JP2023005522A - Osmoregulator - Google Patents

Abstract

To provide a novel polymer composition exhibiting osmoregulatory activity, or particularly, an osmoregulator which can inhibit protein alteration, etc. in lower concentrations.SOLUTION: An osmoregulator comprises a polymer composition including a polymer main chain and a side chain bonded thereto which has a constituent unit comprising an amine oxide structure.SELECTED DRAWING: Figure 1

Description

TECHNICAL FIELD The present invention relates to an osmotic pressure regulator used for the purpose of suppressing protein denaturation.

Proteins are structural proteins such as collagen and keratin that form the cells and extracellular matrices that make up the body. It constitutes a related substance. In recent years, bioactive proteins such as antibodies and enzymes have been widely used in the fields of pharmaceuticals, diagnostic agents, and regenerative medicine.

It is known that the functions expressed in various proteins strongly depend on their higher-order structures. The higher-order structure of the protein is formed due to the equilibrium of weak intermolecular interactions that occur between protein molecules as amino acid polymers, and heating or freezing that changes the equilibrium state of the interactions It is known that when stress such as , pressure, and pH change is applied, the higher-order structure of the protein changes, causing denaturation and deactivation, and losing its function.

On the other hand, deep-sea organisms, which are exposed to particularly high water pressure, retain substances (osmolytes) that exhibit various osmotic pressure-regulating effects in their bodies. is known to suppress the cell volume, prevent denaturation of proteins such as enzymes, and stabilize their structures and functions.
In Patent Document 1, sugars (sucrose, glucose, trehalose, lactose, sodium gluconate, mannitol, etc.) and amino acids (sodium glutamate, lysine, glycine, etc.) are mixed with an enzyme (cholesterol oxidase) together with bovine serum albumin. , that the activity is maintained when the enzyme is lyophilized.

Further, in Patent Document 2, by using a solution containing raffinose, trehalose, sucrose, lactose, trimethylaminooxide, betaine, taurine, sarcosine, glucose, mannose, fructose, ribose, galactose, sorbitol, mannitol, inositol, etc. , that the survival rate of mammalian tissues (cells) is increased when stored in cold storage or frozen storage.

The saccharides, amino acids, and methylammoniums used above all function as osmolytes, and the presence of osmolytes, which are osmotic pressure regulators, causes denaturation, etc. when preserving proteins, cells, tissues, etc. can be suppressed.

Further, Patent Document 3 describes that freeze denaturation of frozen objects such as proteins and cells can be suppressed by using an amine compound having a zwitterionic structure as a freeze denaturation inhibitor.

JP-A-8-187095 Japanese Patent Publication No. 2000-512625 JP 2014-150777 A

As described above, by retaining bio-related substances such as proteins, cells, and tissues in a solution in which a substance exhibiting an osmotic pressure-regulating action is dissolved, denaturation, etc., occurs when stored at a predetermined temperature or frozen. has been observed to be suppressed.

On the other hand, when attempting to obtain a desired effect using a substance that exhibits the osmotic pressure-regulating action, it is generally necessary to use a solution containing the substance at a high concentration of about several mol/L, and the application is limited. This is the reason why it is limited.

An object of the present invention is to provide a novel polymer composition exhibiting an osmotic pressure-regulating action, and in particular, to provide an osmotic pressure-regulating substance capable of suppressing protein denaturation and the like at lower concentrations. and

In order to solve the above problems, the present invention provides a penetrant composition containing a polymer composition having a structure represented by the following formula 8 and containing a structural unit having a structure represented by the following formula 9 or 10 at its tip portion. Provide a pressure regulator. In Formula 8, R ₁ represents hydrogen or a methyl group, and X is a linker moiety that is an ester bond, an ether bond, an amide bond, a disulfide bond, a silylene group, a phenylene group, an alkylene group having 1 carbon atom, or It is selected from a unit structure having a combination, where m is the repeating number of carbon atoms and is in the range of 1 to 6, and n represents the repeating number of structural units. In Formula 9, R ₂ and R ₃ each independently represent an alkyl group having 1 to 12 carbon atoms. Formula 10 represents a 3- to 18-membered saturated or unsaturated heterocyclic amine-N-oxide which can contain a nitrogen atom, an oxygen atom, or a sulfur atom in the ring.

In addition, the present invention provides an osmotic pressure regulator containing a polymer composition containing a structural unit in which the structure shown in Formula 8 above is combined with the structure shown in Formula 9 in particular.
Furthermore, the present invention provides an osmotic pressure regulator used for the purpose of suppressing protein denaturation in an aqueous solution, and is used in combination with one or more substances that exhibit the action of suppressing protein denaturation. Provided is a tonicity modifier that is

Furthermore, the present invention provides an osmotic pressure regulator used as a cell cryopreservation agent for cryopreserving proteins and/or cells, and also provides an osmotic pressure regulator mixed with one or more substances exhibiting other cell cryopreservation effects. Provided are osmotic agents for use in

INDUSTRIAL APPLICABILITY According to the present invention, there is provided an osmotic pressure regulator capable of stabilizing biologically relevant substances, such as suppressing protein denaturation.

