JP5057443B2 - Lipopolymer - Google Patents

Description

  The present invention relates to a novel lipopolymer having a lipid at one end and a binding functional group at the other end, a method for producing the same, and a lipopolymer bound to a substrate surface.

Lipopolymers and liposome compositions containing the same have been studied for therapeutic purposes, and the use of liposomal drug formulations and the like has been actively promoted.
Specifically, polyalkylene oxide-modified phospholipid (for example, Patent Document 1, Patent Document 2), neutral lipopolymer (Patent Document 3), (i) vesicle-forming lipid derivatized with a hydrophilic polymer, and (Ii) One using a suspension of liposomes containing one or more vesicle-forming lipids selected from neutral lipopolymers (Patent Document 4) is known.
The polymer species incorporated into the lipopolymer include polyethylene glycol, polylactic acid, polyglycolic acid, polylactic acid / polyglycolic acid copolymer, polyvinyl alcohol, polyvinylpyrrolidone, polymethoxazoline, polyethyloxazoline, polyhydroxypropylmethacrylamide, Drug delivery systems (Patent Document 5 and Patent Document 6) using a material selected from polymethacrylamide, polydimethylacrylamide and derivatized cellulose are known _. The aim of these inventions is a drug delivery system in which a lysolipid taken up into a cell and released from a liposome is effectively dealt with by a parasite or a parasite, or a drug as an anticancer agent.

  A method for synthesizing a polyethylene oxide-modified phospholipid at that time has been reported by M. C. Woodle et al. (Non-patent Document 1) and S. Zalipsky et al. (Non-patent Document 2). Specifically, after the end of polyethylene glycol is activated with 1,1′-carbonyldiimidazole or disuccinimidyl carbonate in an organic solvent, it is reacted with a phospholipid in the presence of a base such as triethylamine, and then A complicated method for obtaining polyethylene oxide-modified phospholipid by purification by reverse phase silica gel chromatography or dialysis has been used.

The present inventors have been researching a lipopolymer having a lipid molecule at a terminal portion and a biomembrane-like structure obtained by immobilizing a lipopolymer on a substrate. In this case, there is a specific problem required for use as a membrane different from the use as a drug that passes through the cell membrane. Lipopolymers require complicated processes such as the use of a polymer whose molecular weight and degree of dispersion have been controlled in advance by another method as raw materials (polyethylene glycol, etc.), and the molecular weight and degree of dispersion can be controlled, The synthesis scheme is limited to those that cannot be applied only to limited monomer species (such as oxazoline polymer), and it is proposed to use Si substituted with an ethoxy group or the like for bonding to the substrate ( Non-patent document 3, Non-patent document 4).
From the above, as the polymer of lipopolymer, the low degree of dispersion calculated as Mw / Mn (Mw represents the weight average molecular weight, Mn represents the number average molecular weight) means that this is close to monodispersion. It means that the distance between the metal substrate and the film is kept constant to some extent, and it is preferable to control the distance between the metal substrate and the film, This is also preferable from the viewpoint of ease of film forming. In other words, when the lipopolymer is fixed on a metal substrate and considered to be used as a film, it is indispensable to adjust the degree of dispersion and keep it low to some extent. In addition, it has been desired to develop a biomembrane-like structure that can be produced without going through complicated steps when obtaining a lipopolymer, and has a strong binding group to the substrate at the end of the lipopolymer.
JP 2002-37883 A JP 2002-363278 A JP 2003-505401 A Japanese Patent Laid-Open No. 2007-500239 Special table 2003-522193 gazette JP-T-2003-530339 Biochimica et Biophysica Acta, 1105, 193-200 (1992) Bioconjugate Chemistry, 4, 296-299 (1994) Biophysics Journal, 79, 1400-1414 (2000) Macromolecules, 34, 8858-8865 (2001)

  The problem to be solved by the present invention is a lipopolymer, and the degree of dispersion calculated by Mw / Mn (Mw represents a weight average molecular weight, Mn represents a number average molecular weight) is in a specific range. Is preferable as a constituent molecule of the film, the degree of dispersion can be in a specific range, such a lipopolymer can be obtained without going through complicated steps, and the terminal is a substrate. It is to provide a lipopolymer having a linking group and a membrane in which the lipopolymer is fixed on a gold substrate.

