JP7385234B2 - Polyoxazoline-bound hemoglobin and artificial oxygen carrier - Google Patents

Description

The present invention relates to polyoxazoline-bound hemoglobin and artificial oxygen carriers.

When a large amount of blood flows out of blood vessels due to an accident, disaster, or even surgical operation, it is necessary to perform a blood transfusion as soon as possible to restore the oxygen transport function within the body. First, the top priority is to restore circulating blood volume by infusing physiological saline, etc., but since red blood cells play the role of oxygen transport among blood components, administration of red blood cell preparations becomes essential. Red blood cells (length: approximately 8 micrometers) contain approximately 250 million molecules of hemoglobin (a hemoprotein with an active center of heme and a molecular weight of approximately 64,500 that has an oxygen-binding ability), at a high concentration of approximately 35%. It's packed with. Hemoglobin in red blood cells, which binds oxygen in the lungs, releases oxygen in peripheral tissues and supplies oxygen to body tissue cells. In many medically advanced countries, systems have been established that allow blood donations and transfusions to be carried out stably. Blood transfusion therapy can be administered without any problems. Since the virus has been tested, there is almost no risk of infection with known viruses associated with blood transfusions. However, universal challenges to blood transfusion therapy (particularly the administration of red blood cell preparations) always remain. Specifically, there are the following concerns. (i) When administering red blood cells, it is necessary to confirm the blood type (crossmatch) in advance, but this process takes approximately 40 minutes, which may not be enough to treat emergency patients. (ii) Red blood cells have a short storage period of 21 days at 4°C, so if a large-scale disaster or earthquake occurs and a large number of patients are infected at once, it will not be possible to secure a sufficient amount of blood for transfusion. (iii) The risk of infection cannot be excluded from unknown viruses. Furthermore, new problems are expected to arise as social conditions change in the future. (iv) As the birthrate declines and the population ages, the number of elderly people who require blood transfusions increases, and the number of blood donors (young people) decreases, it will become difficult to maintain a stable supply of blood transfusions even in normal medical treatment. . Against this background, there are currently great expectations for the development of artificial blood, especially artificial oxygen carriers that can serve as red blood cell substitutes.

Already in the 1980s, artificial oxygen carriers have been developed that have no blood type (can be administered to humans of all blood types), have no risk of viral infection, can be stored for long periods of time, and can be used whenever and wherever needed. Development has been carried out mainly in Europe, America and Japan. For example, in Japan, a cell-type artificial oxygen carrier has been developed in which hemoglobin is encapsulated in the inner aqueous phase of a bilayer membrane endoplasmic reticulum (liposome) formed by self-assembly of phospholipid molecules in water (for example, patent document (see 1). Although this cell-type artificial oxygen carrier has no problematic side effects and is eagerly awaited for practical use, it has not yet been tested in clinical trials due to concerns about the advanced preparation technology and high initial cost.

On the other hand, in the United States, as artificial oxygen carriers, intramolecularly cross-linked hemoglobin (for example, see Patent Document 2), hemoglobin polymer made by bonding bovine hemoglobin with a crosslinking agent (for example, see Patent Document 3), Hemoglobin polymers in which human hemoglobin is bonded with a crosslinking agent (see, for example, Patent Document 4); PEG-bonded hemoglobin in which polyethylene glycol (PEG), a water-soluble polymer, is bonded to the molecular surface of human hemoglobin (for example, in Patent Document 5); ) have been developed and are proceeding to clinical trials. These artificial oxygen carriers are designed to prevent hemoglobin from dissociating into subunits and being excreted by the kidneys by creating intramolecular crosslinks between subunits or increasing the molecular size (molecular weight). The U.S. Food and Drug Administration (FDA) There are still no approved formulations in clinical use.

PEG is considered a standard biocompatible, bioinert, water-soluble polymer material. However, serum antibodies against PEG have been detected in patients treated with PEG-conjugated asparaginase and PEG-conjugated uricase, and it has been reported that the presence of these anti-PEG antibodies causes the administered PEG-conjugated preparation to be rapidly excreted from the body. (For example, see Non-Patent Documents 1 and 2). Furthermore, anti-PEG antibodies have been found in more than 25% of patients, even those who have not received treatment with PEG-conjugated preparations (eg, Non-Patent Documents 3, 4, 5). This is thought to be due to the fact that PEG is used in various products such as foods and cosmetics on the market. It has also been reported that vacuolization of cells is observed when PEG-conjugated hemoglobin is repeatedly administered (for example, Non-Patent Document 6). Under such circumstances, attention is being focused on the development of new biocompatible water-soluble polymers that can replace PEG.

