WO2017051882A1 - Amphipathic block copolymer, molecule assembly and method for producing same, and protein-included agent - Google Patents

Abstract

The present invention relates to an amphipathic block copolymer in which a polypropylene glycol (PPG) segment (A) is bonded to a sugar chain segment (B) via a linker group, wherein the sugar chain segment (B) contains a maltotriose unit, and the anomeric carbon in maltotriose is bonded to the PPG segment (A) via a bivalent linker group.

Description

Amphiphilic block copolymer, molecular assembly, method for producing the same, and protein inclusion agent

The present invention relates to an amphiphilic block copolymer, a molecular assembly, a method for producing the same, and a protein inclusion agent.

Vesicles (liposomes) made of natural phospholipids have been extensively studied for use as artificial cell models and drug delivery system (DDS) nanocarriers. In recent years, focusing on organelle vesicles that are responsible for the metabolism of unwanted substances, protein synthesis, modification, protein folding and energy production in vivo, artificial organelles using liposomes have been constructed and produced in vivo. Attempts have also been made to use it as a next-generation DDS platform capable of converting unnecessary molecules and producing bioactive substances (Non-Patent Documents 1 and 2).

Since phospholipids exist in biological systems, they have high biocompatibility, and their liposomes have a certain level of substance permeability. On the other hand, it has disadvantages such as weak mechanical strength and unstable under dilution conditions. Therefore, in recent years, attention has been focused on a hollow structure (polymer vesicle) made of a polymer to compensate for these weak points (Non-patent Document 3). The polymer vesicle has an internal aqueous phase similar to the liposome formed by the phospholipid, and also has the characteristics that the surface of the structure and the properties of the membrane can be easily modified. Moreover, since the molecular weight is larger than that of phospholipid, the mechanical strength is strong and stable. On the other hand, in many cases, the structural unit is a synthetic polymer, the biocompatibility is low, and since the molecular weight is large, the vesicle thickness to be formed is large, so that the substance permeability is extremely low. For this reason, a membrane protein having a substance permeability that mimics the function of a living body is incorporated into a polymer vesicle (Non-Patent Documents 4 and 5), or an example using a functional / stimulus-responsive polymer (Non-Patent Documents 6 to 5). 9) High molecular polymers using polyion complex (Non-patent Documents 10 and 11) have been developed. However, these constructions require complicated operations and require multi-step synthesis.

Nat. Nanotech. 2007, 2, 3-7, Acc. Chem. Res., 2011, 44 (10), pp 1039 FEBS Lett. 2011, 585, 1699, Nano Lett. 2013, 13, 2875, Nat. Nanotech. 2007, 2, 3-7 Current Opinion Pharmacology 2014, 18: 104 Nano Lett. 2013, 13 (6), 2875 Nano Lett., 2005, 5 (11), 2220 Adv .Mater., 2009, 21, 2787 Angew. Chem. Int. Ed. 2013, 52, 5070 J. Am. Chem. Soc., 2009, 131, 10557 Angew. Chem. Int. Ed. 2003, 42, 7, 772 Angew. Chem. 2007, 119, 6197 Angew. Chem. Int. Ed. 2009, 48, 329

An object of the present invention is to provide an amphiphilic block copolymer.

Another object of the present invention is to provide a molecular assembly having high biocompatibility, temperature responsiveness and substance permeability, and a method for producing the same. Furthermore, an object of the present invention is to provide a protein inclusion agent.

Therefore, the present inventor examined the addition of a substance permeability to the polymer vesicle membrane itself in order to construct a compartmentalized space having a substance permeability with simpler molecules without requiring the incorporation of a membrane protein. . In the molecular design of polymers, polypropylene glycol (PPG), which has a low glass transition point, is used as a hydrophobic group in order to increase the fluidity of the membrane, and the hydrophilic group takes into consideration the balance with the mass of the hydrophobic group and biocompatibility. A sugar chain segment having a maltotriose unit was used. Such an amphiphilic block copolymer having a PPG segment and a sugar chain segment forms a molecular assembly containing vesicles in water, and its formation can be controlled reversibly by temperature. We found that vesicle membranes permeate water, ions, low-molecular substances, but not high-molecular substances such as proteins, and also have bioreactor functions by enzyme encapsulation and molecular chaperone-like functions that help protein refolding. It was.

The present invention relates to the following amphiphilic block copolymers, molecular assemblies, methods for producing the same, and protein inclusions.
Item 1. An amphiphilic block copolymer obtained by bonding a polypropylene glycol (PPG) segment (A) and a sugar chain segment (B) via a linker group, wherein the sugar chain segment (B) contains a maltotriose unit. An amphiphilic block copolymer comprising an anomeric carbon of maltotriose bonded to a PPG segment (A) via a divalent linker group.
Item 2. Formula (I) below

(Wherein R ¹ and R ² are the same or different and each represents a hydrogen atom or a sugar residue containing at least one glucose. Y ¹ represents a divalent linker group. R represents a hydrogen atom, an alkyl group, An aryl group, an aralkyl group, an alkanoyl group, a hydroxyalkyl group, Y ² -R ³ , Y ² represents a divalent linker group, and R ³ represents the following formula

(Wherein R ¹ and R ² are as defined above.)
N1 is 10 to 500. )
Item 2. The amphiphilic block copolymer according to item 1, represented by:
Item 3. The divalent linker group represented by Y ¹ or Y ² is -NH-,

(In the formula, X represents O, NR ⁴ or S. p represents an integer of 1 to 4. R ⁴ represents a hydrogen atom, an acyl group or an alkyl group.)
Item 3. The amphiphilic block copolymer according to Item 2, which is any one of groups represented by:
Item 4. Item 4. A molecular assembly composed of at least one amphiphilic block copolymer according to any one of Items 1 to 3.
Item 5. Item 5. The molecular assembly according to Item 4, wherein the molecular assembly is a vesicle, a spherical micelle, a rod-like micelle, or a tube structure.
Item 6. Item 6. The molecular assembly according to Item 5, which includes a protein.
Item 7. Item 7. The molecular assembly according to Item 6, wherein the protein is an enzyme.
Item 8. Item 6. The method for producing a molecular assembly according to Item 4 or 5, wherein at least one amphiphilic block copolymer according to any one of Items 1 to 3 is dispersed in water.
Item 9. Item 6. A protein inclusion agent comprising the molecular assembly according to Item 4 or 5.