The inventors of the present invention have been diligently researching the above problems, and found that the above problems can be solved by applying RAFT polymerization (addition-cleavage transfer polymerization) as a method of forming a lipopolymer. The present invention has been completed.
A pyrrolidone polymer having a thiol group (SH group) as an active group at the end of the present invention or a lipopolymer composed of a 2-hydroxy-ethyl acrylate polymer and a lipid comprises steps (a) to (c) using RAFT polymerization. Next, the biological membrane-like structure of the present invention is obtained through the step (d) step of obtaining the biological membrane-like structure.
Specifically, it is as follows.
Step (a) introducing a lipid into the RAFT reagent by reacting the lipid with the RAFT reagent;
Step (b ₁ ) Polymer obtained by introducing a lipid into the RAFT reagent by reacting the compound obtained by introducing the lipid into the RAFT reagent obtained in the step (a) with a monomer N-vinyl-2-pyrrolidone Or a step (b ₂ ) by reacting a compound obtained by introducing a lipid into the RAFT reagent obtained in the step (a) with 2-hydroxy-ethyl acrylate as a monomer. A step of obtaining a polymer to which a hydroxy-ethyl acrylate polymer is added,
Step (c ₁ ) The polymer obtained by adding a polymer to the compound obtained by introducing a lipid into the RAFT reagent obtained in the step b ₁ is decomposed to have a thiol group (SH group) as an active group at the terminal. A step of producing a lipopolymer comprising a pyrrolidone polymer and a lipid , or (c ₂ ) decomposing a polymer obtained by adding a polymer to a compound obtained by introducing a lipid into the RAFT reagent obtained in the step b ₂ Production process of lipopolymer consisting of 2-hydroxy-ethyl acrylate polymer having thiol group (SH group) as an active group at the end and lipid, and process (d) immobilizing this lipopolymer on a gold substrate Obtaining a structure;
The steps (a) to (c ₁ ) or (c ₂ ) are shown in FIG.

  According to the present invention, a lipopolymer synthesizing method capable of dealing with various monomer species, and controlling the degree of dispersion Mw / Mn (Mw represents a weight average molecular weight and Mn represents a number average molecular weight) of the obtained lipopolymer. The lipopolymer can be produced without complicated steps, and a strong binding group can be added to the end of the lipopolymer at the end of the lipopolymer. A biological membrane-like structure can be obtained by fixing to the surface of the substrate.

［式中、Ｒは、下記の式（２）で表される脂質からなる基を表し、Ｒ _１ は、下記の式（３）又は（４）で表される基を表し、ｍは、１０≦ｍ≦１００の範囲の整数を表す。］

（式中、ｎは、１０、１２、１４、１６、１８、２２、２４のいずれかを表す。）

Lipopolymers of the present invention is represented by the following general formula (1), it has one end consisting of a lipid based, comprising a polymer that have a thiol group (SH group) at the other end as the active group It is a lipopolymer characterized by the above.

[Wherein, R represents a group consisting of lipids represented by the formula (2) below, _{R 1} represents a group represented by the following formula (3) or (4), m is 10 Represents an integer in the range of ≦ m ≦ 100. ]

(In the formula, n represents any of 10, 12, 14, 16, 18, 22, and 24.)

The resulting lipopolymer is set so that the degree of dispersion is in the range of 1.0 to 1.5. When the degree of dispersion is larger than this range, for example, more than 1.5, when forming a film on a gold substrate, the distance between the substrate and the film becomes non-uniform. It is not preferable. Preferably, 1.0 or more and 1.4 or less, more preferably 1.0 or more and 1.3 or less are considered suitable.
The molecular weight (Mn and Mw) and dispersity (Mw / Mn) were identified using an SEC column (Shodex KD-802.5 + KD-803) using polyethylene glycol as a standard substance. As a developing solvent, a solution obtained by adding 20 mmol / l of lithium bromide to DMF was used, and a differential refractive index detector was used as a detector.