Japanese Patent Application Publication No. 2004-307404 Japanese Patent Application Publication No. 10-306036 Special Publication No. 11-502821 Special Publication No. 2006-516994 Special Publication No. 2005-515225

J. K. Armstrong et al., Cancer, 2007, 110, 103-111. M. R. Sherman et al., Adv. Drug Delivery Rev. , 2008, 6, 59-68. R. M. Leger et al., Transfusion, 2001, 41, 29S. J. K. Armstrong et al., Blood, 2003, 102, 556A. C. Lubich et al., Pharm. Res. , 2016, 33, 2239-2249. C. Conover et al., Artif. Cells, Blood Subs. , Immob. Biotechnol. , 1996, 24, 599-611.

In the development of artificial oxygen carriers, in addition to PEG, we are using water-soluble high-density oxygen carriers that are highly biocompatible (e.g., no renal excretion, no side effects such as increased blood pressure or antigen-antibody reactions), and are easy to prepare (synthesize). There is a strong desire to develop hemoglobin preparations that bind molecules.

An object of the present invention is to provide polyoxazoline-bound hemoglobin and an artificial oxygen carrier that are highly biocompatible and easy to prepare (synthesize).

The present inventors have discovered that this objective can be achieved by covalently bonding a biocompatible polyoxazoline to hemoglobin, which has an oxygen-binding ability.

Polyoxazoline is a nonionic (uncharged) water-soluble polymer that is highly biocompatible and has a non-immunogenic pseudo-polypeptide structure. It also exhibits many of the favorable properties of PEG while avoiding some of its drawbacks. The noteworthy features of polyoxazoline are listed below (the disadvantages of PEG are in parentheses). (I) It can be easily synthesized by ring-opening polymerization of oxazoline, and various side chain-substituted derivatives can be easily prepared (PEG is difficult to polymerize and has no side chains); (II) it does not generate peroxides (PEG is difficult to polymerize and has no side chains); (forms peroxide), (III) low viscosity (PEG has high viscosity at high concentrations), (IV) stable at room temperature (PEG is stable at low temperatures but unstable at room temperature), (V) in the body. Decomposes and is easily removed (PEG may accumulate). The present inventors have discovered that polyoxazoline can be an alternative material to PEG as a compound that modifies hemoglobin.

That is, the polyoxazoline-bound hemoglobin of the present invention is characterized by having hemoglobin as a core and polyoxazoline as a shell covalently bonded to the hemoglobin via a crosslinking agent.

In the polyoxazoline-bound hemoglobin of the present invention, the binding site with the crosslinking agent in the hemoglobin is preferably lysine, a primary amine at a protein end, or cysteine.

In the polyoxazoline-bound hemoglobin of the present invention, the covalent bond via the crosslinking agent preferably includes the following structure (1).

In the polyoxazoline-bound hemoglobin of the present invention, it is desirable that the covalent bond via the crosslinking agent includes a structure derived from the maleimide group-introducing agent.

（一般式（１）中、Ｒ₂は水素原子又はＳＯ₃ ^-Ｎａ⁺を表し、Ｒ₁は下記一般式（３）、（４）及び下記化学式（１）～（３）のいずれかを表す。また、一般式（２）中、Ｒ₃は下記一般式（３）を表し、Ｒ₄はＯＨ又はＣｌを表す。）

（一般式（３）中、ｎは１～１０の整数を表す。）

（一般式（４）中、ｎは、２、４、６、８、１０又は１２の整数を表す。）

In the polyoxazoline-bound hemoglobin of the present invention, it is desirable that the maleimide group-introducing agent contains at least one selected from the group consisting of compounds represented by the following general formulas (1) and (2).

(In general formula (1), R ₂ represents a hydrogen atom or SO ₃ ^- Na ⁺ , and R ₁ represents any of the following general formulas (3), (4) and the following chemical formulas (1) to (3). (In addition, in general formula (2), R ₃ represents the following general formula (3), and R ₄ represents OH or Cl.)

(In general formula (3), n represents an integer from 1 to 10.)

(In general formula (4), n represents an integer of 2, 4, 6, 8, 10 or 12.)

