JP5019524B2 - Novel poly (meth) acrylate copolymer and delivery method to endoplasmic reticulum and Golgi apparatus - Google Patents

Description

  The present invention relates to novel poly (meth) acrylate copolymers and methods for using them to deliver desired substances to the endoplasmic reticulum and Golgi apparatus.

Glycolipids circulate between the cell membrane and these organelles along with other membrane components. In the Golgi apparatus, sugar chains are added one by one to synthesize new glycolipids, and in the lysosome, glycolipids are removed one by one by removing the sugar chains one by one. In endosomes, glycolipids taken into the cell from the cell membrane by endocytosis are distributed to the Golgi apparatus or lysosome. In addition, glycolipids synthesized by the Golgi apparatus and part of glycolipids taken up by endocytosis pass through here and return to the cell membrane. Depending on the type of cell, half of the glycolipids in the cell membrane circulate in 30 to 40 minutes, maintaining the homeostasis of the glycolipid composition. In most cases, the movement between membranes is performed by forming vesicles that maintain a bilayer membrane structure.
There is no known method for selectively delivering a desired substance to the Golgi apparatus or endoplasmic reticulum important for biosynthesis and modification of glycolipids.

By the way, a copolymer obtained by polymerizing 2-methacryloyloxyethyl phosphorylcholine (hereinafter also referred to as “MPC”) and (meth) acrylate is known as a drug release system (Non-patent Document 1), a blood compatible material (Non-patent Document 1). Reference 2), coating materials for powders for cosmetics (Patent Document 1), and coating materials for sugar chain compound adsorption control experimental instruments (Patent Documents 2 and 3) have been researched and developed.
Japanese Patent Laid-Open No. 2004-189652 JP 2004-275862 A JP 2004-290111 A Kazuhiko Ishihara, Tomoko Ueda, Nobuo Nakabayashi, “Drug Release Characteristics from Hydrogel Membranes with Phospholipid-like Structures”, 1989, Vol. 46, No. 10, p. 591-595 Tomoko Ueda, Kazuhiko Ishihara, Nobuo Nakabayashi, “Effect of hydrophilic group structure on blood compatibility of hydrogel”, Biomaterials, 1991, Vol. 9, No. 6, p.288-295

  The objective of this invention is providing the novel copolymer containing the (meth) acrylic acid derivative monomer which has phospholipid polar groups, such as MPC, and its novel use.

  As a result of earnest research, the present inventor has developed a (meth) acrylamide derivative monomer carrying a desired substance, a specific (meth) acrylic acid derivative monomer, and a (meth) acrylic acid derivative having a phospholipid polar group such as MPC. The present inventors have found that a desired substance can be selectively delivered to the endoplasmic reticulum and the Golgi apparatus by using the novel copolymer.

That is, the present invention
Formula (I):

A repeating unit represented by (hereinafter also referred to as “unit I”),
Formula (II):

A repeating unit represented by (hereinafter also referred to as “unit II”), and formula (III):

A repeating unit represented by (hereinafter also referred to as “unit III”)
Containing a copolymer [wherein
R ¹ , R ² , and R ⁴ are each independently a hydrogen atom or a methyl group;
R ³ represents a C8-24 linear or branched, saturated or unsaturated monovalent hydrocarbon group (this hydrocarbon group is unsubstituted or substituted with a halogen or azide group). Is);
R ⁵ is a divalent hydrocarbon group having 1 to 10 carbon atoms or a (poly) oxyethylene group;
R ⁶ is a C 1-4 divalent hydrocarbon group;
R ⁷ , R ⁸ , and R ⁹ are each independently a hydrogen atom or a hydrocarbon group having 1 to 4 carbon atoms;
A is a divalent bond selected from the group consisting of an ester bond, an amide bond, a urethane bond, and an ether bond;
L is a single bond or a linking group;
X is the residue of the substance to be delivered], and relates to a method of delivering the desired substance to the endoplasmic reticulum and Golgi using its copolymer.

  The copolymer of the present invention has excellent biocompatibility and can selectively deliver a desired substance to the endoplasmic reticulum and the Golgi apparatus.

In the formulas (I) to (III), R ¹ , R ² , and R ⁴ are each independently a hydrogen atom or a methyl group. R ¹ is preferably a hydrogen atom from the viewpoint of polymerization reactivity. R ² and R ⁴ are preferably methyl groups from the viewpoint of the stability of the resulting copolymer.