The formula (1), R ₁ having a thiol group (SH group) at the terminal as the active group _{(R 1} represents a group represented by the structural formula (3) or (4).) And polymer By reacting a lipopolymer comprising lipid with a lipid represented by step (a) ROH (R is the same as R represented by the general formula (5)) and a RAFT reagent represented by the following structural formula (3), Step of introducing lipid into reagent, step (b ₁ ) RAFT reagent by reacting a compound having lipid introduced into the RAFT reagent obtained in the step (a) with N-vinyl-2-pyrrolidone as a monomer A step of obtaining a polymer in which a polymer is added to a compound into which a lipid is introduced, step (b ₂ ), a compound in which a lipid is introduced into the RAFT reagent obtained in the step (a), and 2-hydroxy-ethyl as a monomer Accel Relay A step of obtaining a polymer to which a 2-hydroxy-ethyl acrylate polymer has been added by reacting with a salt, and a step (c ₁ ) wherein the compound obtained by introducing lipid into the RAFT reagent obtained in the step b ₁ is A step of producing a lipopolymer composed of a pyrrolidone polymer having a thiol group (SH group) as an active group and a lipid at the terminal by decomposing the polymer obtained by adding the polymer, and (c ₂ ) obtained in the step b ₂ In addition, a polymer obtained by adding a polymer to a compound in which a lipid is introduced into a RAFT reagent is decomposed to comprise a 2-hydroxy-ethyl acrylate polymer having a thiol group (SH group) as an active group at the terminal and a lipid. Lipopolymer.

  The lipopolymer is fixed on the surface of a substrate such as a gold substrate, thereby constructing a biological membrane-like structure. The gold substrate can be in the form of particles or plates.

In order to produce the lipopolymer of the present invention, a RAFT reagent into which a lipid molecule represented by the structural formula (5) is introduced is used.
Double-chain lipid having a hydroxyl group using diisopropylcarbodiimide (DIC) and N, N′-dimethyl-4-aminopyridine (DMAP) (for example, 1,2-dipalmitoyl-rac-glycerol) and 4-cyanopentanoic Prepared by condensation reaction of acid dithiobenzoate.
The RAFT reagent into which lipid molecules are introduced is represented by the following structural formula (6) (R is the same as in the general formula (2)).

The RAFT reagent into which lipid molecules are introduced is produced by a condensation reaction of a double-stranded lipid having a hydroxyl group (for example, 1,2-dipalmitoyl-rac-glycerol) and 4-cyanopentanoic acid dithiobenzoate. It is described in detail in the following documents.
J. et al. Polym. Sci. Part A: Polm. Chem. 2006.44, 5809-5831, Macromol. Rapid Commun, 2006, 653-692.
The characteristics of the step of obtaining a polymer compound by using this reagent include (1) that it can be applied to various monomer species, and (2) that the molecular weight and degree of dispersion can be controlled. it can.
In the present invention, by using a RAFT reagent into which a lipid molecule is introduced, a polymer having a substantially uniform dispersion degree, a lipid at one end and a thiol group (SH group) as an active group at the other end. A lipopolymer composed of a pyrrolidone polymer and a lipid can be synthesized.

The lipopolymer of the present invention is produced through the following three steps.
Step (a): Step of introducing lipid into RAFT reagent by reacting lipid with RAFT reagent A double-chain lipid (ROH) having a hydroxyl group and 4-cyanopentanoic acid dithiobenzoate (RAFT reagent) are reacted. Specifically, for example, 1,2-dipalmitoyl-rac-glycerol (0.2 mmol) is used as the double-stranded lipid, and 4-cyanopentanoic acid dithiobenzoate (0.4 mmol), N, N′- Dimethyl-4-aminopyridine (DMAP, 0.35 mm0l) and diisopropylcarbodiimide (DIC, 0.8 mmol) are added and allowed to react at room temperature under nitrogen atmosphere with chloroform as solvent.
After completion of the reaction, the solvent is removed, and purification by column chromatography and recycle HPLC can be used to obtain a RAFT reagent into which lipid molecules are introduced.
Specific details of the reaction are described in Example 1 below.