1 is a graph showing thermal denaturation of ribonuclease A in an aqueous solution containing a polymer composition according to the invention. 1 is a graph showing enzymatic activity of horseradish peroxidase in an aqueous solution containing a polymer composition according to the present invention after refrigerated storage. Fig. 2 is a graph showing the recovery rate of cells frozen/thawed in culture medium containing the polymer compositions of the present invention. 4 is a graph showing evaluation results of cytotoxicity of the polymer composition according to the present invention.

The present invention will be described below. In the following description, the terms "monomer" and "monomer" are used interchangeably, and the terms "polymer" and "polymer" are interchangeably used. Moreover, the term "polymer composition" is used to mean a composition containing the polymer and the like.

As described above, bio-related substances such as proteins, cells, and tissues are preserved in a solution in which a substance exhibiting an osmotic pressure-regulating action such as sugars, amino acids, and methylammoniums is dissolved, thereby preserving them at a predetermined temperature. It is observed that denaturation, etc. during freezing and storage is suppressed and stabilized. On the other hand, it is not necessarily clear why various saccharides, amino acids, and methylammoniums that function as osmotic pressure regulators have completely different structures, but have a common protein denaturation inhibitory effect. It has not been.

On the other hand, the present inventor conducted various studies on the correlation between the behavior of various substances in an aqueous solution and the expression of protein denaturation inhibitory action. It is observed that water molecules are bound to the amine oxide molecules to become "bound water" in an aqueous solution, and according to the amount of bound water bound to the amine oxide molecules, the protein is denatured as the amount of bound water increases. It was found that the inhibitory action tends to be strongly expressed.

In the present invention, the term "amine oxide" means an amine in which an oxygen atom is bonded to a nitrogen atom contained in the amine as a result of oxidation of the amine. It also includes heterocyclic amine-N-oxide, which is a nitrogen-containing heterocyclic compound in which a nitrogen atom bonded to the oxygen atom is included in a heterocyclic ring composed together with a carbon atom and the like.

Table 1 shows the amount of bound water measured by terahertz spectroscopy for various amine oxides and urea, and the temperature rise (Tm) that causes protein denaturation observed in an aqueous solution in which the amine oxide is dissolved. shows the relationship between The width of increase in protein denaturation temperature (Tm) was measured by UV-visible spectroscopy in the same manner as in Example 1 below. Further, the structure of each amine oxide, etc. shown in Table 1 is shown in (Chem. 1).
Terahertz spectroscopy is a spectroscopic method using electromagnetic waves in the terahertz band with a frequency of 0.03 to 12 THz, and it is possible to obtain information on the speed of movement of molecules (dynamics) from the absorption of electromagnetic waves in the terahertz band. can. Terahertz spectroscopy enables us to measure the rotational relaxation dynamics of water on the picosecond scale, especially by examining the absorption coefficients attributed to water molecules. It is possible to estimate the amount of water molecules that bind to one water molecule (bound water amount), such as intermediate water that is constrained by

As shown in Table 1, in an amine oxide containing an oxidized nitrogen atom, a maximum of about 7 water molecules are bound per molecule regardless of the form of substituents such as alkyl groups on the nitrogen atom. is observed. In contrast, in urea, which has no bond between nitrogen and oxygen atoms, substantially no bound water was observed, and no effect of raising the denaturation temperature of protein was observed.
From these results, the formation of bound water in the amine oxide is due to the strong polarity of the amine oxide molecule. It was considered to be due to inclusion. It was also considered that protein denaturation is suppressed by the presence of bound water generated by this mechanism.

The present invention is based on the above finding, and by providing a polymer composition containing the above amine oxide in the side chain portion, it is a low-molecular compound while maintaining the effect of inhibiting protein denaturation exhibited by the amine oxide. It makes it possible to impart various properties that cannot be found in amine oxides.

That is, the present invention is characterized by containing a polymer composition having a structure represented by the following formula 8 and containing a structural unit having a structure represented by the following formula 9 or 10 at the tip portion (●). It relates to osmotic pressure regulators.

However, in formula 8, R ₁ represents hydrogen or a methyl group, and X is a linker moiety, such as an ester bond, an ether bond, an amide bond, a disulfide bond, a silylene group, a phenylene group, an alkylene group having 1 carbon atom, or wherein m is the repeating number of carbon atoms in the range of 1 to 6, and n is the repeating number of the structural unit. In Formula 2, R ₂ and R ₃ each independently represent an alkyl group having 1 to 12 carbon atoms. Formula 3 represents a saturated or unsaturated heterocyclic amine-N-oxide having 3 to 18 members which can contain a nitrogen atom, an oxygen atom or a sulfur atom in the ring.