An amphiphilic block copolymer containing a sugar chain segment as a hydrophilic group and a PPG segment as a hydrophobic group forms a molecular assembly in an aqueous solution, and PPG has a lower critical eutectic temperature (LCST) below room temperature. From these results, it was found that the vesicle collapse can be controlled by lowering the temperature of the solution below room temperature. It has also been found that it exhibits low molecular permeability without the need to add membrane proteins. In addition, it was revealed that it was incorporated into the inner aqueous phase of the molecular assembly while maintaining the activity of various enzymes and functioned as an enzyme reaction field. Furthermore, since this polymer is associated by hydrophobic interaction, the present inventors also found a function (artificial chaperone ability) that interacts with a protein in a denatured state and promotes refolding.

From the above, the molecular assembly of the present invention is expected to be used for a new DDS carrier that can encapsulate an enzyme while maintaining its activity stably.

(Left) TEM image of M ₅ P _2.5 vesicle and (Right) DLS particle size distribution. Temperature dependence of turbidity derived from M ₅ P _2.5 aggregates. □ indicates relative transmittance (%) at 500 nm. Release profile of inclusion guests of different molecular weight from M ₅ P _2.5 vesicles. ■: FITC-m PEG 0.5K; ▲: FITC-mPEG 2K; ▼: FITC-mPEG 5K; ○: FITC-BSA 66K. Activity evaluation of chymotrypsin encapsulated in M ₅ P _2.5 . ■: chymotrypsin (without inhibitor); ●: chymotrypsin (with inhibitor); □: chymotrypsin inclusion M ₅ P _2.5 (without inhibitor); ○: chymotrypsin inclusion M ₅ P _2.5 (with inhibitor). Activity evaluation of β-galactosidase encapsulated in M ₅ P _2.5 . ■: β-galactosidase (without proteinase K); ●: β-galactosidase inclusion M ₅ P _2.5 (with proteinase K); □: β-galactosidase (with proteinase K). Activation of anticancer drug prodrugs by β-galactosidase-encapsulated TAT-presenting M ₅ P _2.5 vesicles Correlation of CAB activity recovery by M ₅ P _2.5 and time. □: With M ₅ P _2.5 ; ○: Without M ₅ P _2.5 . Synthesis scheme 3 of M ₅ P _2.5 . ¹ H-NMR spectrum of M ₅ P _2.5 . SAXS profile (◯) of M ₅ P _2.5 vesicles (5 mg / ml, PBS) and fitting results with a bimolecular model (solid line). Schematic diagram of the electron density and size of each bilayer model (right diagram). 2.0 nm corresponds to the length of the hydrophilic part (sugar chain segment), and 9.1 nm corresponds to the length of the hydrophobic part (PPG segment). Distribution of film thickness of M ₅ P _2.5 vesicles obtained by TEM observation. List of chemical structural formulas of synthesized sugar chain polymers. ¹ H-NMR spectrum of a BisM ₅ P ₂ molecular assembly. ¹ H-NMR spectrum of an M ₅ P ₁ molecular assembly. ¹ H-NMR spectrum of M ₈ P _2.5 molecular assembly. ¹ H-NMR spectrum of an M ₃ P ₁ molecular assembly. ¹ H-NMR spectrum of M ₅ P _3.5 molecular assembly. TEM image of BisM ₅ P ₂ molecular assembly. TEM image of M ₅ P ₁ molecular assembly. TEM image of M ₈ P _2.5 molecular assembly. TEM image of M ₃ P ₁ molecular assembly. TEM image of M ₅ P _3.5 molecular assembly. (Left) Change in glucose addition number to M ₅ P _2.5 in the presence of GBE / GPa and GPa (Right) ¹ H-NMR spectrum of the sample after enzymatic polymerization reaction with GPa / GBE. □: in the presence of phosphorylase a; ○: in the presence of phosphorylase a and branching enzyme.

In this specification, “molecular assembly” refers to vesicles, spherical and rod-like micelles, tube structures, and the like. A “vesicle” is a vesicle having a closed membrane structure such as a spherical shape, an ellipsoidal shape, or a rod shape, and has a liquid layer inside when present in a liquid. The vesicle may be lyophilized. The lyophilized vesicle has no internal liquid layer, but the internal liquid layer (aqueous layer) is regenerated by adding the lyophilized vesicle to a liquid such as water. When the polymer substance is encapsulated inside the vesicle, the polymer substance remains in the membrane constituting the vesicle even after lyophilization, and the vesicle encapsulating the polymer substance is regenerated in water.

Vesicles can contain macromolecular substances, such as water, ions (Na ⁺ , K ⁺ , Li ⁺ , Ca ⁺ , Mg ⁺ , Fe ²⁺ / Fe ³⁺ cation, Cl ⁻ , Br ⁻ , NO ₃ ^-, anions such as SO ₄ ^2-), the low molecular substances can permeate through the membrane of the vesicle. The polymer material is confined within the vesicle. The molecular weight threshold that can pass through the vesicle membrane varies depending on the size (size and molecular weight) of the sugar chain segment and PPG of the block copolymer of the present invention, but is usually about 300 to 30000, preferably about 500 to 20000, more preferably. Is about 1000 to 10000, more preferably about 1200 to 8000, particularly about 1500 to 5000. For example, when the molecular weight of the PPG segment is about 2500 and the sugar chain segment is a maltopentaose (A)-(B) type block copolymer, the threshold is about 5000. Examples of the low molecular weight substance include physiologically active substances such as drugs, enzyme substrates, dyes, and fragrances. Examples of the polymer substance included in the vesicle include proteins, nucleic acids (DNA, RNA, etc.), polysaccharides, etc. Proteins are preferred. Examples of proteins include enzymes, antibodies, antigens, receptors, hormones and the like, and enzymes are preferred. Examples of the enzyme include hydrolase, transferase, oxidoreductase, desorption enzyme, isomerase, and synthetic enzyme. The polymer substance contained in the vesicle may be used alone or in combination of two or more. Since the vesicle of the present invention can permeate the substrate of the enzyme, an enzymatic reaction is possible in the vesicle. In addition, since the vesicle of the present invention encapsulating an enzyme has an action of promoting refolding of a protein containing the enzyme, the enzyme activity is maintained for a long time and the enzyme reaction product can be easily separated. As excellent.