Step (b ₁ ): N-vinyl-2-pyrrolidone (in the case of representing the structural formula (3) of R _{1 in} the general formula (1)) obtained by introducing a lipid into the RAFT reagent obtained in the step (a) A step of adding a pyrrolidone polymer to a compound in which a lipid is introduced into a RAFT reagent by reacting with a RAFT reagent into which a lipid molecule has been introduced (a double-stranded lipid having a hydroxyl group (ROH) obtained in the step (a)) A product obtained by reacting 4-cyanopentanoic acid dithiobenzoate (RAFT reagent) with a monomer N-vinyl-2-pyrrolidone to introduce a pyrrolidone polymer into a compound in which lipid is introduced into the RAFT reagent. The added product is obtained.
Specifically, the RAFT reagent into which lipid molecules are introduced is reacted with N-vinyl-2-pyrrolidone as a monomer in the presence of a solvent such as tetrahydrofuran, and the resulting precipitate is purified. A polymer obtained by adding a pyrrolidone polymer to a compound into which is introduced is obtained.
Specific details of the reaction are described in Example 2 below.

Step (b ₂ ): a compound obtained by introducing a lipid into the RAFT reagent obtained in the step (a) and 2-hydroxy-ethyl acrylate (in the case of representing the structural formula (4) of R _{1 in} the general formula (1)), A step of adding 2-hydroxy-ethyl acrylate to a compound in which a lipid has been introduced into the RAFT reagent by reaction. A RAFT reagent into which a lipid molecule has been introduced (the double-stranded lipid having a hydroxyl group (ROH obtained in the step (a)). ) With 4-cyanopentanoic acid dithiobenzoate (RAFT reagent)) and 2-hydroxy-ethyl acrylate, which is a monomer, to react with the compound in which lipid is introduced into the RAFT reagent. -A product with addition of ethyl acrylate polymer is obtained.
Specifically, the RAFT reagent into which lipid molecules have been introduced is reacted with 2-hydroxy-ethyl acrylate as a monomer in the presence of a solvent such as tetrahydrofuran, and the resulting precipitate is purified, whereby the lipid is added to the RAFT reagent. A polymer obtained by adding a 2-hydroxy-ethyl acrylate polymer to the introduced compound is obtained.
Specific details of the reaction are described in Example 5 below.

Step (c ₁ ): By decomposing the pyrrolidone addition polymer obtained by adding N-vinyl-2-pyrrolidone to the compound obtained by introducing lipid into the RAFT reagent obtained in the step (b ₁ ) A step of obtaining a lipopolymer comprising a pyrrolidone polymer having a thiol group (SH group) as an active group at the terminal and a lipid.
Tris (2-carboxyethyl) phosphine hydrochloride obtained by adding a polymer to the compound obtained by introducing a lipid into the RAFT reagent obtained in the step (b ₁ ) in a solvent such as tetrahydrofuran, Decomposition is carried out at room temperature in the presence of propylamine.
Specifically, a polymer obtained by adding N-vinyl-2-pyrrolidone to the compound obtained by introducing a lipid into the RAFT reagent obtained in the step (b ₁ ) (20 mg, Mn≈2000, equivalent to 0.01 mmol) ) With tetrahydrofuran (1 ml), a small amount of tris (2-carboxyethyl) phosphine hydrochloride and propylamine (50 μl), and reacted at room temperature. After the reaction, the precipitate of the reaction product is purified. A lipopolymer comprising a pyrrolidone polymer having a thiol group (SH group) as an active group at the terminal and a lipid is obtained.
Specific details of the reaction are described in Example 3 below.

Step (c ₂ ): By decomposing the 2-hydroxyethyl acrylate addition polymer obtained by adding 2-hydroxyethyl acrylate to the compound obtained by introducing lipid into the RAFT reagent obtained in the step (b ₂ ). And a step of obtaining a lipopolymer comprising a 2-hydroxyethyl acrylate polymer having a thiol group (SH group) as an active group at a target end and a lipid.
Tris (2-carboxyethyl) phosphine hydrochloride obtained by adding a polymer to the compound obtained by introducing a lipid into the RAFT reagent obtained in the step (b ₂ ) in a solvent such as tetrahydrofuran, Decomposition is carried out at room temperature in the presence of propylamine.
Specifically, a polymer (20 mg, Mn≈2000, equivalent to 0.01 mmol) obtained by adding 2-hydroxyethyl acrylate to the compound obtained by introducing a lipid into the RAFT reagent obtained in the step (b ₁ ) is obtained. , Tetrahydrofuran (1 ml), a small amount of tris (2-carboxyethyl) phosphine hydrochloride and propylamine (50 μl) were added and reacted at room temperature. After the reaction, the precipitate of the reaction product was purified to obtain the desired terminal. A lipopolymer comprising a 2-hydroxyethyl acrylate polymer having a thiol group (SH group) as an active group and a lipid is obtained.