The above polymer composition, which mainly constitutes the osmotic pressure regulator according to the present invention, has a (meth)acrylamide skeleton, a (meth)acryl skeleton, etc. formed by vinyl-polymerizing a vinyl monomer, an isopropenyl monomer, or the like. In a dialkylamine having an alkylene group having 1 to 6 carbon atoms (m) (Formula 8) in the side chain portion of the main chain portion and an oxygen atom bonded to the other end of the alkylene group, etc. It has a structure in which a nitrogen atom is bonded (Formula 9) and/or a structure in which a saturated or unsaturated heterocyclic amine-N-oxide structure (Formula 10) having 3 to 18 members is bonded as a structural unit.

In the present invention, the term heterocyclic amine means a heterocyclic compound containing at least one nitrogen atom inside the ring, and in particular the term heterocyclic amine-N-oxide refers to It means a state in which an oxygen atom is bonded to at least one of them.

As a form in which the heterocyclic amine-N-oxide is bound to the terminal of the alkylene group or the like shown in Formula 8, the alkylene group or the like can be bound to any nitrogen atom or carbon atom contained in the heterocyclic amine-N-oxide. . In particular, a structure in which an alkylene group or the like is bonded to a nitrogen atom to which an oxygen atom is bonded corresponds to a cyclized alkyl group of dialkylamine shown in formula (9). In addition, the ring of the heterocyclic amine-N-oxide may contain a heteroatom such as an oxygen atom or a sulfur atom in addition to the nitrogen atom to which oxygen is not bound. Moreover, a ring may be constituted by a saturated bond or an unsaturated bond.

In the structures represented by the above formulas 9 and 10, bound water is generated particularly at the site of direct bonding between the nitrogen atom and the oxygen atom, and high hydrophilicity is exhibited due to this site. On the other hand, the main chain portion of the polymer composition represented by Formula 8 and the portion of the alkylene group bonded to the main chain portion generally exhibit hydrophobicity, so that a hydrophilic portion and a hydrophobic portion are formed in one molecule. By coexisting and changing the balance, it is possible to adjust the affinity for proteins, cells, tissues, etc. dissolved in an aqueous solution in particular, and it is possible to express, in particular, adsorption to proteins and the like. The number of carbon atoms (m) contained in the alkylene group or the like is preferably in the range of m = 1 to 6, and by setting it in the range, it is possible to adjust the degree of hydrophobicity within the range of flexibility. .

Hydrophobicity can also be increased by increasing the number of carbon atoms in the alkyl group that binds to the dialkylamine in formula 9, and similarly the adsorption to proteins and the like can be adjusted. The alkyl groups bonded to the dialkylamine may each independently be a linear or branched alkyl group having 1 to 12 carbon atoms.
Hydrophobicity can also be increased by increasing the number of carbon atoms in the ring of the heterocyclic amine-N-oxide in formula 10, and the number of members can be 3-18. On the other hand, by including an oxygen atom in the ring of the heterocyclic amine-N-oxide, it is possible to generate an ether bond and impart hydrophilicity resulting from the ether bond.

Examples of monomers used in synthesizing a polymer composition having a structural unit containing the structure represented by formula 9 include the molecules shown below. That is, N,N-dimethylaminoethyl (meth)acrylamide-N-oxide, N,N-dimethylaminopropyl (meth)acrylamide-N-oxide, N,N-dimethylaminobutyl (meth)acrylamide-N-oxide, Monomers having a dialkylamino-N-oxide containing a methyl group as an alkyl group, such as N,N-dimethylaminopentyl (meth)acrylamide-N-oxide and N,N-dimethylaminohexyl (meth)acrylamide-N-oxide body can be used.

In addition, N,N-diethylaminoethyl (meth)acrylamide-N-oxide, N,N-diethylaminopropyl (meth)acrylamide-N-oxide, N,N-diethylaminobutyl (meth)acrylamide-N-oxide, N,N - using a monomer having a dialkylamino-N-oxide containing an ethyl group as an alkyl group, such as diethylaminopentyl (meth)acrylamide-N-oxide, N,N-diethylaminohexyl (meth)acrylamide-N-oxide, etc. can be done.

In addition, N,N-dipropylaminopropyl (meth)acrylamide-N-oxide, N,N-dipropylaminobutyl (meth)acrylamide-N-oxide, N,N-dipropylaminopentyl (meth)acrylamide-N -oxide, N,N-dipropylaminohexyl(meth)acrylamide-N-oxide, etc., can be used. In addition, a monomer capable of introducing a structure corresponding to the dialkylamino oxide shown in Formula 2 above into the side chain portion can be used.