（式中、Ｒ₁は、下記一般式（３）、（４）又は下記化学式（１）～（３）のいずれかを表す。）

（一般式（３）中、ｎは１～１０の整数を表す。）

（一般式（４）中、ｎは、２、４、６、８、１０又は１２の整数を表す。）

In the polyoxazoline-bound hemoglobin of the present invention, the covalent bond via the crosslinking agent preferably includes the following structure (2).

(In the formula, R ₁ represents any of the following general formulas (3), (4) or the following chemical formulas (1) to (3).)

(In general formula (3), n represents an integer from 1 to 10.)

(In general formula (4), n represents an integer of 2, 4, 6, 8, 10 or 12.)

（一般式（５）中、ｎは１～１０の整数を表す）

（一般式（６）中、Ｒ₁はＯＨ又はＣｌを表し、ｎ、ｍは、１～１０の整数を表す。） In the polyoxazoline-bound hemoglobin of the present invention, the covalent bond via the crosslinking agent further includes a structure derived from a thiol group-introducing agent, and the thiol group-introducing agent has the following chemical formula (4), the following general formula (5). ), and at least one compound selected from the group consisting of compounds represented by the following general formula (6).

(In general formula (5), n represents an integer from 1 to 10)

(In general formula (6), R ₁ represents OH or Cl, and n and m represent integers from 1 to 10.)

（構造（３）、（４）中、ｍは１～１０の整数を表す。） In the polyoxazoline-bound hemoglobin of the present invention, the covalent bond via the crosslinking agent preferably includes the following structure (3) or structure (4).

(In structures (3) and (4), m represents an integer from 1 to 10.)

The artificial oxygen carrier of the present invention is characterized by containing the polyoxazoline-bound hemoglobin.

According to the present invention, it is possible to provide a novel polyoxazoline-bound hemoglobin and an artificial oxygen carrier that have high biocompatibility and are easy to prepare (synthesize).

FIG. 1 is a diagram showing an example of polyoxazoline-bound hemoglobin of the present invention.

Embodiments of the present invention will be specifically illustrated below with reference to the drawings as necessary.
<Polyoxazoline-bound hemoglobin>
The polyoxazoline-bound hemoglobin of this embodiment has at least hemoglobin as a core and polyoxazoline as a shell, and further has other parts as necessary. The hemoglobin and the polyoxazoline are covalently bonded via a crosslinking agent.

For example, as shown in FIG. 1, the polyoxazoline-bound hemoglobin 100 of this embodiment may have a hemoglobin 10 as a core and six polyoxazolines 20 as a shell. In FIG. 1, hemoglobin 10 and polyoxazoline 20 are covalently bonded via a crosslinking agent.

[hemoglobin]
The hemoglobin, when composed of four hemoglobin subunits, has a molecular weight of about 64,500 daltons.
Preferably, the hemoglobin is composed of four subunits, each subunit having one protoheme. Oxygen binds to the iron atom within the protoheme. That is, four oxygen molecules can bind to one molecule of hemoglobin, which is composed of four subunits. The hemoglobin may be a tetramer consisting of four subunits, or may be a 3-, 2-, or 1-mer.
In the polyoxazoline-bound hemoglobin of this embodiment, it is sufficient that the polyoxazoline is bound to at least one hemoglobin subunit contained in hemoglobin via a crosslinking agent. For example, when hemoglobin is composed of multiple hemoglobin subunits (e.g., dimeric, trimeric, tetrameric, etc.), polyoxazoline may be bonded to all hemoglobin subunits via a crosslinking agent, or Hemoglobin subunits to which polyoxazoline is not bound via an agent may also be included.

The hemoglobin is not particularly limited and can be appropriately selected depending on the purpose, and hemoglobin purified from red blood cells derived from vertebrates including humans can be used. The hemoglobin may be at least one selected from the group consisting of human hemoglobin, bovine hemoglobin, porcine hemoglobin, horse hemoglobin, canine hemoglobin, feline hemoglobin, recombinant hemoglobin, intramolecularly crosslinked hemoglobin, and hemoglobin polymer. desirable. These may be used alone or in combination of two or more. Among these, human hemoglobin is preferred because of its high biocompatibility. Further, the hemoglobin can be easily produced by protein synthesis (culture).

-Human hemoglobin-
The human hemoglobin includes those purified from human-derived red blood cells, and is not particularly limited and can be appropriately selected depending on the purpose.

