted of the polymerizable phospholipid. When bacteriorhodopsin which is a membraneous protein transporting proton with light is used as the protein to be supported, the function of the protein cannot be developed because the proton concentration gradient formed by the light-irradiation of bacteriorhodopsin is not maintained in a state of monomer before polymerization. The function is first developed by polymerization and the development of the function and the extent of developed function of the protein can be controlled by controlling the polymerization degree. The particle size of liposome is also controllable by starting the polymerization when the particle diameter reaches the desired level.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a polymerizable liposome having a phosphorylcholine-type phospholipid having a diene group as a functional group, which is polymerizable by light and a radical initiator, and carrying a protein having various functions. It relates to proteoliposomes.

[0002]

BACKGROUND ART Liposomes, which are vesicles closed by lipid membranes, can contain various substances in their lumens or membranes. Therefore, many studies have been conducted to utilize the liposome as an in-vivo drug carrier by utilizing this property. In addition, liposomes are also useful as a model of biological membranes, and as liposomes alone or as proteoliposomes in which various membrane proteins are immobilized on lipid membranes,
It is widely used to study the biophysical properties of biological membranes such as mass transport, energy conversion, and information transmission. More recently, its application has been promoted in the fields of cosmetics and foods.

However, the mechanical and physical stability of liposomes has become a problem in the application of liposomes. In other words, it is only the hydrophobic cohesive force of amphipathic substances such as phospholipids that forms the structure of liposomes, and the binding force for maintaining the membrane structure is weak and unstable. It's a problem. In order to widely apply the liposomes to the various fields mentioned above, it is indispensable to improve this instability, and a technique for that purpose is strongly demanded.

In response to the demand for improving the instability of such liposomes, a photopolymerizable functional group is introduced into the phospholipid molecule constituting the liposome to form the liposome, which is then polymerized by irradiation with light to form a polymer. Has been proposed and a technique for stabilizing the structure has been attracting attention.

Specifically, a typical phospholipid, lecithin, has a diacetylene group (J. Polmy. Sci., Polym. Lett. Ed.,
19,95 (1981)), diene group (Angew.Chem.Int.Ed.Engl.,
20,90 (1981)), styrene group (J.Am.Chem.Soc., 104,456
(1982)), methacrylic (J. Am. Chem. Soc., 104, 791 (198
Those in which a photopolymerizable functional group such as 2)) is introduced have been devised. Furthermore, a phospholipid in which a diene group, which is a polymerizable functional group, is introduced into only one of the hydrophobic alkyl chains has been proposed (JP-A-62-81394).

[0006] As an example of supporting a protein on such a polymerizable liposome, a polymerizable proteoliposome (FEBS Lett.154,1) composed of a phospholipid having a diacetylene group introduced thereinto and a bacteriorhodopsin which is a photoresponsive protein is used. ,Five
(1983)), and a polymerizable proteoliposome (FEBS Lett. 132, 2, 313 (1983)), which also comprises a phospholipid into which a diacetylene group is introduced and ATP synthase.

[0007]

The above-mentioned polymerizable proteoliposomes both contain a diacetylene derivative, and the polymerizable functional group is located at the center of the alkyl group,
Moreover, since a conjugated unsaturated bond is generated, the liposome structure becomes rigid. Therefore, when the protein is retained in the liposome, the function may be adversely affected.

The present invention has been made to solve the above problems in the prior art. That is, it is an object of the present invention to provide a polymerizable proteoliposome which is provided with stability by introducing a polymerizable functional group and which does not impair the function of the retained protein.

[0009]

The above object can be achieved by the present invention described below. That is, the present invention is a polymerizable proteoliposome containing a polymerizable phospholipid represented by the following general formula (1) and a protein that can be supported on a liposome membrane constituted by the polymerizable phospholipid.

[0010]

[Chemical 2] [Wherein _one or both of R ₁ and R ₂ are R′-CH═C
HCH = CHCO- (wherein R ′ represents a C _{5 to} C ₁₇ alkyl group) represents an acyl group represented by R ₁ and R ₂
When only one of the above is an acyl group, the other one is a saturated or unsaturated C _{10 to} C ₂₂ fatty acid residue. Further, the present invention is a polymerizable proteoliposome in which the protein that can be supported on the liposome membrane is bacteriorhodopsin.