On the other hand, when the main chain structure of the polymer composition represented by the above formula 9 is composed of a (meth)acrylic skeleton, N,N-dimethylaminoethyl (meth)acrylate-N-oxide, N,N-dimethylamino Propyl (meth)acrylate-N-oxide, N,N-dimethylaminobutyl (meth)acrylate-N-oxide, N,N-dimethylaminopentyl (meth)acrylate-N-oxide, N,N-dimethylaminohexyl ( (Meth)acrylic acids having dialkylamino-N-oxides such as meth)acrylate-N-oxide, N,N-diethylaminoethyl (meth)acrylate-N-oxide, etc. can be used as monomers.

Further, when synthesizing a polymer composition having a structural unit containing the structure represented by formula 10, a monomer having the structure described below can be used. That is, aziridine-N-oxide, azetidine-N-oxide, pyrrolidine-N-oxide, pyrrole-N-oxide, imidazole-N-oxide, imidazolidine-N-oxide, which are oxides of nitrogen-containing heterocyclic compounds, pyrazolidine-N-oxide, pyrazole-N-oxide, oxazolidine-N-oxide, piperidine-N-oxide, morpholine-N-oxide, oxazine-N-oxide, azepan-N-oxide, azepine-N-oxide, azocan- A monomer obtained by bonding a nitrogen atom contained in N-oxide, azonin-N-oxide or the like to (meth)alkylamide, (meth)acrylic acid or the like via an alkylene group can be used.

The above polymer composition, which mainly constitutes the osmotic pressure regulator according to the present invention, uses monomers having the structures of formulas 9 and 10, and particularly uses only a single type of monomer. It can be produced by polymerizing the monomers by an appropriate method, using a mixture of a plurality of types of monomers.

Further, for example, according to the purpose of adjusting the properties such as hydrophilicity/hydrophobicity exhibited by the polymer composition, the structures represented by the above formulas 9 and 10 are not included within the range that does not impair the effects of the present invention. Other monomers exhibiting radical polymerizability such as (meth)alkylamides and (meth)acrylic acid can be used by mixing with the monomers containing the structures of the above formulas 9 and 10 at a predetermined ratio. is.
The blending ratio of other monomers that do not contain structures represented by formulas 9 and 10 can be appropriately determined according to the use of the polymer composition obtained by polymerization. For example, when aiming to suppress protein denaturation, the blending ratio of other monomers that do not contain the structures shown in the above formulas 9 and 10 is based on the total amount of monomers constituting the polymer composition. It is preferably 30 mol % or less, more preferably 10 mol % or less, or 5 mol % or less.

Polymerization of the above monomers is performed by adding an appropriate initiator to a solution obtained by dissolving at least one type of monomer in a predetermined solvent, and performing random polymerization, ionic polymerization, photopolymerization, polymerization using macromers, and the like. It can be carried out by a general method. As the polymerization initiator, for example, a peroxide radical initiator (benzoyl peroxide, ammonium persulfate, etc.) or an azo radical initiator (azobisisobutyronitrile (AIBN), 2,2′-azobis- dimethylvaleronitrile (ADVN), 2,2′-azobiscyanovaleric acid (ACVA), azobis[2-(2-imidazolin-2-yl)propane] dihydrochloride (VA-044), water-soluble or oil-soluble redox radical initiator (composed of dimethylaniline and benzoyl peroxide) and the like can be used.

The amount of the polymerization initiator to be used can be appropriately determined within the range in which the desired polymer can be obtained. More preferably, it is 0.01 to 5 parts by mass. The polymerization temperature and polymerization time can be appropriately selected and determined depending on the type of polymerization initiator and the presence and type of other monomers. For example, when AIBN is used as a polymerization initiator, the polymerization temperature is 40 to 90°C, preferably 50 to 80°C, more preferably 60 to 70°C. Polymerization time can be 1 to 48 hours, preferably 1 to 24 hours, more preferably 2 to 24 hours.

The pressure for the polymerization reaction is not particularly limited, but normal pressure is preferred. In the polymerization reaction using a plurality of types of monomers, the respective monomers may be mixed and random copolymerization may be performed, or the respective monomers may be polymerized to some extent and then mixed. Block copolymerization may also be used.
The solvent used for the polymerization reaction is not particularly limited as long as it dissolves the monomers to be polymerized, and common solvents can be used. For example, polar aprotic solvents such as acetone, dioxane, dimethylformamide (DMF), dimethylsulfoxide (DMSO) and tetrahydrofuran (THF), and polar protic solvents such as propanol, ethanol, methanol, and water are appropriately selected and used. be able to.