-Bovine hemoglobin-
The bovine hemoglobin includes those purified from bovine red blood cells, and is not particularly limited and can be appropriately selected depending on the purpose.

-Porcine hemoglobin-
Examples of the porcine hemoglobin include those purified from porcine red blood cells, and there are no particular limitations, and it can be appropriately selected depending on the purpose.

-Equine hemoglobin-
Examples of the horse hemoglobin include those purified from horse-derived red blood cells, and there are no particular limitations, and it can be appropriately selected depending on the purpose.

-Canine hemoglobin-
Examples of the canine hemoglobin include those purified from dog-derived red blood cells, and there are no particular limitations, and it can be appropriately selected depending on the purpose.

-Feline hemoglobin-
Examples of the cat hemoglobin include those purified from cat-derived red blood cells, and there are no particular limitations, and it can be selected as appropriate depending on the purpose.

-Recombinant hemoglobin-
The recombinant hemoglobin is not particularly limited as long as it is produced by conventional genetic recombination operations, culture operations, etc., and can be appropriately selected depending on the purpose.

-Intramolecularly cross-linked hemoglobin-
The intramolecularly crosslinked hemoglobin is not particularly limited and can be appropriately selected depending on the purpose, such as diaspirin crosslinked hemoglobin, bismaleimide crosslinked hemoglobin, etc. in which subunits of hemoglobin are bonded to each other via a crosslinking agent. can be mentioned.

-Hemoglobin polymer-
Specific examples of the hemoglobin polymer are not particularly limited and can be appropriately selected depending on the purpose, such as a hemoglobin polymer in which a plurality of hemoglobins are bonded with a glutaraldehyde crosslinking agent.

[Crosslinking agent]
Examples of the crosslinking agent include a crosslinking agent containing a maleimide group-introducing agent, a crosslinking agent containing a thiol group-introducing agent, and more specifically, a maleimide group-introducing agent, a thiol group-introducing agent, and the like. The crosslinking agents may be used alone or in combination of two or more.

一般式（１）中、Ｒ₂は水素原子又はＳＯ₃ ^-Ｎａ⁺のいずれかを表し、Ｒ₁は下記一般式（３）、（４）又は下記化学式（１）～（３）のいずれかを表す。また、一般式（２）中、Ｒ₃は下記一般式（３）を表し、Ｒ₄はＯＨ又はＣｌを表す。

一般式（３）中、ｎは１～１０の整数を表す。

一般式（４）中、ｎは２、４、６、８、１０又は１２の整数を表す。

(Maleimide group introduction agent)
The maleimide group-introducing agent is not particularly limited as long as it is a reagent capable of introducing a maleimide group into hemoglobin or polyoxazoline, and can be appropriately selected depending on the purpose. Compounds having a maleimide group are preferred, and reaction efficiency From this point of view, a group consisting of bifunctional compounds such as the following general formula (1), in which one end is a succinimidyl group and the other end is a maleimide group, and a compound represented by the following general formula (2). At least one compound selected from is preferred.
When introducing a maleimide group using a compound represented by the following general formula (2) in which R ₄ is OH, it is used together with a condensing agent to introduce a maleimide group to the NH ₂ terminal or OH terminal of hemoglobin or polyoxazoline. It is preferable to do so. Preferred examples of the condensing agent include N,N'-dicyclohexylcarbodiimide (DCC) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC). Furthermore, the compound represented by the following general formula (2) in which R ₄ is OH may be used after converting the OH of R ₄ to Cl by reacting with a compound such as thionyl chloride or oxalyl chloride. good.
The above-mentioned maleimide group-introducing agent can be used to introduce a maleimide group into the NH ₂ group of the lysine residue of hemoglobin, the NH ₂ group of the primary amine at the terminal of a protein, and the terminal NH ₂ group of polyoxazoline. 1) or the compound represented by the following general formula (2) is preferable, and the compound represented by the following general formula (2) is preferable for introducing a maleimide group into the terminal OH group of the polyoxazoline. These may be used alone or in combination of two or more.

In general formula (1), R ₂ represents either a hydrogen atom or SO ₃ ^- Na ⁺ , and R ₁ represents any of the following general formulas (3), (4) or the following chemical formulas (1) to (3). represents. Further, in the general formula (2), R ₃ represents the following general formula (3), and R ₄ represents OH or Cl.

In general formula (3), n represents an integer of 1 to 10.

In general formula (4), n represents an integer of 2, 4, 6, 8, 10 or 12.