In said formula (II), R < ³ > is a C8-C24 linear or branched saturated or unsaturated monovalent hydrocarbon group. The hydrocarbon group may be unsubstituted or substituted with a halogen or azido group, but is preferably unsubstituted. Examples of the hydrocarbon group substituted with a halogen or an azido group include a 12-bromododecyl group and a 12-azidododecyl group.
The number of carbon atoms of the hydrocarbon group is usually 8 to 24, but is preferably 12 to 24 because the desired substance can be suitably delivered. In addition, the delivered substance has a small size to which the delivered substance has been delivered. 18-24 are preferable from the point that it can take into consideration that it becomes difficult to leak from a vesicle or a Golgi body.
The unsaturated hydrocarbon group preferably has 1 to 3, more preferably 1 to 2 carbon-carbon double bonds and / or carbon-carbon triple bonds in addition to carbon-carbon single bonds. Most preferably, it may contain one. The hydrocarbon group is preferably saturated because it can impart good cell membrane permeability to the resulting copolymer.
As the hydrocarbon group, linear higher alkyl groups such as lauryl group, tridecyl group, myristyl group, pentadecyl group, palmityl group, margaryl group, stearyl group, nonadecyl group, arachidyl group, and behenyl group; branched such as isostearyl group Examples are higher alkyl groups in the chain; unsaturated higher hydrocarbon groups such as myristolyl group, palmitoleyl group, oleyl group, linole group, linolel group, arachidol group, 12-docosel group and the like. Among these, a myristyl group, a palmityl group, a stearyl group, and particularly a palmityl group are preferred because the resulting copolymer can be given good affinity with a cell membrane.

In the formula (III), R ⁵ is a divalent hydrocarbon group having 1 to 10 carbon atoms, or (poly) oxyethylene group: — (OCH ₂ CH ₂ ) _n — (wherein n represents the present invention). There is no particular limitation as long as it does not impair the function of the unit III that imparts appropriate water solubility and biocompatibility to the copolymer of, for example, 1 to 10. The polyoxyethylene group is via —O—. And is preferably a divalent hydrocarbon group having 1 to 6 carbon atoms, particularly alkylene. Examples of such alkylene include methylene, ethylene, propylene, butylene, pentylene, hexylene, and the like, preferably ethylene, propylene, butylene, hexylene, and particularly ethylene.
R ⁶ is, for example, a divalent hydrocarbon group having 1 to 4 carbon atoms, preferably alkylene having 1 to 4 carbon atoms such as methylene, ethylene, propylene, butylene and the like, and ethylene is particularly preferable.
R ⁷ , R ⁸ , and R ⁹ are each independently a hydrogen atom or a hydrocarbon group having 1 to 4 carbon atoms, such as methyl or ethyl, and in particular, R ⁷ , R ⁸ , and R ⁹ are simultaneously A hydrogen atom or methyl, particularly methyl is preferred.
A is a divalent bond selected from the group consisting of an ester bond, an amide bond, a urethane bond, and an ether bond, and an ester bond, an amide bond, and particularly an ester bond is preferable.

  In the formula (I), L is a single bond or a linking group, and examples of the linking group include linear or branched saturated or unsaturated hydrocarbon groups. The number of carbon atoms of the hydrocarbon group is preferably 1 to 12, more preferably 2 to 12, and most preferably 2 to 6 from the viewpoint that high water solubility can be imparted to the resulting copolymer. This hydrocarbon group may be interrupted at one or more positions, preferably at one position, by oxygen atoms and / or sulfur atoms that are not adjacent to each other. The unsaturated hydrocarbon group may contain one or more double bonds, triple bonds, or both.

In the formula (I), X is a residue of the substance to be delivered. The substance to be delivered may be directly bonded to the L group, or may be an ester bond, an amide bond, a urethane bond, an ether bond, a disulfide bond, a diazo bond, a thiocarbamide bond, an acetal bond, a ketal bond, etc. They may be bonded via one or more bonds selected from the group consisting of: The bond is formed, for example, by reacting the functional group Z ¹ present in the substance to be delivered or the functional group Z ² newly introduced into the substance with the functional group Z ³ introduced into the L group. May be. Examples of these functional groups Z ^{1 to} Z ³ include amino group, carboxyl group, hydroxyl group, mercapto group, carboxamide group, imidate group, isothiocyanato group, and isocyanato group.
When the bond is physiologically cleaved in the endoplasmic reticulum and the Golgi apparatus, for example, by hydrolysis or enzyme, the substance to be delivered is released from the copolymer of the present invention, and in the endoplasmic reticulum and Golgi apparatus It may be able to exert activity and function. The bond is preferably an ester bond or an amide bond from the viewpoint of allowing such liberation and expressing the activity of the substance to be delivered. Conversely, in some cases, it is possible to suppress intracellular sugar chain synthesis by suppressing release of a substance to be delivered. From the viewpoint of suppressing such liberation, the bond is preferably an ether bond.

  The substance to be delivered is not particularly limited, and nucleotides; peptides, proteins, antibodies, enzymes; lipids; monosaccharides (glucose, galactose, fructose, mannose, etc.), disaccharides (maltose, isomaltose, lactose, sucrose) Oligosaccharides such as trisaccharides (maltotriose, etc.) and polysaccharides; labels such as radiolabels, magnetic labels, spin labels, fluorescent labels; inhibitors of sugar chain biosynthesis; glycosidase inhibitors (deoxynojirimycin) Etc.). In particular, glycosidase inhibitors (such as deoxynojirimycin) are useful.

  The copolymer of the present invention contains two or more kinds of units I each carrying residues of two or more kinds of substances to be delivered, and thereby carries two or more kinds of substances to be delivered. Can do. For example, in addition to the desired substance to be delivered, a label is also carried as the substance to be delivered, so that the delivered site can be conveniently labeled.