The molecular weight of the polymer composition that mainly constitutes the osmotic pressure regulator according to the present invention is not particularly limited, and can be appropriately determined depending on the application. Generally, the number average molecular weight (Mn) can be about 1,000 to 100,000, preferably about 2,000 to 50,000. By increasing the molecular weight, it is possible to improve the adsorptivity of the polymer composition to the surfaces of cells and tissues, particularly when the polymer composition is mixed with cells and tissues.

By retaining proteins, cells, tissues, etc. in the aqueous solution produced by dissolving the osmotic pressure regulator according to the present invention, it is possible, for example, to reduce the degree of decrease in enzymatic activity over time. etc., the osmotic pressure regulator according to the present invention can be used as a stabilizer for proteins and the like in an aqueous solution.
In addition, by freezing proteins, cells, tissues, etc. in a state of being held in an aqueous solution produced by dissolving the osmotic pressure regulator according to the present invention, functional deterioration, viability deterioration, etc. when thawed later can be prevented. can be suppressed, and the osmotic pressure regulator according to the present invention can be used as a cryopreservation agent.

Targets that are stabilized by the osmotic pressure regulator according to the present invention and that are suppressed in functional deterioration, viability deterioration, etc. when frozen include, for example, enzymes, antibodies, structural proteins, transport proteins, storage proteins, and other physiological proteins. Proteins such as active proteins, cells such as animal cells, plant cells, microorganisms, etc., especially fibroblasts, mesenchymal stem cells, germ cells, fertilized eggs, embryos, etc. Isolated cells and cultures Examples include cells, unestablished cells obtained from biological tissues, cell aggregates such as pancreatic islets of Langerhans, tissues, and tissue fragments.

When retaining proteins, cells, tissues, etc. in an aqueous solution containing the osmotic pressure regulator according to the present invention as a protein stabilizer, etc., water that mainly constitutes the aqueous solution may be purified water, pure water, ion Exchanged water or the like can be used, and it is particularly preferable to dissolve the osmotic pressure regulator according to the present invention in a commonly used buffer. Examples of buffers used include phosphate buffers, Tris buffers, Good's buffers, glycine buffers, borate buffers and the like, and these may be mixed and used.

When the osmotic pressure regulator according to the present invention is used in the form of an aqueous solution as a protein stabilizer or the like, the polymer constituting the osmotic pressure regulator according to the present invention is adjusted so that the concentration is suitable for the application. The composition is dissolved in a buffer or the like before use. In general, increasing the concentration of the polymer composition enhances the effect of suppressing denaturation, deterioration of function, decrease in survival rate, etc. of the retained material, but may affect the subsequent use of the retained material. produce sexuality. For this reason, it is preferable to use an aqueous solution containing a polymer composition at a lower concentration within a range in which a predetermined purpose can be achieved, depending on the type of the object to be held, its application, and the like.

Specifically, when used mainly for the purpose of stabilizing proteins, the concentration of the osmotic pressure regulator according to the present invention in the aqueous solution of the polymer composition should be 10 wt % or less, more preferably 5 wt % or less. preferably. In addition, by setting the concentration of the polymer composition in the aqueous solution to 0.01 wt% or more, the effect as a protein stabilizer is observed, particularly in the range of 0.05 wt% or more, or 0.1 wt% or more. As a result, a remarkable effect as a protein stabilizer can be expressed.

When used mainly for the purpose of cryopreservation of proteins, cells, etc., the concentration of the osmotic pressure regulator according to the present invention in the aqueous solution of the polymer composition should be 20 wt % or less, more preferably 15 wt % or less. preferably. In addition, when the concentration of the polymer composition in the aqueous solution is 0.5 wt% or more, the effect as a cryopreservation agent is observed, and in particular, the concentration is in the range of 1.0 wt% or more, or 2.0 wt% or more. Therefore, a remarkable effect as a cryopreservation agent can be exhibited.

When retaining proteins, cells, tissues, etc. in an aqueous solution containing the osmotic pressure regulator according to the present invention, for the purpose of enhancing the protein stabilization effect and the effect as a cryopreservation agent, For example, sugars, proteins other than the protein to be stabilized, salts, surfactants, etc. can be mixed at a predetermined concentration and used.

Examples of the sugars include lactose, sucrose, trehalose and the like. Proteins other than the protein to be stabilized include, for example, bovine serum albumin, gelatin, and casein. Examples of salts include amino acids and amino acid salts such as glycine, alanine, serine, threonine, glutamic acid, aspartic acid, glutamine, asparagine, lysine and histidine; peptides such as glycylglycine; Salts, inorganic salts such as tris salts, flavins, organic acids such as acetic acid, citric acid, malic acid, maleic acid and gluconic acid, and salts of organic acids. Surfactants include, for example, substances exhibiting biocompatibility such as polyoxyethylene alkyl ethers.