The size of the molecular assembly is about 10 nm to 5 μm, preferably about 20 nm to 3 μm, more preferably about 30 nm to 1 μm, still more preferably about 30 nm to 500 nm, and particularly preferably about 40 nm to 300 nm.

The molecular assembly is prepared by melting the block copolymer of the present invention in dimethoxypolyethylene glycol (mw = 500) and adding a buffer solution stepwise or by cooling the cooled block copolymer of the present invention. It can be obtained by dissolving it in the solution and raising the temperature.

The block copolymer of the present specification is composed of a hydrophobic PPG segment (A) and a hydrophilic sugar chain segment (B), and (A)-(B) or (B)-(A)-(B ) Type block copolymer is preferably exemplified.

For the PPG segment (A), commercially available polypropylene or a derivative thereof may be bonded to the glucose residue of the sugar chain segment via a linker group. The PPG segment (A) is bonded to the anomeric carbon of the glucose residue of the sugar chain segment via a linker group, but can also be bonded to the hydroxyl group of a glucose residue other than the anomeric carbon.

Examples of commercially available or known polypropylene or derivatives thereof include the following.

(In the formula, n1 represents 10 to 500. R ^b represents a hydrogen atom, an alkyl group or an acyl group.) The molecular weight of the PPG segment is preferably about 500 to 30000, more preferably about 1000 to 20000, and still more preferably. Is about 2000 to 15000, particularly preferably about 3000 to 10000. Since the molecular weight of the PPG segment is related to the threshold value for the permeation of the substance inside the vesicle, a preferable molecular weight may be selected in relation to the threshold value. The molecular weight includes a number average molecular weight.

The alkyl group has a straight or branched chain having 1 to 6 carbon atoms such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, n-hexyl, etc. An alkyl group is mentioned.

Examples of the aryl group include phenyl, naphthyl, fluorenyl, anthryl, biphenylyl, tetrahydronaphthyl, chromanyl, 2,3-dihydro-1,4-dioxanaphthalenyl, indanyl and phenanthryl.

Aralkyl groups include benzyl, naphthylmethyl, fluorenylmethyl, anthrylmethyl, biphenylylmethyl, tetrahydronaphthylmethyl, chromanylmethyl, 2,3-dihydro-1,4-dioxanaphthalenylmethyl, indanyl Examples include methyl, phenanthrylmethyl, phenethyl, naphthylethyl, fluorenylethyl.

Examples of the alkanoyl group include formyl, acetyl, propionyl, butyryl, isobutyryl, valeryl, isovaleryl, pivaloyl, lauroyl, myristoyl, palmitoyl, stearoyl and the like.

Hydroxyalkyl groups include carbons such as hydroxymethyl, hydroxyethyl, hydroxy n-propyl, hydroxyisopropyl, hydroxy n-butyl, hydroxyisobutyl, hydroxy sec-butyl, hydroxy tert-butyl, hydroxy n-pentyl, hydroxy n-hexyl, etc. Examples thereof include a linear or branched hydroxyalkyl group having a number of 1 to 6.

Acyl groups include C _1-6 alkylcarbonyl, arylcarbonyl, aryl-substituted C _1-4 alkylcarbonyl.

Specific examples of C _1-6 alkylcarbonyl include methylcarbonyl, ethylcarbonyl, n-propylcarbonyl, isopropylcarbonyl, n-butylcarbonyl, isobutylcarbonyl, tert-butylcarbonyl, n-pentylcarbonyl, isopentylcarbonyl, hexylcarbonyl Is mentioned.

Specific examples of arylcarbonyl include phenylcarbonyl, naphthylcarbonyl, fluorenylcarbonyl, anthrylcarbonyl, biphenylylcarbonyl, tetrahydronaphthylcarbonyl, chromancarbonyl, 2,3-dihydro-1,4-dioxanaphthalenyl Examples include carbonyl, indanylcarbonyl and phenanthrylcarbonyl.

Specific examples of the aryl-substituted C _1-4 alkylcarbonyl include benzylcarbonyl, naphthylmethylcarbonyl, fluorenylmethylcarbonyl, anthrylmethylcarbonyl, biphenylylmethylcarbonyl, tetrahydronaphthylmethylcarbonyl, chromanylmethylcarbonyl, 2,3 -Dihydro-1,4-dioxanaphthalenylmethylcarbonyl, indanylmethylcarbonyl, phenanthrylmethylcarbonyl, phenethylcarbonyl, naphthylethylcarbonyl, fluorenylethylcarbonyl.
p is an integer of 1 to 4, preferably 1, 2 or 3, more preferably 1 or 2, especially 1.
X is O, NR ⁴ or S, preferably NR ⁴ .
R ⁴ is a hydrogen atom, an acyl group or an alkyl group, preferably a hydrogen atom or an alkyl group.

The sugar chain segment (B) contains a maltotriose residue, and the anomeric carbon of maltotriose is bonded to the PPG segment (A) via a divalent linker group.

The sugar chain segment (B) may be a single one, or two or more sugar chain segments (B) may be mixed. Preferred sugar chain segments (B1) and (B2) are shown below.

(Wherein, n2 is .R ² in which represents an integer of 2 or more is .R ^2a is as defined above shows a sugar residue containing at least one glucose.)
The number of glucose residues in the sugar chain segment is 3 or more, for example, 3 to 100, preferably 3 to 50, more preferably 3 to 30, even more preferably 3 to 20, particularly 3 to 10. The sugar chain segment may be composed only of glucose residues, or may contain sugar residues other than glucose. Examples of sugar residues other than glucose include galactose, mannose, and fructose. The proportion of glucose in the sugar chain segment is more than 50% in molar ratio, preferably 60% or more, more preferably 70% or more, still more preferably 80% or more, particularly preferably 90% or more, and most preferably 100%. .