The unit III is preferably from the viewpoint of imparting good water solubility and biocompatibility to the copolymer of the present invention.

It is.

  The copolymer of the present invention may be a random copolymer, a block copolymer, or a highly alternating copolymer, and the units I, II, and III are averagely distributed throughout the copolymer molecule. Random copolymers are preferred because the reaction field uniformity is maintained when a biological reaction to the group X occurs in the Golgi apparatus or endoplasmic reticulum.

In the copolymer of the present invention, the unit I has a role of carrying the substance to be delivered. Unit II has a role of imparting cell membrane penetrability and target directivity to the endoplasmic reticulum and Golgi apparatus to the copolymer of the present invention. Unit III has a role of imparting water solubility and biocompatibility to the copolymer of the present invention. Due to the role of each unit, the copolymer of the present invention can penetrate the cell membrane while ensuring water solubility and biocompatibility. As a result, the substance to be delivered is endoplasmic reticulum and Golgi apparatus. To be delivered selectively. By changing the ratio of these units, the cell membrane penetrability, target directivity, water solubility, and biocompatibility of the copolymer of the present invention can be appropriately controlled.
In the copolymer of the present invention, the content ratio of the unit I, the unit II, and the unit III is a molar ratio from the viewpoint of imparting appropriate cell membrane penetrability, water solubility, biocompatibility and the like to the copolymer of the present invention. It is preferably 0.1 to 2: 0.1 to 2: 1, particularly 0.1 to 1: 0.1 to 1: 1, especially 0.1 to 1: 0.2 to 1: 1.
The method for changing the ratio of the above units is not particularly limited, and examples thereof include a method of adjusting the mixing ratio of monomers as raw materials.

Furthermore, the number average molecular weight of the copolymer of the present invention is 1 × 10 ³ to 1 × 10 ⁵ from the viewpoint of imparting appropriate cell membrane penetrability, water solubility, biocompatibility and the like to the copolymer of the present invention. In particular, it is preferably 1 × 10 ³ to 1 × 10 ⁴ , particularly preferably 1 × 10 ^{3 to} 5 × 10 ³ . The number average molecular weight is measured by gel permeation chromatography (GPC).

The method for delivering a desired substance to the endoplasmic reticulum and the Golgi apparatus according to the present invention is not particularly limited. For example, when a desired substance is to be delivered to cultured cells, the desired substance is delivered to the culture medium of the cells. The desired substance can be delivered to the endoplasmic reticulum and the Golgi apparatus by adding the copolymer of the present invention carrying the desired substance desired and continuing the culture for an appropriate time, for example, 5 to 120 minutes.
The above culture conditions may be appropriately selected by those skilled in the art depending on the type of cells to be cultured. However, when serum is contained in the medium, selective delivery of a desired substance to the endoplasmic reticulum and Golgi apparatus is possible. It is preferable from the point of increase. From this point, it is preferable that 5 to 10% of serum is contained in the culture solution. The serum is not particularly limited as long as it is usually used for cell culture, and examples include fetal bovine serum (FBS), newborn bovine serum, calf serum, and horse serum.

  As a target to which the copolymer of the present invention is applied, eukaryotic cells having a Golgi apparatus or an endoplasmic reticulum are exemplified. Examples are cancer cells such as cervical cancer-derived cells (Hela), melanoma cells, kidney cells, brain cells and the like.

The copolymer of the present invention is expected to self-associate when the concentration in the solution exceeds a certain level since the unit II exhibits hydrophobicity and the unit III exhibits hydrophilicity. When the copolymer of the present invention is administered to cultured cells, a concentration that does not form micelles (boundary micelle concentration), for example, 1 to 1000 μg / ml, in order to successfully deliver a desired substance to the endoplasmic reticulum and Golgi apparatus, It is preferable to add to the liquid in which the cells are cultured or dispersed at 1 to 200 μg / ml, particularly 1 to 100 μg / ml.
The boundary micelle concentration (cmc) of such a copolymer can be measured by a conventional method.
For example, pyrene may be used as a fluorescent probe to determine the aggregation concentration of the copolymer of the present invention. Pyrene shows an emission peak at 334.5 nm in the hydrophilic field, shifts to a higher wavelength side in the hydrophobic field, and shows an emission peak at 336.5 nm. When the copolymer is aggregated and pyrene is taken in, the aggregation concentration can be determined by utilizing the fact that the emission peak shifts. Varying the copolymer concentration in the solution, observing the excitation spectrum of pyrene associated therewith, obtaining its fluorescence intensity ratio (334.5nm / 336.5nm), and calculating this fluorescence intensity ratio as a log of the copolymer concentration Plot against values. The aggregation concentration can be obtained by calculating the inflection point.

  Although the manufacturing method of the copolymer of this invention is demonstrated below, the copolymer of this invention is not necessarily limited to what is obtained by the following manufacturing method.