The polymerizable phospholipid used in the present invention has a diene group, which is a polymerizable functional group, introduced at the root of the hydrophobic alkyl chain, that is, at the most hydrophilic group side. Compared to acetylene derivatives, it has less restrictions on long-chain alkyl groups and has a more flexible structure. Therefore, even in a polymerizable proteoliposome in which a protein is retained in such a polymerizable liposome, the steric load on the protein structure is small, and the adverse effect on the functional expression of the protein can be reduced.

Further, when a polymerizable phospholipid in which a diene group which is a polymerizable functional group is introduced into only one of the two hydrophobic alkyl chains is used, the load on the retained protein can be further reduced. Even when a diene group is introduced into only one of such alkyl chains, the polymerization proceeds due to light irradiation or the like to allow polymerization, and the liposome structure can be stabilized.

The polymerizable phospholipid of the present invention has an advantage that less unsaturated bonds remain in addition to the above-mentioned characteristics.

Further, the polymerizable proteoliposome of the present invention differs from conventional polymerizable proteoliposomes using a diacetylene derivative in the case of retaining bacteriorhodopsin which is a membrane protein that transports protons by light in the liposome. In the pre-polymerization monomer state, the concentration gradient of protons formed by light irradiation to bacteriorhodopsin cannot be maintained, and therefore the function cannot be exerted, and the function can be expressed only by polymerization. Therefore, the functional expression of a protein and its expression level can be controlled by controlling the presence or absence of polymerization by light or the like and the degree of polymerization.

[0015] In general, liposomes, particularly liposomes containing a membrane protein such as bacteriorhodopsin, tend to fuse with the passage of time, and their particle size may increase over time. In such a case, when a polymerizable lipid is used as the lipid, the fusion can be stopped by polymerizing at the time when the required particle size is reached, and the particle size of the liposome can be controlled.

As the method for polymerizing the proteoliposome of the present invention, ultraviolet or gamma ray irradiation or A
Addition of a radical initiator such as IBN may be mentioned, and the polymerization conditions thereof are appropriately set within a range that does not impair desired properties and functions of the proteoliposome.

In the present invention, in addition to the above-mentioned polymerizable lipid, other lipids capable of forming liposomes can be added within a range that does not impair the desired characteristics. The phospholipids, glycolipids, and quaternary ammonium salts having the amphipathic property described above can be used.

These lipid molecules capable of forming a membrane are constituted by having a long-chain alkyl group having 8 or more carbon atoms and a hydrophilic group, and the hydrophilic group is, for example,

[0019]

[Chemical 3] Cations, for example

[0020]

[Chemical 4] Anions such as, for example

[0021]

[Chemical 5] Non-ion such as, for example

[0022]

[Chemical 6] Any zwitterion such as

Of these lipid materials, glycerophospholipids such as phosphatidylcholine (lecithin), phosphatidylethanolamine and diphosphatidylglycerol; sphingolipids such as sphingomyelin and ceramideciliatin; celebrideside, sulfatide, ceramide oligohexoside and the like. Glyceroglycolipids such as glycosphingolipids and glycosyldiacylglycerols containing carbohydrate as a hydrophilic group are preferred, and phospholipids such as glycerophospholipids and sphingophospholipids are particularly preferred.

If desired, various substances may be added to the liposome-forming mixed solution within a range that does not impair the formation of liposomes. For example, a substance that is used in the membrane or lumen of a liposome to utilize its function;
Various metal ions; Membrane stabilizers such as cholesterol and sterols such as cholestanol; Charged substances such as stearylamine, phosphatidic acid and dicetylphosphate;
An antioxidant such as tocopherol may be used. Examples of substances that are used in the lipid membrane of liposomes or in the lumen thereof to utilize their functions include antibacterial compounds, antiviral compounds, antifungal compounds, antiparasitic compounds, and antitumor compounds. Examples thereof include various compounds such as sex compounds, vitamins, membrane proteins, various proteins such as enzymes, hormones, radiolabeling substances, fluorescent compounds, polysaccharides, nucleic acids and the like. The content of these additives is determined according to the purpose of use. Regarding the membrane stabilizer, the charged substance, and the antioxidant, for example, 1 part by weight of the liposome-forming material,
It is preferable that the sterols are 0 to 2 parts by weight, the charged substance is 0 to 0.5 parts by weight, and the antioxidant is 0 to 0.2 parts by weight.