Step (a) is as follows.
Double-chain lipid having a hydroxyl group in a two-necked eggplant flask (eg, 1,2-dipalmitoyl-rac-glycerol, 0.2 mmol), 4-cyanopentanoic acid dithiobenzoate (0.4 mmol), N, N ′ -Dichloro-4-aminopyridine (DMAP, 0.35 mmol) was added, dried under high vacuum for 2 hours, then purged with nitrogen to form a nitrogen atmosphere and dehydrated with calcium hydride (10 ml) and Diisopropylcarbodiimide (DIC, 0.8 mmol) was added, and the mixture was stirred at room temperature under a nitrogen atmosphere for 3 days. After completion of the reaction, the solvent was removed under reduced pressure, and purification was performed using column chromatography using silica gel (developing solvent hexane: ethyl acetate = 2: 1), and further purification was performed using recycle HPLC to introduce lipid molecules. RAFT reagent was obtained. The yields are n = 12 (36%), n = 14 (63%), n = 16 (31%). Confirmation data of NMR (n = 14) are as shown in FIG.

Step (b ₁ ) is as follows.
Tetrahydrofuran (THF) dehydrated with RAFT reagent (0.05 mmol), α, α'-azobis (isobutyronitrile) (AIBN, 0.02 mmol), calcium hydride, etc., with lipid molecules introduced into a pressure-resistant reaction tube that can be sealed , 40 mmol), N-vinyl-2-pyrrolidone (NVP, 40 mmol) from which the stabilizer has been removed by performing vacuum distillation twice, and coagulation and decompression using a high vacuum pump (oil diffusion pump) and liquid nitrogen The melting operation was repeated three times to remove oxygen. After coagulation with liquid nitrogen again, it was sealed under reduced pressure. After melting, the polymerization reaction was carried out by stirring in an oil bath at 70 ° C or 80 ° C. After an arbitrary reaction time, the reaction product was purified by precipitation three times with diethyl ether to obtain a pyrrolidone polymer obtained by adding a polymer to a compound in which a lipid was introduced into the RAFT reagent.
The number average molecular weight (Mn), weight average molecular weight (Mw), and dispersity (Mw / Mn) of a pyrrolidone polymer obtained by adding a polymer to a compound obtained by introducing a lipid into the obtained RAFT reagent are shown in Table 1 below. To Table 4 below.

Step (c ₁ ) is as follows.
A pyrrolidone polymer (20 mg, Mn≈2000, equivalent to 0.01 mmol) obtained by adding a polymer to the compound obtained by introducing a lipid into the RAFT reagent obtained in the step (b) was placed in an eggplant flask, and tetrahydrofuran (THF 1 ml) and a small amount of tris (2-carboxyethyl) phosphine hydrochloride and propylamine (50 μl) were added and stirred at room temperature for 12 hours. After the reaction, the reaction product was purified by precipitation three times using diethyl ether to obtain a lipopolymer composed of a pyrrolidone polymer having a thiol group (SH group) as an active group at the terminal and a lipid.
The confirmation of the product was confirmed by the disappearance of UV absorption derived from the dithiobenzoate group at the terminal portion of the pyrrolidone polymer before decomposition. The change of the UV spectrum is as shown in FIG.
The results obtained are summarized as follows.

An example of fixing the lipopolymer to the gold substrate is as follows.
A lipopolymer comprising a lipopolymer (n = 12,1,2-dimyristoyl-rac-glycerol) comprising a pyrrolidone polymer having a thiol group (SH group) as an active group at the terminal and a lipid is passed through a syringe filter (20 μm). After removing the dust, the lipopolymer having a dry weight of 15.47 mg was obtained by drying on a high vacuum line for 2 hours. Next, the obtained dried lipopolymer was dehydrated with CaH ₂ and methanol (1.547 ml) purified by distillation was added and stirred to obtain a methanol solution of lipopolymer. In this lipopolymer methanol solution, a gold substrate obtained by depositing gold on mica in a vacuum deposition apparatus is immersed, and left overnight, so that a thiol group is formed as an active group at the end on the surface of the gold substrate. A lipopolymer composed of a pyrrolidone polymer having (SH group) and a lipid was immobilized. Scanning probe micrographs of a gold substrate on which a pyrrolidone polymer having a thiol group (SH group) as an active group at the end and a lipopolymer composed of lipids are fixed are shown in FIGS. It was confirmed that the lipopolymer composed of a pyrrolidone polymer having a thiol group (SH group) as an active group and a lipid was immobilized at a thickness of about 4 nm or less from the mica surface.