The ratio of the molecular weight of the PPG segment to the sugar chain segment is PPG segment / sugar chain segment = about 0.1 to 50, preferably about 0.5 to 30, more preferably about 1 to 20.

3 or more glucose residues such as maltotriose, maltotetraose, maltopentaose, maltohexaose, maltoheptaose, maltooctaose, maltononaose, maltodecaose as raw materials for obtaining sugar chain segments The maltooligosaccharide is preferably used. A polypropylene glycol residue is bonded to maltooligosaccharide via a linker. Thereafter, if necessary, glycosyltransferase may be further reacted to further add glucose to the 4-position of the end of the maltooligosaccharide or the 6-position of the glucose residue of the maltooligosaccharide. Examples of such glycosyltransferases include starch synthase and blanching enzyme, and one or more of these can be used in combination.

The block copolymer of the present invention has sugar chain segments at one or both ends. A copolymer having PPG segments at both ends is not included in the block copolymer of the present invention.

The maltooligosaccharide used as a raw material can be obtained by hydrolyzing starch or cyclodextrin (α, β, γ) with an acid. In addition, starch is decomposed by maltooligosaccharide-generating amylase to generate maltooligosaccharides in which glucose such as maltotriose, maltotetraose, maltopentaose, maltohexaose is α-1.4-linked.

The divalent linker group that connects the PPG segment and the sugar chain segment is -NH-,

(Wherein X, p and R ⁴ are as defined above.)
The group represented by these is mentioned.

The block copolymer of the present invention can be produced according to the following scheme 1 and scheme 2.

(In the formula, X represents O, NR ⁴ or S. R ¹ , R ² , R, R ^b , n1, and p are as defined above.)
In Scheme 1, compound (2a) is used in an excess amount of 1 mol per 1 mol of compound (1a), and a solvent such as dimethylformamide (DMF), dimethylacetamide, dimethylsulfoxide (DMSO), N-methylpyrrolidone (NMP), etc. In the presence of a catalytic amount to an excess amount of CuSO ₄ , the target compound (IA) can be obtained by reacting for 1 to 24 hours from room temperature to the boiling temperature of the solvent.

In scheme 2, 2-chloro-4,5-dihydro-1,3-dimethyl-1H-imidazolium chloride (DMC), diisopropylethylamine (DIPEA), sodium azide in the presence of 1 mol of compound (1b) Stir in pure water at room temperature. After the reaction, the solvent is removed, ethanol is added, and the resulting precipitate is removed by filtration. After adding ultrapure water to the filtrate and washing the aqueous solution with ethyl acetate, DIPEA is removed with an ion exchange column and freeze-dried to obtain compound (3b).

Propegylamine is used in an excess amount from 1 mol with respect to compound (3b), and in the presence of N- (3-dimethylaminopropyl) -N'-ethylcarbodiimide hydrochloride (EDC), dichloromethane (DCM), tetrahydrofuran (THF) ) And dimethylformamide (DMF) in a solvent such as dimethylformamide (DMF) at room temperature to the boiling temperature of the solvent for 1 to 24 hours, compound (4b) can be obtained.

1 mol of compound (2b) is used in an excess amount of 1 mol of compound (4b) and a catalytic amount in a solvent such as dimethylformamide (DMF), dimethylacetamide, dimethyl sulfoxide (DMSO), N-methylpyrrolidone (NMP) In the presence of an excess amount of CuSO ₄ , the target compound (IB) can be obtained by reacting from room temperature to the boiling temperature of the solvent for 1 to 24 hours.

EXAMPLES Hereinafter, although this invention is demonstrated in detail using an Example, it cannot be overemphasized that this invention is not limited to an Example.
Example 1
In this study, we tried to give the polymer vesicle membrane itself a substance permeability in order to construct a compartmentalized space with a simple molecule and a substance permeability without requiring the incorporation of membrane proteins. In the molecular design of polymers, PPG, which has a low glass transition point, is used as a hydrophobic group in order to increase the fluidity of the membrane, and the hydrophilic group has five glucose groups in consideration of the balance with the mass of the hydrophobic group and biocompatibility. A series of maltopentaose was used.

Maltopentaose modified PPG (M ₅ P _2.5 ) was prepared according to Scheme 3 shown in FIG.

First, the hydroxyl group of poly (propylene glycol) monobutyl ether (PPO, m = 14) (11) with a number average molecular weight of 2500 is converted to a mesyl group, and then treated with sodium azide to modify the azido group modified PPO (12). Obtained. Subsequently, Huisgen cycloaddition reaction of alkyne-modified maltopentaose (14) and azide-modified PPO (12) was carried out in the presence of a copper catalyst (CuSO ₄ ) to obtain the target block copolymer. The modification rate was about 90%. Hereinafter, the block copolymer Maltopentaose-b-PPO (m = 44) is abbreviated as M ₅ P _2.5 .

(1) Association behavior of M ₅ P _2.5 in aqueous solution M ₅ P _2.5 vesicles were prepared according to the Direct Hydration method developed by Hubbell et al. The obtained vesicle was subjected to TEM observation and DLS measurement, and a TEM image is shown in the left figure of FIG. 1 and a DLS measurement result is shown in the right figure. From the TEM image, it was found that the vesicles are spherical and have an edged aggregate. From this, it was found that M ₅ P _2.5 formed a vesicle structure in an aqueous solution. From the DLS measurement, the particle size was about 120 nm. The PDI was 0.16, indicating that vesicles with relatively uniform particle sizes were formed.

(2) Temperature responsiveness of M ₅ P _2.5 vesicles Low molecular weight (MW1000 to 3000) PPG has a critical lower limit eutectic temperature (LCST) in the temperature range of 5 to 40 ° C. PPG is known to dissolve. Therefore, turbidity measurement was performed while changing the temperature of the M ₅ P _2.5 molecular assembly solution, and whether there was a cloud point was evaluated from the change in turbidity. The result is shown in FIG.
From FIG. 2, an increase in transmittance was observed in the temperature lowering process. From this, it was found that M ₅ P _2.5 shows LCST. The cloud point of the M ₅ P _2.5 aggregates from the primary differential curve (dT λ _/ dT) was 7 ℃. From this, it was found that M ₅ P _2.5 vesicles can be destroyed by cooling to 7 ° C or lower.