Formula (IV):

(Hereinafter also referred to as “monomer I”),
Formula (V):

And a compound represented by formula (VI):

(Hereinafter also referred to as “monomer III”)
To obtain a copolymer of the present invention [wherein R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , R ⁸ , R ⁹ , A, L] And X are as defined above].
That is, the present invention also relates to a copolymer obtained by this method.

It is necessary to polymerize the monomers I, II, and III to obtain the copolymer of the present invention by using a known method such as solution polymerization, bulk polymerization, emulsion polymerization, suspension polymerization, etc. Depending on the above, it can be produced by a method of radical polymerization by substituting with an inert gas such as nitrogen, carbon dioxide or helium. In this case, in the presence of an initiator in a solvent, 0 to 100 ° C., preferably 60 to 65 ° C., 10 minutes to 48 hours, preferably 4 to 20 hours, more preferably 4 to 8 hours. The copolymer of this invention can be obtained by making a body react. At this time, what can be used as a solvent is not particularly limited as long as it can dissolve the above monomer, and specifically, water, methanol, ethanol, propanol, t-butanol, benzene, toluene, dimethylformamide, dimethyl sulfoxide. , Tetrahydrofuran, chloroform, and mixed solvents thereof, for example, a mixed solvent of ethanol and tetrahydrofuran of DMSO and water.
As the initiator, any normal radical initiator can be used, and 2,2′-azobis (2-amidinopropane) dihydrochloride, 4,4′-azobis (4-cyanovaleric acid), 2,2′-azobis (2- (5-methyl-2-imidazolin-2-yl) propane) dihydrochloride, 2,2′-azobis (2- (2-imidazolin-2-yl) propane) dihydrochloride Salt, 2,2′-azobisisobutyramide dihydrate, ammonium persulfate (APS), potassium persulfate, benzoyl peroxide, diisopropyl peroxydicarbonate, t-butylperoxy-2-ethylhexanoate, t-butyl Peroxypivalate, t-butylperoxydiisobutyrate, lauroyl peroxide, 2,2'-azobisisobutyronitrile (AIBN), 2,2'-azobis 2,4-dimethylvaleronitrile), fatty acid azo compounds such as azobis maleenonitrile, t-butyl peroxyneodecanoate (trade name “Perbutyl ND”, manufactured by NOF Corporation, hereinafter abbreviated as P-ND) Or a mixture thereof. Various redox accelerators may be used as the polymerization initiator. The amount of the polymerization initiator used is preferably 0.01 to 5.0 parts by weight with respect to 100 parts by weight of the total of monomers I, II and III.
The copolymer thus obtained can be purified by a general purification method such as a reprecipitation method, a dialysis method, or an ultrafiltration method.

The monomer I is not particularly limited and can be produced using a known method, and the following method is exemplified. First, a compound having a linking group and an amino group: H ₂ NLZ ³ (wherein L is a linking group, and the Z ³ group is the functional group such as a hydroxyl group, an amino group, or a carboxyl group). Z ³ group may be protected by a protecting group such as Fmoc group) and is synthesized using a known method. This is then condensed with acrylic acid (or methacrylic acid): CH ₂ ═CR ¹ —COOH using a commercially available condensing agent to obtain CH ₂ ═CR ¹ —CO—HN-LZ ³ . If the Z ³ group is protected, it is deprotected, and then the bond is obtained by reacting the Z ³ group of the obtained compound with the functional group Z ¹ or Z ² present in the desired substance to be delivered. was allowed to form, the monomer _I: ^CH 2 = CR 1 can be obtained -CO-HN-L-X. Further, when L is a single bond, the monomer I is formed by amide bonding the carboxyl group of the acrylic acid (or methacrylic acid) and the amino group present in the substance to be delivered: H ₂ N—X. : it is possible to obtain ^{_{CH 2 = CR 1 -CO-HN}} -X.

  Monomer II can be produced by a known method.

The monomer III can be produced by a known method such as the method described in JP-A-2005-239988. As the monomer III, 2-methacryloyloxyethyl phosphorylcholine, 2-acryloyloxyethyl phosphorylcholine, 3- (meth) acryloyloxypropyl phosphorylcholine, 4- (meth) acryloyloxybutyl phosphorylcholine, 6- (meth) acryloyloxyhexyl phosphorylcholine 10- (meth) acryloyloxydecyl phosphorylcholine, ω- (meth) acryloyl (poly) oxyethylene phosphorylcholine, 2-acrylamidoethyl phosphorylcholine, 3-acrylamidopropylphosphorylcholine, 4-acrylamidobutylphosphorylcholine, 6-acrylamidohexylphosphorylcholine, 10- Acrylamide decyl phosphorylcholine, ω- (meth) acrylamide (poly) oxyethylenephospho Rylcholine, 2-methacryloyloxyethyl phosphorylethanolamine, 2-acryloyloxyethyl phosphorylethanolamine, 3- (meth) acryloyloxypropyl phosphorylethanolamine, 4- (meth) acryloyloxybutyl phosphorylethanolamine, 6- (meth) acryloyl Examples include oxyhexyl phosphorylethanolamine, 10- (meth) acryloyloxydecyl phosphorylethanolamine, and ω- (meth) acryloyl (poly) oxyethylene phosphorylethanolamine.
The number of repeating oxyethylene units of the (poly) oxyethylene moiety in the monomer is particularly limited as long as the function of the unit III imparting appropriate water solubility and biocompatibility to the copolymer of the present invention is not impaired. For example, it is 1-10.