In addition, when the osmotic pressure regulator according to the present invention is used as a cryopreservation agent for proteins, cells, etc., dimethyl sulfoxide (DMSO), glycerol, and L-ectoin, which have been conventionally used as freeze denaturation inhibitors, are used. , glycine betaine, trimethylamine-N-oxide, methylcellulose, polyvinylpyrrolidone, etc. can be mixed at a predetermined concentration and used.

When the osmotic pressure regulator according to the present invention is used as a protein stabilizer as described above, it should be maintained at a temperature higher than the temperature at which the components dissolved in the protein-retaining aqueous solution do not freeze or precipitate. Specifically, the temperature is preferably 2° C. or higher, or 4° C. or higher. In addition, the denaturation of proteins can be suppressed by the presence of the osmotic pressure regulator according to the present invention regardless of the temperature of the aqueous solution. It is desirable to keep the aqueous solution at a temperature of 70°C to 60°C or less, more preferably 50°C to 40°C or less.

In addition, when the osmotic pressure regulator according to the present invention is used as a cryopreservation agent for proteins, cells, etc. as described above, for example, when the material to be frozen is protein, the temperature is about -30°C to 4°C. When the object to be frozen is a cell or the like, the temperature is preferably about -200°C to -20°C.
Hereinafter, the osmotic pressure regulator according to the present invention will be described in more detail based on Examples, but the present invention is not limited to the Examples.

(Synthesis example)
According to the following synthesis scheme, polydimethylaminopropyl (meth)acrylamide-N-oxide (formula 12) is produced by a polymerization reaction using N,N-dimethylaminopropyl (meth)acrylamide-N-oxide (formula 11) as a monomer. ) was synthesized.
The polydimethylaminopropyl (meth)acrylamide-N-oxide is based on trimethylamine-N-oxide (TMAO), which is conventionally known to act as an osmolyte, and one of its methyl groups is bound to acrylamide. It corresponds to those substituted with the alkylene group and included in the side chain portion relative to the polymer backbone. Hereinafter, polydimethylaminopropyl(meth)acrylamide-N-oxide may be referred to as polydimethylamine-N-oxide (PDMAO).

The reaction was carried out under nitrogen atmosphere. N,N-dimethylaminopropyl acrylamide (manufactured by Tokyo Kasei Kogyo Co., Ltd.) was added to 1-propanol as a reaction solvent maintained at 70 ° C. in a 1 L three-necked flask equipped with a stirrer, thermometer, and nitrogen inlet tube. After adding and dissolving N,N-dimethylaminopropyl acrylamide-N-oxide (4.0 g) obtained by oxidation, azobisisobutyronitrile (AIBN) (manufactured by Tokyo Chemical Industry Co., Ltd., 38 mg) was added, and the mixture was stirred at 70° C. for 8 hours to carry out polymerization. After completion of the reaction, an excess amount of methanol was added to reprecipitate the polymer, and the precipitate was washed again with methanol to obtain a sample.

¹ H NMR (D ₂ O) identified the precipitate as PDMAO. Also, the number average molecular weight (Mn=15000) was measured by gel permeation chromatography (Prominence GPC system manufactured by Shimadzu Corporation) based on polyethylene glycol (PEG).

Table 2 shows the amount of intermediate water and the amount of antifreeze water contained when PDMAO synthesized above is saturated with water, in comparison with poly 2-methacryloyloxyethylphosphorylcholine (PMPC). Antifreeze water is water molecules that are strongly restrained by binding to molecules such as polymers, and do not exhibit freezing and thawing behavior as water molecules. Intermediate water, on the other hand, is a water molecule that has a certain degree of freedom and is constrained by various molecules. It is a water molecule in a state where it can be attached. Intermediate water is also found in polymers that exhibit biocompatibility and in various biological substances, and is believed to play an important role in the development of biocompatibility.

The content of the intermediate water in the hydrous polymer or the like is determined by measuring the amount of latent heat transfer observed in the hydrous polymer using a differential scanning calorimeter (DSC). It is calculated by dividing the calorific value observed due to ordering by the latent heat of solidification of water molecules. In addition, the amount of water molecules for which transfer of latent heat associated with melting or solidification is not observed among the water molecules contained in water is defined as the amount of non-freezing water. The intermediate water and the antifreeze water are considered to constitute the bound water observed by the terahertz spectroscopy.
The PMPC used for comparison in Table 2 is a polymer containing phospholipids contained in living organisms, and is known to contain a particularly large proportion of intermediate water. It is a polymer that exhibits a high antifouling effect such that proteins and the like are difficult to adsorb to its surface, and also exhibits high biocompatibility.

As shown in Table 2, it was observed that PDMAO contained intermediate water in the saturated water content state. On the other hand, when compared with PMPC, it was observed that the intermediate water content was low, while the non-freezing water content was high.