(3) M ₅ P _2.5 in substance permeability then M ₅ P _2.5 vesicles vesicles encasing an FITC-labeled molecular weight different PEG and bovine serum albumin (BSA), the permeability by examining its release behavior Evaluation was performed. The results are shown in FIG.
From FIG. 3, BSA (molecular weight: 66000) having a large molecular weight is hardly released after 3 hours. On the other hand, it can be seen that the encapsulated molecules having a small molecular weight (PEG: molecular weight 550 to 5000) are released. Further, the permeation rate depends on the molecular weight of the encapsulated molecule, and is rapidly released when the molecular weight is 5000 or less. From these results, it was found that M ₅ P _2.5 vesicles can selectively permeate low-molecular substances.

(4) Vesicle function as an enzyme reaction field
(4-1) Function of chymotrypsin-encapsulated M ₅ P _2.5 vesicle Enzyme was encapsulated in the inner aqueous phase of vesicle composed of M ₅ P _2.5, and it was evaluated whether it functions as an enzyme reaction field. The encapsulated enzyme was chymotrypsin, a hydrolase, and the substrate was N-benzoyl-l-tyrosine p-nitroanilide. In addition, since the permeation of vesicle membranes was extremely slow for those with a molecular weight of 5000 or more, the selective substance permeability of M ₅ P _2.5 vesicles was also evaluated using aprotinin (6.5K), an inhibitor of chymotrypsin. . The results are shown in FIG. As shown in FIG. 4, chymotrypsin encapsulated in M ₅ P _2.5 shows an activity although its reaction rate is slower than that of chymotrypsin in the natural state. In addition, chymotrypsin encapsulated in vesicles shows similar activity even in the presence of an inhibitor, indicating that the bilayer membrane of vesicles selectively permeates molecules. Since there is no change in the reaction rate in the presence or absence of an inhibitor, it is considered that the diffusion of the substrate into the vesicle membrane is rate limiting as a factor that causes a slower reaction rate than chymotrypsin in the natural state. From these facts, it was found that the vesicle composed of M ₅ P _2.5 functions as an enzyme reaction field without inhibiting the enzyme activity.
(4-2) Function of β-galactosidase-encapsulated M ₅ P _2.5 vesicle To confirm its usefulness as an enzyme reaction field, β-galactosidase is encapsulated in M ₅ P _2.5 vesicle and the activity is evaluated in the same manner as above. It was. From FIG. 5, β-galactosidase encapsulated in M ₅ P _2.5 vesicles shows an activity although the reaction rate is slower than that of β-galactosidase in the natural state. In addition, β-galactosidase encapsulated in vesicles is active even in the presence of protein hydrolase (proteinase K) with a large molecular weight, and β-galactosidase retained in the inner aqueous phase is protected by the bilayer membrane of vesicles. I found out.
From the above results, it was found that the β-galactosidase-encapsulating M ₅ P _2.5 vesicle maintained the enzyme activity in an aqueous solution.
(4-3) Function of β-galactosidase-encapsulated M ₅ P _2.5 vesicle in cells From the above results, an attempt was made to activate an anticancer drug prodrug in cells for the purpose of pharmaceutical use. In order to confer affinity to M ₅ P _2.5 vesicle cells, PAT modified with TAT peptide was synthesized, and 1 mol% was added to M ₅ P _2.5 to prepare TAT peptide-presenting vesicles. The size and surface charge of the obtained vesicles were similar to those of M ₅ P _2.5 vesicles, and β-galactosidase inclusion was possible. Therefore, β-galactosidase-encapsulated TAT-presenting M ₅ P _2.5 vesicles were added to HeLa cells and incorporated into the cells. Thereafter, the medium was changed, and the prodrug 5-Fluorouridine-5′-ObD-galactopyranoside (FURG) as a substrate was added. After 24 hours of culture, cytotoxicity was evaluated using cell counting kit-8 (FIG. 6). . From FIG. 6, the addition of only the prodrug (FURG) does not show cytotoxicity, whereas in the presence of β-galactosidase-encapsulated TAT-presenting M ₅ P _2.5 vesicle, the prodrug (FURG) is an anticancer agent (5-FUR). The same level of cytotoxicity was observed. From this, after the prodrug (FURG) was taken up into the cell, the prodrug was converted by β-galactosidase-encapsulated TAT-presenting M ₅ P _2.5 vesicle, which became an anticancer drug (5-FUR) and showed significant cytotoxicity. Conceivable. From the above, it has been clarified that β-galactosidase-encapsulating vesicles exhibit selective permeation of low molecular weight substrates and maintenance of the function of the encapsulating enzyme even in cells.

(5) Chaperone activity of M ₅ P _2.5 Next, it was examined whether the regenerative function of denatured protein, which is one of the functions of the endoplasmic reticulum, which is an in vivo organelle, exists in the M ₅ P _2.5 vesicle. The enzyme used was carbonic acid dehydrogenase (CAB), the substrate was N-benzoyl-l-tyrosine p-nitroanilide, and the activity was evaluated for 1 hour after mixing with M ₅ P _2.5 vesicle. The results are shown in Table 1 below.

From Table 1, the solution in which CAB in a natural state and M ₅ P _2.5 vesicle were mixed did not substantially inhibit the enzyme activity of CAB. In addition, the activity is recovered to about 80% by adding M ₅ P _2.5 vesicle to the chemically modified CAB solution. On the other hand, in the system in which M ₅ P _2.5 vesicle was not added, the activity recovered only to about 20%.

Next, the relationship between the refolding rate and the time change was examined as to how long the M ₅ P _2.5 vesicle was refolding the chemically modified CAB (FIG. 7).
From FIG. 7, it was found that the activity of CAB recovered to about 80% within 1 hour after mixing. From the above, it was found that M ₅ P _2.5 has a molecular chaperone-like function that promotes the refolding of chemically modified proteins.