Particularly preferably, from the viewpoint of imparting good water solubility and biocompatibility to the copolymer of the present invention, the following formula:

2-methacryloyloxyethyl phosphorylcholine represented by the formula:

EXAMPLES Hereinafter, although an Example demonstrates this invention in detail, this invention is not limited by this.
The NMR measurement was performed using an ECP-600 nuclear magnetic resonance spectrometer ( ¹ H 600 MHz; ¹³ C 150 MHz) manufactured by JEOL. The number average molecular weight (Mn) and the weight average molecular weight (Mw) of the copolymer are those manufactured by Shimadzu Corporation (LC-9A, RID-6A, SPD-10A). For measurement, Tosoh column, TSK-gel G4000PW _XL , G3000PW _XL , G2000PW _XL and G-Oligo PW _XL were used and measured by the GPC method. Pullulan was used as a standard. Reagents were purchased from Wako Pure Chemical Industries, Ltd. and Tokyo Chemical Industry and used without purification. MERCK silica gel (70-230 mesh) was used as the silica gel for column chromatography. For thin layer chromatography, MERCK TLC plate silica gel (60 F2540.25 mm) was used.

Synthesis of lactose-carrying monomer 1

5-Amino-1-pentanol was acrylated before glycosylation. Glycosylation of acetyl lactose (3) introduced with an imidate group and N-acrylpentanol (10) obtained by acrylating 5-amino-1-pentanol yielded a yield of about 10%. Compound 7 was obtained. Next, deacetylation was performed to obtain lactose-carrying monomer 1.

4-O- (2 ′, 3 ′, 4 ′, 6′-tetra-O-acetyl-β-D-galactopyranosyl) -2,3,6-tri-O-acetyl-β-D-glucopyranoside -Α-Trichloroacetimidate (3) synthesis

Under an argon atmosphere, acetyl lactose (30 g, 29.5 mol) was dissolved in 80 ml of DMF, and hydrazine monohydrate (1.43 ml, 1.0 eq) was added in an ice bath. In order to prevent the removal of a plurality of acetyl groups, the solution was concentrated in a state where unreacted substances remained. Subsequently, the mixture was dissolved in 1,2-dichloroethane (15 ml) under an argon atmosphere, trichloroacetonitrile (15 ml, 5.1 eq) was added, and the mixture was stirred at room temperature. DBU (1.8 ml, 0.4 eq) was added dropwise under ice cooling, and the mixture was stirred overnight at room temperature. After completion of the reaction, the mixture was concentrated, purified by silica gel column chromatography (hexane: ethyl acetate = 1: 1), freeze-dried with benzene, and then the title compound 3 (12.8 g, 2 steps, yield 55.8%) as a white yellow solid. )

Synthesis of 5-acrylamido-1-pentanol (10)

5-Amino-1-pentanol (5 ml, 46.0 mmol) and sodium carbonate (5.85 g, 1.2 eq) are dissolved in methanol (50 ml), and acrylic acid chloride (4.5 ml, 1.2 eq) is added dropwise in an ice bath. And stirred at room temperature for 1 hour. After completion of the reaction, the mixture was concentrated and purified by open column chromatography (chloroform: methanol = 4: 1) to obtain Compound 10 (4.79 g, 62.8%).

1- (5-N-acrylamide-1-pentanyl) -4-O- (2 ′, 3 ′, 4 ′, 6′-tetra-O-acetyl-β-D-galactopyranosyl) -2,3 Of 6,6-tri-O-acetyl-β-D-glucopyranoside (7)

Under an argon atmosphere, compound 3 (3 g, 3.84 mmol) was dissolved in dichloromethane (90 ml), compound 10 (0.91 mg, 1.5 eq) and molecular sieves (9 g) were added, cooled to −30 ° C., and stirred for 30 minutes. . Boron trifluoride-ether complex (420 μl, 0.4 eq) was diluted to 0.1 M with dichloromethane, added dropwise in several portions, and stirred at −30 ° C. for 3 hours. After completion of the reaction, the mixture was extracted with chloroform and washed with a saturated aqueous sodium hydrogen carbonate solution. The chloroform layer was dried over anhydrous sodium sulfate, concentrated and purified by open column chromatography (hexane: ethyl acetate = 1: 4) to obtain the title compound 7 (0.315 g, 10.6%).

Synthesis of 1- (5-N-acrylamide-1-pentanyl) -4-O- (β-D-galactopyranosyl) -β-D-glucopyranoside (1)

Compound 7 (995 mg, 1.5 mmol) was dissolved in methanol (30 ml), sodium methoxide (4.0 mg, 0.75 eq) was added, and the mixture was stirred overnight at room temperature. After completion of the reaction, a cation exchange resin was added to neutralize the reaction solution, and the resin was removed by filtration and then concentrated to obtain the title compound 1 (720 mg) as a lactose-carrying monomer 1.