(Example 1)
Using the ribonuclease A (RNase A) enzyme as a sample in the presence of guanidinium chloride (GdmCl) that exhibits protein denaturation, the following method was used to evaluate the protein stabilization effect of PDMAO synthesized above under heating. .
In the absorption cell, RNase A (RNase A), an enzyme derived from bovine pancreas, was added to 20 mM phosphate-buffered saline (PBS, pH 7.0) containing GdmCl (1.5 M, Fujifilm Wako Pure Chemical Corporation, Osaka, Japan). Nacalai Tesque, Inc., Kyoto, Japan) was added and the absorbance at 287 nm was recorded while the temperature was raised from room temperature to 90°C at a rate of 1°C/min. In addition, in the measurement, the same measurement was performed when the PDMAO synthesized above was added at concentrations of 0.1 wt % and 1.0 wt %.

FIG. 1 shows the change in absorbance (ΔA) at 287 nm measured at each temperature in the above. In FIG. 1, the absorbance at the start of measurement is taken as the reference (0), and the lowest absorbance in each measurement is set to "-1" to show the standardized changes in absorbance. Table 3 also shows the thermal denaturation midpoint temperature (Tm) in the above measurement for each concentration of PDMAO.

As shown in FIG. 1, in the above measurements, changes in absorbance at 287 nm were observed in all samples due to denaturation of RNase A with the lapse of time and temperature rise. On the other hand, addition of PDMAO was observed to shift the temperature at which denaturation occurs to a higher temperature, suggesting that added PDMAO inhibits RNase A denaturation. In addition, it was considered that PDMAO effectively suppresses the denaturation of RNase A from the fact that the heat denaturation midpoint temperature (Tm) shifts to the high temperature side by increasing the concentration of PDMAO added.

The above results show that PDMAO, which is incorporated into a polymer composition by substituting one of the methyl groups of TMAO, which is known as an osmotic pressure regulator, suppresses denaturation of proteins derived from PTMA. It was thought to suggest that

(Example 2)
By the method shown below, suppression of decrease in enzymatic activity due to the addition of PDMAO synthesized above was evaluated when the horseradish peroxidase enzyme (HRP) was stored in a refrigerator.
HRP (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was dissolved in 20 mM phosphate-buffered saline (PBS, pH 7.0) kept at 4°C to a concentration of 1.0 (µg/ml). After storage at 4° C. for 0.5 h and 5 days, 2,2′-azinobis[3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt (ABTS) was added and the diammonium salt was observed at 410 nm. HRP activity was assessed by measuring absorbance.

In addition, in the measurement, when the PDMAO synthesized above was added at a concentration of 1.0 wt%, and for comparison, TMAO, 2-methacryloyloxyethylphosphorylcholine (MPC), and its polymer polyMPC (Mn=100 kDa) was added to give a monomer unit concentration of 60 mM, and similar measurements were performed. The monomer unit concentration of the acrylamide N-oxide structure in the 1.0 wt % PDMAO solution is about 60 mM.

Moreover, the MPC is a zwitterion having a biased charge within one molecule, and is known to have high biocompatibility due to its extremely low protein adsorption frequency.

FIG. 2 shows the HRP activity (enzyme activity) observed in each of the above samples. In FIG. 2, HRP activity observed when HRP was dissolved in PBS and stored at 4° C. for 0.5 hours was used as the standard (100%), and the degree of HRP activity observed under each of the above conditions was calculated. show.
As shown in FIG. 2, it was observed that the HRP activity was greatly reduced by refrigerating for 5 days at 4° C. in a state of being mixed with PBS alone. It was also observed that the samples added with TMAO, MPC, and polyMPC decreased in HRP activity to the same extent as in the case of PBS alone. On the other hand, it was observed that the HRP enzymatic activity was significantly maintained in the sample to which the PDMAO synthesized above was added as compared with the other samples.

The above results show that PDMAO, which is one of the osmotic pressure regulators according to the present invention, suppresses the denaturation of proteins such as enzymes and stabilizes them even when they are stored in an aqueous solution under cold storage. be. In addition, the above results show that the osmotic pressure regulator according to the present invention containing TMAO or the like, which is a low-molecular compound exhibiting high hydrophilicity, as part of the polymer composition is more effective than TMAO. This indicates that the effect of maintaining the enzyme activity is exhibited.

As described above, when compared with TMAO and the like, the mechanism by which the osmotic pressure regulator according to the present invention effectively exerts the protein-stabilizing effect is that a low-molecular-weight compound such as TMAO, which exhibits high hydrophilicity, is added to the hydrophobic site. It is speculated that this is the result of increasing the frequency of adsorption with enzymes such as HRP and locally increasing the density of TMAO units by making it part of the polymer composition having

(Example 3)
The cryoprotective effect of using the PDMAO synthesized above was evaluated using human lung cancer cell A549 by the method shown below.
A solution of cells dissolved in a culture medium (DMEM/F12 medium) at a density of 1×10 ⁵ (cells/ml) was frozen at −80° C. and stored for one week. Cells were plated on plastic plates after changing to fresh culture medium and the percentage of cells recovered from freezing was assessed using the WST-8 assay using tetrazolium salts.