(6) Synthesis Method of Block Copolymer A synthesis method of the block copolymer (M ₅ P _2.5 ) is shown in FIG.
Synthesis of N3-PPO (12) Poly (Propylene glycol) monobutylether (11) (4.98 g, 2.0 mmol) was dissolved in dehydrated Pyridine (5 mL), and then mesyl chloride (0.46 g, 4.0 mmol) was added. After stirring at 30 ° C. for 20 hours, the reaction solution was added to 50 mL of ultrapure water. The aqueous phase was extracted with DCM (dichloromethane), dried over magnesium sulfate, filtered through filter paper, and dried under reduced pressure. The obtained mesylated compound was used in the next reaction without purification. The obtained pale yellow oily compound was dissolved in dehydrated DMF (5 mL), NaN ₃ (1.3 g, 20.0 mmol) was added, and the mixture was stirred at 45 ° C. for 48 hours. After cooling to room temperature, the reaction solution was added to ultrapure water (50 mL), and the aqueous phase was extracted with ethyl acetate. The organic layer was washed with ultrapure water and brine, dried over magnesium sulfate, filtered through filter paper, and dried under reduced pressure to obtain N3-PPO (12).

Synthesis of alkyne-modified maltopentaose (14) Maltopentaose (13) (1.0 g, 1.22 mmol) was dissolved in propargylamine (1.34 g, 24.4 mmol) and stirred at room temperature for 72 hours. Methanol 10mL was added to the reaction solution, and it was dripped at the dichloromethane solution 100mL, and white solid was obtained. The obtained solid was washed with a dichloromethane / methanol mixed solution = 9: 1 (100 mL). The washed solid was dissolved in dehydrated methanol (100 mL), acetic anhydride (7.23 g, 96.4 mmol) was added, and the mixture was stirred at room temperature for 24 hours. The solvent was removed, 20 mL of ultrapure water was added and lyophilized to obtain the target compound (14).

Synthesis of M ₅ P _2.5 The obtained N3-PPO (12) (0.53 g, 0.21 mmol), alkyne-modified maltopentaose (13) (0.29 g, 0.32 mmol), copper sulfate pentahydrate (5 mg, 0.02 mmol), sodium ascorbate (8 mg, 0.04 mmol) was added to dehydrated DMF (5 mL), and the mixture was stirred at 50 ° C. for 17 hours. After cooling to room temperature, dialysis was performed (spectra por 7 MWCO1000, 25 mM EDTA, water, 4 days). After dialysis, lyophilization was performed to obtain a block polymer M ₅ P _2.5 as a white powder. The polymer was identified by measuring ¹ H-NMR spectrum (FIG. 9).

(7) Preparation and characterization of M ₅ P _2.5 vesicles Preparation method of vesicles About 5 mg of M ₅ P _2.5 and 10 times (weight ratio) PEG dimethoxyether (MW550) were added to a 1.5 mL safe-lock tube. Thereafter, the mixture was heated at 95 ° C. for 20 minutes and then cooled at room temperature for 15 minutes. Thereafter, 100 μL, 200 μL, and about 700 μL of PBS (4 mM, pH = 7.4) were added so that the final concentration of MMP was 5 mg / ml. Further, each buffer solution was mixed by vortexing for 30 seconds. After preparation, the solution was passed through a 0.22 μm PVDF filter to prepare an M ₅ P _2.5 vesicle solution.

TEM Observation Method 5 μL of the vesicle solution prepared above was dropped on an elastic carbon support membrane (ELS-C10 STEM, Oken Shoji). Excess water was wiped off with filter paper, and a 1 wt% phosphotungstic acid solution was dropped onto the grid. Again, excess water was wiped off with filter paper and dried overnight in a desiccator. TEM observation was performed using Hitachi HT-7700.

DLS Measurement Method The particle size of the vesicle was measured at 25 ° C. using Malvern zetasizer nano ZSP for the vesicle solution prepared above.

(8) Preparation of PEG-encapsulated vesicles and measurement of PEG release amount Encapsulation of substances in vesicles About 5 mg of M ₅ P _2.5 and 10-fold (weight ratio) PEG dimethoxyether (MW550) in a 1.5 mL safe-lock tube Was added. After heating at 95 ° C. for 20 minutes and cooling at room temperature for 15 minutes, the guest molecule solution was added to 10 wt% of M ₅ P _2.5 and mixed by vortexing for 30 seconds. Subsequently, 100 μL, 200 μL, and about 700 μL of PBS (4 mM, pH = 7.4) were added so that the final concentration of MMP was 5 mg / ml. In addition, each buffer was mixed by vortexing for 30 seconds. The prepared solution was allowed to stand for 1 hour, and then purified by dialysis using a MWCO 300 kDa dialysis membrane or Sephadex g-50. 100 μL of the purified sample and DMSO were mixed with 200 μL to obtain a sample for calculating the encapsulation rate.

Release from M ₅ P _2.5 vesicles The purified vesicle solution (1.0 mL) was added to a MWCO 300 kDa dialysis membrane and dialyzed in 20 mL PBS (4 mM, pH = 7.4). The temperature during dialysis was 25 ° C. 20 μL of the solution in the dialysis tube was taken at regular intervals up to 48 hours and mixed with 40 μL of DMSO. This solution was subjected to UV-Vis measurement, and the release amount was calculated from the UV absorption of the guest molecules remaining in the tube.

(9) Preparation method of chymotrypsin-encapsulated M ₅ P _2.5 vesicle and measurement method of enzyme activity About 5 mg of M ₅ P _2.5 and 10 times amount (weight ratio) of polyethylene glycol dimethoxyether (MW 500) were added to a safe-lock tube. After heating at 95 ° C. for 20 minutes, the mixture was cooled at room temperature for 15 minutes. A 15 mg / ml chymotrypsin stock solution was added to a final concentration of 10 wt% of the weight of M ₅ P _2.5 and mixed by vortexing for 30 seconds. 100 μL, 200 μL, and about 700 μL of PBS (4 mM, pH = 7.4) were added so that the final concentration of M ₅ P _2.5 was 5 mg / ml. Further, each buffer solution was mixed by vortexing for 30 seconds. The prepared solution was purified using a MWCO 300 kDa dialysis membrane, and chymotrypsin-encapsulated M ₅ P _2.5 vesicle solution was obtained except for non-encapsulated chymotrypsin. The concentration of α-chymotrypsin contained was quantified by UV-Vis and BCA assay.