Synthesis of fluorescent label-carrying monomer 13 Fluorescent label-carrying monomer 13 was synthesized using 1,5-diaminopentane protected on one side with an Fmoc group and the fluorescent substance fluorescein isothiocyanate (FITC) (Scheme 2).

Synthesis of (9H-fluoren-9-yl) methyl 5- (acrylamide) pentylcarbamate (15)

(9H-fluoren-9-yl) methyl 5- (ammonium chloride) pentylcarbamate (200 mg, 0.55 mmol), N, N-diisopropylethylamine (DIEA, 193 μl, 2.0 eq) was dissolved in DMF (4 ml), Acrylic acid chloride (50 μl, 1.1 eq) was added dropwise in an ice bath, and the mixture was stirred at room temperature for 1 hour. After completion of the reaction, reprecipitation was performed by dropping the reaction solution into water. The precipitate was collected by centrifugation, washed with a 10% aqueous citric acid solution and a saturated aqueous sodium bicarbonate solution, and then freeze-dried to obtain the white title compound 15.

Synthesis of N- (5-aminopentyl) acrylamide (16)

Compound 15 (200.1 mg, 529 μmol) was dissolved in diethylamine (10 ml) and stirred overnight. After completion of the reaction, the mixture was concentrated and extracted with chloroform, and the aqueous phase was collected and lyophilized to obtain the white title compound 16 (119.2 mg, 144%). NMR confirmed that compound 16 and diethylamine were mixed in about 1: 1.

Synthesis of 5- (3- (5- (acrylamide) pentyl) thioureido) -2- (3-hydroxy-6-oxo-6H-xanthen-9-yl) benzoic acid (13)

Compound 16 (19.6 mg, 2.0 eq) and triethylamine (9.9 μl, 1.1 eq) were dissolved in DMF (250 μl) and methanol (50 μl), and FITC (25.6 mg, 64.2 μmol) was added and stirred overnight in the dark. . After completion of the reaction, the product was purified by preparative TLC (chloroform: methanol = 2: 1) to obtain the title compound 13 (7.2 mg, 20.5%) as the fluorescent label-carrying monomer 13.

Synthesis of copolymer (Examples 1 to 6)

Stearyl methacrylate (11) was purchased from Tokyo Kasei (M0593), and MPC (12) was synthesized according to the literature (Patent 2870727 or Polymer Journal, 22 (5), 355-360 (1990)).
Under an argon atmosphere, the lactose-carrying monomer 1 synthesized above, stearyl methacrylate (11), MPC (12), and the fluorescent label-carrying monomer synthesized above showing the position of the resulting copolymer in the cell. The monomer 13 was dissolved in DMSO: water = 1: 1 solvent at a molar ratio shown in Table 1 so that the total concentration of these monomers was 0.5 M. To this, APS was added as an initiator so as to be 2.5 × 10 ⁻² M, and the mixture was stirred at 60 ° C. for 20 hours. After purification by a reprecipitation method using acetone, the mixture was dialyzed for 3 days using a 3500 cut-off dialysis membrane and then freeze-dried to obtain the copolymers of Examples 1 to 6. Table 1 shows the molecular weight of the obtained copolymer and the composition of each unit in the copolymer.

Administration to cells The copolymer synthesized above was administered to cells, and the results were observed under a microscope to observe the behavior of the copolymer in cells. Conventional synthetic modifiers modify the cell membrane surface when administered to cells. Therefore, it was confirmed whether the copolymer synthesized this time was not taken up into cells, localized in cell membranes, or taken up into cells.

Medium / Reagent / Labware
DMEM (Gibco), DMEM / F12 (Gibco), fetal bovine serum (FBS), glutamine (trade name “Nissui”, Nissui Pharmaceutical), trypsin-EDTA solution (containing 0.25% trypsin, 1 mM EDTA-4Na) Dulbecco PBS (-) powder (trade name "Nissui", manufactured by Nissui Pharmaceutical), HBSS (manufactured by Gibco)

Preculture of Hela cells Human cervical cancer-derived cells (Hela) were used. Using DMEM / F1 medium (containing 2 mM glutamine and 10% FBS), the cells were cultured at 37 ° C. under 5% CO ₂ and subcultured every 2-3 days. At the time of passage, after removing the medium, 2 ml of trypsin-EDTA solution was added, the cell surface layer was washed, and after removal, 2 ml of trypsin-EDTA solution was added again, and the mixture was added at 37 ° C., 5% CO ₂ for 5 minutes. Incubated about 5 minutes. This trypsin-EDTA solution is added to a 15 ml centrifuge tube to which about 10 ml of DMEM / F12 medium has been added in advance, and the cells are precipitated by centrifugation (1000 rpm, 5 minutes, room temperature), and the supernatant is removed. Cells were collected. Thereafter, 10 ml of DMEM / F12 medium was again added and suspended, and the cells were counted with a hemocytometer and seeded at 1.0 × 10 ⁶ cells / 7 ml / dish.