For example, by reacting the succinimidyl group in the maleimide group-introducing agent with the amino group (NH ₂ group) of the lysine residue in hemoglobin or the amino group (NH ₂ group) at the end of the protein, the amino group (NH 2 group) of the lysine residue ( -NH ₂ ) or an amino group (NH ₂ group) at the end of a protein.
Examples of the method for introducing the maleimide group include stirring hemoglobin and a maleimide group-introducing agent at 5° C. to 30° C. for 1 hour to 40 hours.

（一般式（５）中、ｎは１～１０の整数を表し、架橋反応のしやすさの観点から好ましくは２～５、更に好ましくは２～３、特に好ましくはｎ＝２である。）

（一般式（６）中、Ｒ₁はＯＨ又はＣｌを表し、ｎ、ｍは、１～１０の整数を表す。それぞれのＲ¹は同じであってもよいし異なっていてもよい。Ｒ¹はＯＨであることが好ましく、２つのＲ¹が共にＯＨであることがより好ましい。ｎ、ｍは同じであってもよいし異なっていてもよい。ｎ、ｍは、架橋反応のしやすさの観点から好ましくは２～５、更に好ましくは２～３、特に好ましくはｎ＝ｍ＝２である。） (thiol group introducing agent)
The thiol group-introducing agent is not particularly limited as long as it is a reagent capable of introducing a thiol group into hemoglobin or polyoxazoline, and can be appropriately selected depending on the purpose. From the viewpoint of reaction efficiency, at least one compound selected from the group consisting of compounds represented by the following chemical formula (4) (2-iminothiolane hydrochloride), the following general formula (5), and the following general formula (6) is preferable. .
When introducing a thiol group using a compound represented by the following general formula (6) in which R ₁ is OH, it is used together with a condensing agent to introduce a thiol group to the NH ₂ terminal or OH terminal of hemoglobin or polyoxazoline. It is preferable to do so. Preferred examples of the condensing agent include N,N-dicyclohexylcarbodiimide (DCC) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC). Further, the compound represented by the following general formula (2) in which R ₁ is OH may be used after converting the OH of R ₁ to Cl by reacting with a compound such as thionyl chloride or oxalyl chloride. good.
The above-mentioned thiol group-introducing agent is used to introduce a thiol group into the NH ₂ group of the lysine residue of hemoglobin, the NH ₂ group of the primary amine at the terminal of a protein, and the terminal NH ₂ group of polyoxazoline. , a compound represented by the general formula (5) or a general formula (6) is preferable, and a compound represented by the general formula (6) is preferable for introducing a thiol group into the terminal OH group of the polyoxazoline. These may be used alone or in combination of two or more.

(In general formula (5), n represents an integer of 1 to 10, preferably 2 to 5, more preferably 2 to 3, particularly preferably n=2 from the viewpoint of ease of crosslinking reaction.)

(In general formula (6), R ₁ represents OH or Cl, and n and m represent integers of 1 to 10. Each R ¹ may be the same or different. R ¹ is preferably OH, and more preferably both R ¹ are OH. n and m may be the same or different. n and m represent ease of crosslinking reaction. (From the viewpoint of n = m = 2, preferably 2 to 5, more preferably 2 to 3, particularly preferably n = m = 2.)

[Polyoxazoline]
The polyoxazoline is a nonionic water-soluble polymer having a highly biocompatible and non-immunogenic pseudo-polypeptide structure. It can be synthesized in large quantities, various functional groups can be introduced, and it can be safely used in animals. It exhibits many of the superior properties of PEG while avoiding some of its drawbacks. Furthermore, polyoxazolines are excreted through the kidneys without accumulating in body tissues.
The above polyoxazolines may be used alone or in combination of two or more.

（一般式（７）中、Ｒ₁は炭素数１～８の炭化水素基を表し、好ましくはメチル基、エチル基、プロピル基、イソプロピル基である。Ｒ₂は、ヒドロキシル基、アミノ基、又は－ＮＨ－（ＣＨ₂）₂－ＯＨを表す。ｎは反復単量体単位の数を表す。）
前記ポリオキサゾリンは、ホモ重合体であってもよいし、ヘテロ重合体であってもよい。 Examples of the polyoxazoline include compounds represented by the following general formula (7).