Evaluation of enzyme activity of chymotrypsin -in the presence of inhibitors-
100 μL of the chymotrypsin-encapsulated M ₅ P _2.5 solution obtained above, substrate N-benzoyl-l-tyrosine p-nitroanilide, 20 μL inhibitor (aprotinin) (0.81 mg / ml stock solution, final concentration 100 μM), PBS 280 μL (1 × , pH = 7.4). The change in absorbance derived from 410 nm p-nitrophenaniline was followed for 15 minutes with an ultraviolet spectrophotometer, and the enzyme activity was measured.

-In the absence of inhibitors-
100 μL of the chymotrypsin-encapsulated M ₅ P _2.5 solution obtained above was mixed with 20 μL of substrate (0.81 mg / ml stock solution, final concentration 100 μM) and 260 μL of PBS (1 ×, pH = 7.4). The change in absorbance at 410 nm was followed for 15 minutes with an ultraviolet spectrophotometer, and the enzyme activity was measured.
(10) β-galactosidase encapsulated M ₅ P _2.5 vesicle M ₅ P _2.5 vesicles preparation and was encapsulated enzyme activity assay beta-galactosidase of were prepared by chymotrypsin containing M ₅ P _2.5 vesicles similar manner.
Β-galactosidase-encapsulated M ₅ P _2.5 solution ([M ₅ P _2.5 ] = 5.0 mg / ml, [β-galactosidase] = 25 μg / mL) obtained above, 60 μL, PBS 1936 μL, TokyoGreen β-gal solution (25 μg / ml ml) 4 μL was mixed, and the change in fluorescence intensity at 510 nm was followed with a fluorescence spectrophotometer.
Moreover, activation of the anticancer drug prodrug in the cells was performed as follows. Β-galacotsidase-encapsulated TAT-presenting vesicles ([β-galactosidase] = 25 μg / ml) were added to HeLa cells pre-cultured in a 96-well plate, the medium was removed after 24 hours, washed with PBS, and 5-Fluorouridine-5 ′ -ObD-galactopyranoside (FURG, 100 μM) was added. Furthermore, after culturing for 24 hours, 10 μL of Cell counting kit-8 was added and incubated at 37 ° C. for 2 hours, and then the absorbance at 450 nm was measured with a plate reader to evaluate cytotoxicity.

(11) Chaperone activity measurement method Refolding of chemically denatured protein 12 μL was taken from the chemically denatured carbonic anhydrase solution (6 mg / ml) and diluted 200-fold with 2400 μL of the polymer solution (5.0 mg / ml) or buffer. When methyl β cyclodextrin was added, 117 μL of solution was added here (CAB final concentration was 1.0 μM (0.03 mg / ml), polymer was 1.5 mM). Refolding was performed by stirring at 26 to 28 ° C. and 30 to 40 rpm. 400 μL of the refolding solution was taken at regular intervals. 11 μL of pNPA solution (50 mM acetonitrile solution) was added to the solution, and the hydrolysis rate of pNPA was evaluated using UV-Vis spectrum. (UV-Vis measurement: JASCO, V-660, 10 mm x 2 mm cell, 400 nm increase in absorbance was measured for 60 seconds, and the increase in absorbance was used as an activity index)

Example 2
Samples for structural analysis and experimental conditions of M ₅ P _2.5 vesicles by X-ray small angle scattering measurement: M ₅ P _2.5 vesicles prepared by direct hydration and cooling (5 mg / ml in PBS (1 ×))
SAXS measurement: Aichi Synchrotron Light Center BL8S3
Camera length: 2120mm, wavelength: 0.92mm, detector: Rigaku R-axis IV (Imaging plate type) sample cell: quartz capillary (Hilgenberg, φ = 2mm), irradiation time: 600 seconds Fit 2D, Igor's program was used for data analysis.

Measurement Results and Vesicle Structure FIG. 10 shows the obtained SAXS profile. Since there is a high possibility that a vesicle-like structure is formed from a TEM image, the following bilayer model (J. Phys. Chem. B 2007, 111, 10357, J. Phys. Chem. B 1998, 102, 5737), SAXS profile fitting was performed.

Here, the electron densities (J. Phys. Chem. B 2011, 115, 11318) of the solvent and the hydrophobic layer were 334 to 335 and 330 to 331 e / nm ³ from literature values, respectively. In addition, fitting was performed considering that the lengths of sugar chain and PPO in full stretch were 2.5 and 10 nm, respectively. In addition, it is assumed that there is a normal distribution in the thickness of the hydrophobic layer because it is considered that there is some distribution in the thickness of the film.

From FIG. 10, it was found that the SAXS profile obtained by the experiment using the theoretical formula of the bilayer model can be almost fitted. From this, it was confirmed that a bilayer structure was formed. The thickness of the film was 13.1 nm, which was found to be almost the same as the result of TEM observation (FIG. 11).

Example 3
(1) Synthesis of block copolymers with different sugar chains and PPG segments and structural analysis of molecular assemblies Using a method similar to the synthesis scheme of M ₅ P _2.5, a series of compounds with different sugar chains and PPG segments were synthesized. (FIG. 12). Thereafter, a compound in which both ends of PPO (Mn = 2000) are modified with maltopentaose is BisM ₅ P ₂ , one end of PPO (Mn = 1000) is modified with maltopentaose in M ₅ P ₁ , One end of PPO (Mn = 2500) modified with maltooctaose is M ₈ P _2.5 , one end of PPO (Mn = 1000) is modified with maltotriose, M ₃ P ₁ , PPO ( A compound in which one end of (Mn = 3500) is modified with maltopentaose is defined as M ₅ P _3.5 . The obtained compound was identified by ¹ H-NMR, and the NMR spectra of the block copolymers (BisM ₅ P ₂ , M ₅ P ₁ , M ₈ P _2.5 , M ₃ P ₁ , M ₅ P _3.5 ) were shown respectively. It is shown in FIGS.