Experimental procedure The experiment was performed according to the following procedure. Further, the position in the cell where the copolymer was taken in was identified by detecting the fluorescence from the fluorescent label contained in the copolymer. The position of the nucleus was identified by staining with propidium iodide (PI), and the position of the Golgi apparatus and endoplasmic reticulum was identified by staining with WGA lectin-Alexa647.
1) The sterilized cover glass was put into a 6-well plate and pre-cultured for 24 hours (37 ° C., 5% CO ₂ , 3.0 × 10 ⁵ cells / well).
2) The medium was removed, and the synthesized copolymer solution adjusted to 50 μg / ml was added (2 ml / well) and incubated for 1 hour (37 ° C., 5% CO ₂ ).
3) Washed with HBSS (2 ml / well x 2).
4) Methanol was added (0.5 ml / well) and incubated for 5 minutes (room temperature) to fix.
5) Washed with HBSS (2 ml / well x 2).
6) WGA lectin-Alexa647 diluted to 5 μg / ml with HBSS was added (2 ml / well) and incubated for 10 minutes (room temperature).
7) Washed with HBSS (2 ml / well x 2).
8) PI diluted to 0.5 μg / ml with HBSS was added (1 ml / well) and incubated for 5 minutes (37 ° C., 5% CO ₂ ).
9) Washed with HBSS (2 ml / well x 2).
10) One drop of anti-fading agent was dropped on one slide glass on the slide.
11) Using tweezers, the cover glass was taken out, dropped on the fading inhibitor and placed on the observation surface.
12) The cover glass was covered with manicure.
13) The cells were observed.

Results Differences due to the presence or absence of lipids in the copolymer By administering to the cells a copolymer having a stearyl group (Example 4) and a copolymer not having it (Example 5) Examined. The results are shown in FIG. From FIG. 1, paying attention to the photograph of A in which the copolymer of Example 4 was administered to Hela cells, it was confirmed that the copolymer was incorporated into any part of the cells. Therefore, when compared with Photograph B and Photograph C, which stained the nucleus and the Golgi apparatus / endoplasmic reticulum, respectively, it was found that many copolymers coincided with the localization of the stained sites of the Golgi apparatus and endoplasmic reticulum. From this, it was found that the incorporated copolymer was localized in the Golgi apparatus and endoplasmic reticulum and was not transported to the nucleus. Next, when compared with Photo E in which the copolymer of Example 5 was administered to cells, it was found that the copolymer of Example 5 was hardly taken up by the cells.

Administration of Copolymer Having Carbohydrate The copolymers of Examples 1 to 3 having lactose and stearyl groups were respectively administered to cells. The results are shown in FIG. From FIG. 2, focusing on the photographs of A, E, and I in which the copolymers of Examples 1 to 3 were administered to Hela cells, the copolymer containing a large amount of lactose was not introduced into the cells, and the lactose content was decreased. It was found that it was easily taken up by cells. In addition, it was found that many polymers incorporated into the cells coincided with the localization of the markers of the Golgi apparatus / endoplasmic reticulum when compared with the photos stained for the nucleus and the Golgi apparatus / endoplasmic reticulum. It is thought that when the lactose content increases, the hydrophilicity of the polymer increases, making it difficult for cells to be taken up by cells.

Incubation time and incorporation into cells Using the copolymer of Example 3, the incubation time and localization of the copolymer were confirmed. The results are shown in FIG. From FIG. 3, it was found that the cells were already taken up by the cells at the time of incubation for 5 minutes and localized in the Golgi apparatus / endoplasmic reticulum. Until the incubation time was 1 hour, the copolymer was localized in the Golgi apparatus and the endoplasmic reticulum, but after 3 and 6 hours of incubation, the polymer spread throughout the cell.

Serum effects
The effect of serum on the uptake of the copolymer was examined by adjusting the copolymer solution of Example 3 using a medium containing ITS-X (GIBCO) instead of a medium containing 10% FBS. The results are shown in FIG. Compared to the medium containing ITS-X, the medium containing 10% FBS was found to have a higher uptake of the copolymer into the cells. It is possible that the copolymer is not taken up into the cell by simple concentration diffusion but is taken up into the cell via carrier and endocytosis.

According to the present invention, a desired substance can be delivered to the endoplasmic reticulum or the Golgi apparatus. Thereby, for example, the copolymer of the present invention containing a label can be used as a label for these endoplasmic reticulum and Golgi apparatus. In addition, for example, by delivering a sugar as a glycosyltransferase inhibitor or a raw material to the endoplasmic reticulum and the Golgi apparatus, it is possible to suppress or promote specific sugar chain biosynthesis performed there. Alternatively, the desired peptide can be delivered to the endoplasmic reticulum and the Golgi where the desired glycopeptide can be synthesized.
In this way, it is possible to control the biosynthesis of sugars and proteins in the cell, thus suppressing the production of glycoproteins and glycolipids with abnormal sugar chains, promoting the biosynthesis of normal glycoproteins and glycolipids, Diseases caused by glycoproteins and glycolipids, such as cancer and brain diseases, can be treated or prevented.
Moreover, it can also be used in the production of glycopeptide drugs or polysaccharides by adding sugar chains to peptides and proteins. In particular, since the copolymer of the present invention is selectively delivered by the endoplasmic reticulum or the Golgi apparatus when administered to Hela, it is preferable to use Hela cells for such substance production.