(In general formula (7), R ₁ represents a hydrocarbon group having 1 to 8 carbon atoms, preferably a methyl group, ethyl group, propyl group, or isopropyl group. R ₂ represents a hydroxyl group, an amino group, or -NH-(CH ₂ ) ₂ -OH. n represents the number of repeating monomer units.)
The polyoxazoline may be a homopolymer or a heteropolymer.

The weight average molecular weight of the polyoxazoline is preferably 500 to 100,000 Daltons.

<Production method of polyoxazoline-bound hemoglobin>
Examples of the method for producing the polyoxazoline-bound hemoglobin of the present embodiment include a method of reacting using at least a hemoglobin derivative derived from the hemoglobin and a polyoxazoline derivative derived from the polyoxazoline.

(hemoglobin derivative)
The hemoglobin derivative is preferably at least one selected from the group consisting of thiol group-introduced hemoglobin, maleimide group-introduced hemoglobin, and unmodified hemoglobin.

Among these, when using a terminal maleimide group polyoxazoline as the polyoxazoline derivative, it is desirable to use thiol group-introduced hemoglobin or unmodified hemoglobin as the hemoglobin derivative.
The thiol group-introduced hemoglobin has the above thiol group-introducing agent (for example, 2-iminothiolane hydrochloride) bound to the amino group (NH ₂ group) of the lysine residue of hemoglobin or the amino group (NH ₂ group) at the terminal of the protein. The obtained thiol group-introduced hemoglobin is desirable. The thiol group-introduced hemoglobin can be obtained, for example, by stirring hemoglobin and a thiol group-introducing agent such as 2-iminothiolane at 5° C. to 30° C. for 0.2 to 3 hours.
As unmodified hemoglobin, hemoglobin treated with a reducing agent may be used.

Further, when using polyoxazoline with a terminal thiol group as the polyoxazoline derivative, it is desirable to use maleimide group-introduced hemoglobin as the hemoglobin derivative.
The maleimide group-introduced hemoglobin has the above-mentioned maleimide group-introducing agent (for example, a compound represented by the general formula (1)) on the lysine residue (NH ₂ group) or the amino group (NH ₂ group) at the protein terminal of hemoglobin. A maleimide group-introduced hemoglobin obtained by bonding is preferable. The maleimide group-introduced hemoglobin can be obtained, for example, by stirring hemoglobin and a maleimide group-introducing agent at 0° C. to 30° C. for 0.1 to 3 hours.

(Polyoxazoline derivative)
The polyoxazoline derivative is preferably at least one selected from the group consisting of terminal maleimide group polyoxazoline and terminal thiol group polyoxazoline, and is selected from compounds represented by the following general formulas (8) to (13). It is more desirable that at least one of the above is used.
Among these, when the hemoglobin derivative is thiol group-introduced hemoglobin or unmodified hemoglobin, terminal maleimide group polyoxazoline is preferable, and when the hemoglobin derivative is maleimide group-introduced hemoglobin, terminal thiol group polyoxazoline is preferable.
The polyoxazoline derivative can be used, for example, as a crosslinking agent when introducing polyoxazoline into a compound (for example, a compound having a thiol group at the end, a compound having a maleimide group at the end, etc.).

一般式（８）中、ｎは反復単量体単位の数を表し、ｍは１～１０の整数を表す。Ｒ₁はメチル基、エチル基、プロピル基、イソプロピル基のいずれかを示す。

一般式（９）中、ｎは反復単量体単位の数を表し、ｍは１～１０の整数を表す。Ｒ₂はメチル基、エチル基、プロピル基、イソプロピル基のいずれかを示す。

一般式（１０）中、ｎは反復単量体単位の数を表し、ｍは１～１０の整数を表す。Ｒ₃はメチル基、エチル基、プロピル基、イソプロピル基のいずれかを示す。 -Terminal maleimide group polyoxazoline-
The terminal maleimide group polyoxazoline is not particularly limited and can be appropriately selected depending on the purpose, and includes, for example, compounds represented by the following general formulas (8) to (10). These may be used alone or in combination of two or more. The weight average molecular weight of the terminal maleimide group polyoxazoline is preferably 500 to 100,000 daltons.

In general formula (8), n represents the number of repeating monomer units, and m represents an integer of 1 to 10. R ₁ represents any one of a methyl group, an ethyl group, a propyl group, and an isopropyl group.

In the general formula (9), n represents the number of repeating monomer units, and m represents an integer of 1 to 10. R ₂ represents any one of a methyl group, an ethyl group, a propyl group, and an isopropyl group.