Step (b ₂ ) is as follows.
Tetrahydrofuran (THF) dehydrated with RAFT reagent (0.05 mmol), α, α'-azobis (isobutyronitrile) (AIBN, 0.01 mmol), calcium hydride, etc. with lipid molecules introduced into a pressure-resistant reaction tube that can be sealed , 80 mmol), 2-hydroxy-ethyl acrylate (HEA, 40 mmol) from which the stabilizer was removed by performing vacuum distillation twice, and using a high vacuum pump (oil diffusion pump) and liquid nitrogen, coagulation, reduced pressure, The melting operation was repeated three times to remove oxygen. After coagulation with liquid nitrogen again, it was sealed under reduced pressure. After melting, the polymerization reaction was carried out by stirring in an oil bath at 70 ° C or 80 ° C. After an arbitrary reaction time, the reaction product was purified by precipitation three times using diethyl ether to obtain an addition polymer obtained by adding a polymer to a compound in which a lipid was introduced into the RAFT reagent.
The number average molecular weight (Mn), weight average molecular weight (Mw), and dispersity (Mw / Mn) of the addition polymer obtained by adding a polymer to the compound obtained by introducing lipid into the obtained RAFT reagent are shown in Table 1 below. As shown in

It is a figure which shows NMR of the compound which introduce | transduced the lipid into the RAFT reagent by making a lipid and a RAFT reagent react. It is a figure which shows the change of UV spectrum before and after amino-decomposing the addition polymer obtained by adding a polymer to the compound which introduce | transduced the lipid into the RAFT reagent obtained at the said process (b). It is a scanning probe microscope photograph figure (bird's-eye view) of the gold substrate surface which fixed the lipopolymer. It is the scanning probe microscope photograph figure (the figure seen from the perpendicular direction with respect to a substrate surface (top), and its sectional drawing (bottom)) of the gold substrate surface which fixed the lipopolymer.

Claims (4)

Formula represented by (1), lipoic polymer characterized by comprising a polymer that have a thiol group (SH group) lipids at one end as the active group at the other end.

[Wherein, R represents a group which Ru formula (2) below, _{R 1} represents a group represented by the following formula (3) or (4), m is 10 ≦ m ≦ Represents an integer in the range of 100. ]

(In the formula, n represents any of 10, 12, 14, 16, 18, 22, and 24.)

Engineering as (a) ROH (R is the same as R represented by the formula (2)) introducing a lipid into RAFT reagent by reaction with RAFT reagent represented by lipids and the following formula represented by (5) The process of
In step (b) RAFT reagent introducing lipid obtained in the step (a), N-vinyl-2-pyrrolidone or 2-hydroxy - by reacting ethyl acrylate, RAFT reagent polymer obtained by introducing the lipid Obtaining a polymer to which is added ,
2. The lipopolymer according to claim 1, wherein the lipopolymer is obtained through a step of decomposing the polymer obtained in the step (c) and converting the terminal to a thiol group .

A biological membrane-like structure, which is constructed by fixing the lipopolymer according to claim 1 or 2 to the surface of a gold substrate. A method for producing a lipopolymer according to claim 1,
Engineering as (a) ROH (R is the same as R represented by the formula (2)) introducing a lipid into RAFT reagent by reaction with RAFT reagent represented by lipids and the following formula represented by (5) The process of
In step (b) RAFT reagent introducing lipid obtained in the step (a), N-vinyl-2-pyrrolidone or 2-hydroxy - by reacting ethyl acrylate, RAFT reagent polymer obtained by introducing the lipid Obtaining a polymer to which is added ,
Step (c) Step of decomposing the polymer obtained in the step (b) to make the terminal a thiol group
A process for producing a lipopolymer, characterized in that

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