On the other hand, in a state in which the PDMAO synthesized above was added to the cell solution, the cells were similarly frozen and thawed, and then the number of cells recovered from freezing was evaluated in the same manner as above, and the results were compared.

FIG. 3 shows the percentage of cells recovered from freezing under each of the above conditions (cell viability). As shown in FIG. 3, human lung cancer cells A549 (Cell+media) frozen in cell solution using only culture medium recovered very few cells from freezing. On the other hand, it was observed that the number of cells recovered from freezing was significantly increased in the cells frozen in the cell solution containing the PDMAO (7%) synthesized above dissolved in the culture medium (7% PDMAO). , confirmed that the PDMAO exhibits a cryoprotective effect.
In addition, by using DMSO (2.5%), which has been conventionally used as a cell cryoprotectant, compared to adding only DMSO (2.5% DMSO) or only PDMAO at the same concentration, An increase in the number of cells recovered from freezing was observed.

When the PDMAO synthesized above was used on cells, the cytotoxicity exhibited by PDMAO was evaluated by the following method.
A549 cells were seeded in 96 wells at a density of 1×10 ⁴ (cells/well) using DMEM/F12 medium, and after 1 day of culture, PDMAO was added to the medium so that the concentration was 2 to 7 wt %. Cell viability was determined by measuring absorbance at 450 nm using WST-8 assay after 1 day, 1 day, and 4 days. In addition, for comparison, cell viability was evaluated after similarly culturing in a medium containing 5% DMSO.

FIG. 4 shows the cell viability when cultured as described above. In addition, in FIG. 4, the cell viability when various additions are performed is shown based on the cell viability when culturing for 1 day or 4 days in a culture medium to which PDMAO or the like is not added.
As shown in FIG. 4, when DMSO, which has been conventionally used as a cell cryoprotectant, was added, the cell viability after culturing was greatly reduced. In contrast, the percentage reduction in cell viability upon addition of PDMAO in the range of up to 7 wt% was shown to be lower than DMSO at comparable concentrations, indicating that the cytotoxicity of the cryoprotectant is low. It was shown that it can be used as

The osmotic pressure regulator according to the present invention can effectively suppress denaturation of proteins, etc., and also functions as a cryoprotectant for cells, and stabilizes bio-related substances such as preservation of proteins, cells, and biological tissues. It is useful as an osmotic pressure regulator that can

Claims (10)

An osmotic pressure regulator characterized by containing a polymer composition having a structure represented by the following formula 1 and containing a structural unit having a structure represented by the following formula 2 or 3 at the tip portion (●) thereof.

However, in Formula 1, R ₁ represents hydrogen or a methyl group, and X is a linker moiety such as an ester bond, an ether bond, an amide bond, a disulfide bond, a silylene group, a phenylene group, an alkylene group having 1 carbon atom, or wherein m is the repeating number of carbon atoms in the range of 1 to 6, and n is the repeating number of the structural unit. In Formula 2, R ₂ and R ₃ each independently represent an alkyl group having 1 to 12 carbon atoms. Formula 3 represents a saturated or unsaturated heterocyclic amine-N-oxide having 3 to 18 members which can contain a nitrogen atom, an oxygen atom or a sulfur atom in the ring. 2. The osmotic pressure regulator according to claim 1, comprising a polymer composition comprising a structural unit in which the structure represented by the above formula 2 is bonded to the structure represented by the above formula 1. 3. The osmotic pressure regulator according to claim 1, wherein said _R1 is a hydrogen atom. 4. The osmotic pressure regulator according to claim 2, wherein both _R2 and R3 _are methyl groups. 5. The osmotic pressure regulator according to any one of claims 1 to 4, wherein said X is an amide bond. 6. The osmotic pressure regulator according to any one of claims 1 to 5, which is used for the purpose of suppressing protein denaturation in an aqueous solution. 7. The osmotic pressure regulator according to claim 6, which is used in combination with one or more substances exhibiting an action of inhibiting denaturation of other proteins. 6. The osmotic pressure regulator according to any one of claims 1 to 5, which is used as a cell cryopreservation agent for cryopreserving proteins and/or cells. 9. The osmotic pressure regulator according to claim 8, which is used in combination with one or more other substances exhibiting cell cryopreservation action. The above-mentioned other substance exhibiting cell cryopreservation action is a substance selected from dimethylsulfoxide (DMSO), glycerol, trehalose, L-ectoin, glycine betaine, trimethylamine-N-oxide, methylcellulose, and polyvinylpyrrolidone. The osmotic pressure regulator according to claim 9.

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