It is a figure which shows the intracellular localization of the copolymer of this invention. A to D show Example 4 and E to H show Example 5 cells. A and E show FITC fluorescence of the administered copolymer, B and F show nuclei stained with PI, and C and G show Golgi and endoplasmic reticulum stained with WGA lectin-Alexa647, respectively. D is a diagram in which A to C are superimposed, and H is a diagram in which E to G are superimposed. It is a figure which shows the intracellular localization of the copolymer of this invention. A to D show Example 1, E to H show Example 2, and I to L show Example 3 respectively. A, E, and I are FITC fluorescence of the administered copolymer, B, F, and J are nuclei stained with PI, C, G, and K are Golgi and endoplasmic reticulum stained with WGA lectin-Alexa647 Are shown respectively. D is a diagram in which A to C are superposed, H is E to G, and L is I to K. Sugar indicates the copolymer composition (mol%) of lactose-carrying monomer 1. It is a figure which shows the change of the intracellular localization by the incubation time of the copolymer of Example 3. FIG. Before administering Example 3 (A to D), 5 minutes after Example 3 was administered and incubation was started (E to H), 60 minutes (I to L), 180 minutes (M to P), Cells after 360 minutes (Q-T) are shown. A, E, I, M, and Q are FITC fluorescence of the administered copolymer, B, F, J, N, and R are nuclei stained with PI, C, G, K, O, and S indicates the Golgi apparatus and endoplasmic reticulum stained with WGA lectin-Alexa647, respectively. D is a view in which A to C, H is in the range of E to G, L is in the range of I to K, P is in the range of M to O, and T is in the range of Q to S. It is a figure which shows the change of the intracellular localization of the copolymer of Example 3 by the presence or absence of serum. A to D are media containing 10% FBS, E to H are media containing ITS-X, and each shows cells to which Example 3 was prepared. A and E show FITC fluorescence of the administered copolymer, B and F show nuclei stained with PI, and C and G show Golgi and endoplasmic reticulum stained with WGA lectin-Alexa647, respectively. D is a diagram in which A to C are superimposed, and H is a diagram in which E to G are superimposed.

Claims (10)

Formula (I):

A repeating unit represented by
Formula (II):

A repeating unit represented by formula (III):

The content ratio of the unit represented by formula (I), the unit represented by formula (II), and the unit represented by formula (III) is 0.1 to 2: 0 in terms of molar ratio. .1～2: 1, number average molecular weight of ^{1 × 10 3 ~1 × 10 5} , a copolymer wherein
R ¹ , R ² , and R ⁴ are each independently a hydrogen atom or a methyl group;
R ³ represents a C8-24 linear or branched, saturated or unsaturated monovalent hydrocarbon group (this hydrocarbon group is unsubstituted or substituted with a halogen or azide group). Is);
R ⁵ is a divalent hydrocarbon group having 1 to 10 carbon atoms or a (poly) oxyethylene group;
R ⁶ is a C 1-4 divalent hydrocarbon group;
R ⁷ , R ⁸ , and R ⁹ are each independently a hydrogen atom or a hydrocarbon group having 1 to 4 carbon atoms;
A is a divalent bond selected from the group consisting of an ester bond, an amide bond, a urethane bond, and an ether bond;
L is a single bond or a linking group;
X is the residue of the substance to be delivered]. The repeating unit represented by the formula (III) is

The copolymer according to claim 1, wherein Formula (IV):

A compound represented by
Formula (V):

And a compound of formula (VI):

Obtained by polymerizing a compound represented by formula (I), a unit represented by formula (II) and a unit represented by formula (III) [wherein formula (I), (II) and The unit represented by (III) is as defined in claim 1] and the molar ratio is 0.1 to 2: 0.1 to 2: 1, and the number average molecular weight is 1 ×. 10 is a ³ to 1 × 10 ^5, a copolymer ^{wherein, R 1, R 2, R} 3, R 4, R 5, R 6, R 7, R 8, R 9, a, L, and X Is as defined in claim 1].   The copolymer according to claim 3, wherein the compound represented by the formula (VI) is 2-methacryloyloxyethyl phosphorylcholine. The copolymer according to any one of claims 1 to 4, wherein R ³ is a hydrocarbon group having 12 to 24 carbon atoms.   L is a hydrocarbon group having 1 to 12 carbon atoms (this hydrocarbon group is uninterrupted or interrupted at one or more positions by oxygen atoms or sulfur atoms or both not adjacent to each other), Item 6. The copolymer according to any one of Items 1 to 5. The copolymer according to any one of claims 1 to 6 , wherein the substance to be delivered is a label. A reagent for labeling endoplasmic reticulum and Golgi apparatus, comprising the copolymer according to claim 7 . Use of a copolymer according to any one of claims 1 to 6 for delivering a desired substance to the endoplasmic reticulum and the Golgi apparatus. Use according to claim 9 , wherein the desired substance is a label.