As the protein capable of forming the proteoliposome of the present invention, a protein called "membrane protein" such as bacteriorhodopsin and membrane-bound ATP-degrading enzyme present in purple membrane, and other than the membrane protein, A protein or the like having a hydrophobic portion on a part of its surface and capable of binding to a lipid membrane can be used. The protein content is 0.5 to 20 mM, preferably 1 to 10 mM. When using a membrane protein, it does not have to be purified to a pure form,
For example, it can be used in a state in which a lipid membrane fragment is attached.

[0026]

The present invention will be described in more detail with reference to the following examples.

<Example 1> (Preparation of bacteriorhodopsin) P. Oesterhe
lt and W. The method of Stoeckenius (Met
hod. Enzymol. , 31, (1974), 66
7-678), the highly halophilic Halobacte
The purple membrane was extracted from the rum galloium RJ strain,
K. Huang et al. (Proc. Natl. Aca
d. Sci. , USA, 77, (1980), 323).
According to the surfactant (trade name: Triton X-
100, manufactured by Wako Pure Chemical Industries, Ltd. to delipidate the purple membrane to obtain the membrane protein bacteriorhodopsin (hereinafter abbreviated as bR).

(Preparation of Proteoliposome)

[0029]

[Chemical 7] 0.5 ml of a chloroform solution containing 20 mM of the above-mentioned polymerizable phospholipid was put in an eggplant-shaped flask, the solvent was distilled off using a rotary evaporator under light blocking, and then placed in a desiccator to completely remove the solvent using a vacuum pump. To prepare a lipid thin film. Next, 1m of 3M potassium chloride solution
l was added, and the mixture was treated with a vortex mixer for 2 minutes to disperse the lipid thin film, and then a water bath type ultrasonic oscillator (trade name:
Sonifier-B-15 type, using hop horn, Bra
(manufactured by Nson) for 30 minutes to obtain a liposome dispersion.

Next, bR12 was added to the liposome dispersion.
The operation of adding 0 ug, freezing with liquid nitrogen, thawing at 20 ° C., and vortex mixer treatment for 30 seconds was repeated 3 times. After this, the treatment solution is transferred to a dialysis tube,
Dialyze against 4 L of M potassium chloride aqueous solution for 2 days,
Bacteriorhodopsin giant proteoliposome (hereinafter,
bR-GUV) was prepared.

(Polymerization of proteoliposome) bR-
Put 0.5 ml of GUV in a 1 cm square cell, and put an ultraviolet lamp (T-15C, no filter, VILBER-L
(Made by OURMAT) to polymerize the proteoliposomes. The degree of polymerization of proteoliposomes was checked by measuring the absorbance at 256 nm after diluting the liposome dispersion liquid 1000 times. The relationship between ultraviolet irradiation time and absorbance is shown in FIG.

(Measurement of Proton Pumping Capacity) It is known that bR has a function of transporting protons across a membrane when irradiated with light. Therefore, the bR-G
After UV was diluted 100-fold with a 10 mM potassium chloride aqueous solution, a halogen light source (TECHNO LIGHT, KLS-) was passed through a bandpass filter (multilayer film filter, Asahi spectroscopy, central wavelength 555 nm, value width 30 nm).
2150, manufactured by Kenko), and irradiated with bR-GUV
The proton pumping ability was evaluated by measuring the pH change of the external liquid of the dispersion liquid. FIG. 2 shows the relationship between the degree of polymerization of bR-GUV and the proton pumping ability. As is clear from the results shown in FIG. 2, by controlling the degree of polymerization of proteoliposomes, bR
It was possible to control the proton pumping ability of the.

(Stability of Proteoliposome) bR
-A polymerized product of GUV (hereinafter abbreviated as bR-GUV-P) and a non-polymerized product (hereinafter referred to as bR-GUV).
-M) was stored at 4 ° C for 1 month. After saving, b
R-GUV-M shows an increase in liposome particle size and a decrease in proton pumping ability, whereas bR-GUV-P does not show an increase in liposome particle size and a decrease in proton pumping ability, and has good stability. Met.

<Example 2> (Preparation of ATP-degrading enzyme TF ₀ F ₁ ) The Jour
nal of BIOLOGICAL CHEMIST
RYVol, 250, No. 19, p7910-791
6, and p7917-7923 (1975), culturing thermophilic bacterium PS3 and extracting and purifying ATP-degrading enzyme TF ₀ F ₁ were carried out to prepare TF ₀ F ₁ .