(2) Structural analysis of molecular assembly The block copolymer obtained above was dispersed in an aqueous solution, and the structural analysis of the molecular assembly in the aqueous solution was performed using TEM. From the TEM image analysis results shown in FIGS. 18 to 22 below, block copolymers (M ₅ P ₁ , M ₈ P _2.5 , M ₃ P ₁ , M ₅ P _3.5 ) form vesicles as molecular aggregates in water. I found out that

While cooling the BISM ₅ P ₂ structural analysis BISM ₅ P ₂ molecules aggregate to obtain BISM ₅ P ₂ molecules aggregate by returning slowly to room temperature dissolved in PBS buffer. The TEM observation result of the obtained molecular assembly is shown in FIG.

From FIG. 18, many spherical particles having a diameter of 10 to 20 nm were observed. From this, it was found that BisM ₅ P ₂ formed spherical micelles.

While cooling the structural analysis M ₅ P ₁ of M ₅ P ₁ molecular aggregates are dissolved in PBS buffer to obtain M ₅ P ₁ molecular assembly solution by slowly returned to room temperature. A TEM image of the molecular assembly is shown in FIG.

From FIG. 19, particles having a diameter of about 100 nm and the edges of the particles stained black with the dyeing agent were observed. From this, it is considered that M ₅ P ₁ forms a vesicle.

Structural analysis of M ₈ P _2.5 molecular assembly M ₈ P _2.5 was dissolved in PBS buffer while cooling, and slowly returned to room temperature to obtain a molecular assembly solution. A TEM image of the obtained molecular assembly is shown in FIG.

From FIG. 20, particles having a diameter of about 100 to 150 nm and having edges can be confirmed. From this, it can be considered that M ₈ P _2.5 forms a vesicle.

Structural analysis of M ₃ P ₁ molecular assembly M ₃ P ₁ was dissolved in PBS buffer while cooling, and slowly returned to room temperature to obtain a molecular assembly solution. FIG. 21 shows a TEM image of the obtained molecular assembly.

From FIG. 21, particles having a diameter of about 100 nm and having edges can be confirmed. From this, it can be considered that M ₈ P _2.5 forms a vesicle.

Structural analysis of M ₅ P _3.5 molecular assembly M ₅ P _3.5 was dissolved in PBS buffer while cooling, and slowly returned to room temperature to obtain a molecular assembly solution. A TEM image of the obtained molecular assembly is shown in FIG.

From FIG. 22, particles having a diameter of 50 to 200 nm and having edges can be confirmed. From this, it is considered that M ₅ P _3.5 forms vesicles.

Example 4
Synthesis of block copolymers with linear and hyperbranched sugar chains by enzymatic polymerization using M ₅ P _2.5 as a sugar chain primer Glycopolymerization with phosphorylase a (GPa) using M ₅ P _2.5 as a primer Then, an enzymatic synthesis reaction of a multi-branched sugar chain was performed by synthesizing a linear sugar chain, and further by a tandem enzyme reaction with GPa and glycogen branching enzyme (GBE). In the linear sugar chain polymerization reaction, the final primer concentration is adjusted to 0.5 mM, glucose monophosphate is added at 0.36 mmol, phosphorylase a is added at 0.77 nmol, and the reaction is performed at 37 ° C. in 0.1 M Bis tris buffer. It was. In the branched sugar chain polymerization reaction, the reaction was carried out by adding 1.2 nmol of GBE in addition to the above compounds. 10 μL was sampled at regular time intervals, free phosphoric acid was quantified, and the progress of polymerization was confirmed. After the reaction, ¹ H-NMR measurement was performed to confirm the presence or absence of protons derived from α1,6-position anomer. The results are shown in FIG.

23. From the left diagram of FIG. 23, it is clear that the sugar chain polymerization reaction is proceeding from the fact that any reaction can observe an increase in the number of glucose. In GPa / GBE tandem polymerization, it can be seen that the initial polymerization rate is increased about 6 times compared to amylose polymerization reaction using only GPa. The reason for this is thought to be that the reaction rate increased because the substrate was changed to a hyperbranched sugar chain and the ability to recognize the enzyme was increased. From the right figure of FIG. 23, the proton derived from the α1,6 position anomer was confirmed at 4.8 ppm, and in the GBE / GPa enzyme polymerization reaction, there were many α (1,4) and α (1,6) bonds. It was found that branched sugar chains can be synthesized.

Claims (9)

An amphiphilic block copolymer obtained by bonding a polypropylene glycol (PPG) segment (A) and a sugar chain segment (B) via a linker group, wherein the sugar chain segment (B) contains a maltotriose unit. An amphiphilic block copolymer comprising an anomeric carbon of maltotriose bonded to a PPG segment (A) via a divalent linker group. Formula (I) below

(Wherein R ¹ and R ² are the same or different and each represents a hydrogen atom or a sugar residue containing at least one glucose. Y ¹ represents a divalent linker group. R represents a hydrogen atom, an alkyl group, An aryl group, an aralkyl group, an alkanoyl group, a hydroxyalkyl group, Y ² -R ³ , Y ² represents a divalent linker group, and R ³ represents the following formula

(Wherein R ¹ and R ² are as defined above.)
N1 is 10 to 500. )
The amphiphilic block copolymer of Claim 1 represented by these. The divalent linker group represented by Y ¹ or Y ² is —NHR ⁴ —,

(In the formula, X represents O, NR ⁴ or S. p represents an integer of 1 to 4. R ⁴ represents a hydrogen atom, an acyl group or an alkyl group.)
The amphiphilic block copolymer according to claim 2, which is any one of groups represented by: A molecular assembly composed of at least one amphiphilic block copolymer according to any one of claims 1 to 3. The molecular assembly according to claim 4, wherein the molecular assembly is a vesicle, a spherical micelle, a rod-like micelle, or a tube structure. 6. The molecular assembly according to claim 5, which contains a protein. The molecular assembly according to claim 6, wherein the protein is an enzyme. The method for producing a molecular assembly according to claim 4 or 5, wherein at least one amphiphilic block copolymer according to any one of claims 1 to 3 is dispersed in water. A protein inclusion agent comprising the molecular assembly according to claim 4 or 5.

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