(Preparation of proteoliposome) After forming a lipid thin film in the same manner as in Example 1, 1 ml of 10 mM potassium chloride, 2% sodium cholate, 10 mM Tris-hydrochloric acid buffer solution (pH 8.0) was added. After being treated with a vortex mixer for 2 minutes to disperse the lipid thin film, it was treated with a water bath type ultrasonic oscillator (Sonifire-B-15 type, using Hophorn, manufactured by Branson) for 30 minutes to obtain a liposome dispersion liquid. It was

Next, TF ₀ F was added to the liposome dispersion.
₁ 125 μg was added and sonication was performed for 5 minutes. Then, the treatment solution was transferred to a dialysis tube, and 2 liters of 1 were added.
1 day against 0 mM potassium chloride, 10 mM Tris-HCl buffer (pH 8.0), followed by 2 liters of 1
Dialyze against 0 mM potassium chloride aqueous solution for 1 day
F ₀ F ₁ small liposomes (abbreviated as TF ₀ F ₁ -SUV) were prepared.

(Polymerization of Proteoliposome) TF ₀
0.5 ml of F ₁ -SUV was placed in a 1 cm square cell and irradiated with an ultraviolet lamp (T-15C, no filter, manufactured by VILBER-LOURMAT) in the same manner as in Example 1 to polymerize the proteoliposome.

(Stability of Proteoliposome) TF
Polymerization of ₀ F ₁ -SUV (hereinafter referred to as TF ₀ F ₁ -S
UV-P) and unpolymerized (hereinafter,
TF ₀ F ₁ -SUV-M) was stored at 4 ° C. for 1 month. After storage, TF ₀ F ₁ -SUV-M showed an increase in liposome particle size, whereas TF ₀ F ₁ -SUV-
In P, the liposome particle size did not increase, and the stability was good.

(Freeze-thaw resistance of proteoliposomes) The above-mentioned TF ₀ F ₁ -SUV-P and TF ₀ F ₁ -SUV-M were frozen in liquid nitrogen and thawed at 20 ° C. to perform proteolysis against freeze-thawing. The stability of liposomes was evaluated by turbidity before and after freeze-thawing. The result is shown in FIG. As is clear from FIG. 3, TF ₀ F ₁ -SUV-M shows an increase in turbidity due to an increase in particle size of proteoliposomes before and after freeze-thawing, whereas TF ₀ F ₁ -SUV-P shows freeze-thawing. No change in turbidity was observed before and after, and the stability against freeze-thawing was good.

[0040]

EFFECTS OF THE INVENTION According to the present invention, it is possible to provide a polymerizable proteoliposome in which a photopolymerizable functional group is introduced to impart stability and which does not impair the function of the retained protein. It will be possible.

Furthermore, the polymerizable proteoliposomes of the present invention, particularly the proteoliposomes carrying bacteriorhodopsin, can control the functional expression of proteins and the expression level thereof by controlling the presence or absence of polymerization by light or the like and the degree of polymerization. It is possible.

[Brief description of drawings]

FIG. 1 is a graph showing the relationship between the UV irradiation time and the absorbance of a liposome dispersion liquid measured in Example 1 to determine the degree of polymerization of proteoliposomes.

FIG. 2 is a graph showing the relationship between the degree of polymerization and the proton pumping ability of the bacteriorhodopsin giant proteoliposome obtained in Example 1.

FIG. 3: Freeze-thaw resistance of the polymerized TF ₀ F ₁ small liposomes obtained in Example 2 (TF ₀ F ₁ -SUV-P) and those not polymerized (TF ₀ F ₁ -SUV-M). It is a graph which shows the change of the turbidity before and after freeze-thawing measured for the evaluation of the sex.

 ─────────────────────────────────────────────────── ─── Continued Front Page (72) Inventor Tomoko Maruyama 3-30-2 Shimomaruko, Ota-ku, Tokyo Canon Inc.

Claims (2)

[Claims] 1. A polymerizable proteoliposome containing a polymerizable phospholipid represented by the following general formula (1) and a protein that can be supported on a liposome membrane constituted by the polymerizable phospholipid. [Chemical 1] [Wherein _one or both of R ₁ and R ₂ are R′-CH═C
HCH = CHCO- (wherein R ′ represents a C _{5 to} C ₁₇ alkyl group) represents an acyl group represented by R ₁ and R ₂
When only one of the above is an acyl group, the other one is a saturated or unsaturated C _{10 to} C ₂₂ fatty acid residue. ] 2. The polymerizable proteoliposome according to claim 1, wherein the protein that can be supported on the liposome membrane is bacteriorhodopsin.