In the general formula (10), n represents the number of repeating monomer units, and m represents an integer of 1 to 10. R ₃ represents any one of a methyl group, an ethyl group, a propyl group, and an isopropyl group.

The structures of the repeating units in the terminal maleimide group polyoxazoline may be the same or different.
The terminal maleimide group polyoxazoline can be used as a crosslinking agent for adding polyoxazoline to a compound having an SH group at the terminal (for example, a cysteine residue in a protein, a protein modified with a thiol group, etc.).
The terminal maleimide group polyoxazoline can be obtained by, for example, reacting the polyoxazoline with a maleimide group-introducing agent such as the compound of the above general formula (2) (for example, if R 4 is Cl, it is used as it is; if R ₄ is OH, it is reacted with a condensing agent). and stirring at 25°C for 10 to 20 hours). The mixing ratio may be 5 to 15 moles of the compound of general formula (2) per 1 mole of polyoxazoline. After the reaction, purification may be performed by centrifugation, filter filtration, gel filtration, or the like.
The structure of the resulting terminal maleimide polyoxazoline can be analyzed by ¹ H-NMR or the like.

一般式（１１）中、ｎは反復単量体単位の数を表す。Ｒ₁はメチル基、エチル基、プロピル基、イソプロピル基のいずれかを示す。

一般式（１２）中、ｎは反復単量体単位の数を表し、ｍは１～１０の整数を表す。Ｒ₁はメチル基、エチル基、プロピル基、イソプロピル基のいずれかを示す。

一般式（１３）中、ｎは反復単量体単位の数を表し、ｍは１～１０の整数を表す。Ｒ₁はメチル基、エチル基、プロピル基、イソプロピル基のいずれかを示す。 -Terminal thiol group polyoxazoline-
The terminal thiol group polyoxazoline is not particularly limited and can be appropriately selected depending on the purpose, and includes, for example, compounds represented by the following general formulas (11) to (13). These may be used alone or in combination of two or more. The weight average molecular weight of the terminal thiol group polyoxazoline is preferably 500 to 100,000 Daltons.

In general formula (11), n represents the number of repeating monomer units. R ₁ represents any one of a methyl group, an ethyl group, a propyl group, and an isopropyl group.

In general formula (12), n represents the number of repeating monomer units, and m represents an integer of 1 to 10. R ₁ represents any one of a methyl group, an ethyl group, a propyl group, and an isopropyl group.

In general formula (13), n represents the number of repeating monomer units, and m represents an integer of 1 to 10. R ₁ represents any one of a methyl group, an ethyl group, a propyl group, and an isopropyl group.

The structures of the repeating units in the terminal thiol group polyoxazoline may be the same or different.
The terminal thiol group polyoxazoline can be used as a crosslinking agent for adding polyoxazoline to a compound having a maleimide group at the terminal (for example, a protein modified with a maleimide group).
The terminal thiol group polyoxazoline can be obtained by, for example, reacting polyoxazoline with a thiol group-introducing agent such as the compound of general formula (5) (for example, by stirring at 25° C. for 10 to 20 hours). Obtainable. The mixing ratio may be 10 to 20 moles of the compound of general formula (5) per 1 mole of polyoxazoline. After the reaction, treatment with a reducing agent or purification by centrifugation, filter filtration, gel filtration, etc. may be performed.
The structure of the obtained thiol group polyoxazoline can be analyzed by ¹ H-NMR or the like.

In the general formulas (8) to (13) _, those in which R ₁ , R ₂ , and R ₃ are all methyl groups tend _to exhibit higher water solubility than PEG; When ₃ is a propyl group, the solubility in water tends to decrease upon heating. Therefore, those in which R ₁ , R ₂ , and R ₃ are all ethyl groups are preferable in that the hydrophilic and hydrophobic properties are well-balanced.

In all of the above general formulas (8) to (13), m is preferably 2, which has high chemical stability and is the minimum.

More specific examples of the method for producing the polyoxazoline-bound hemoglobin include the following methods (a) to (c).
-Production example (a) of polyoxazoline-bound hemoglobin-
By reacting the terminal maleimide group polyoxazoline with the thiol group-introduced hemoglobin, the maleimide group in the terminal maleimide group polyoxazoline forms a covalent bond with the thiol group of the thiol group-introduced hemoglobin.
Examples of the method for introducing the polyoxazoline include stirring the thiol group-introduced hemoglobin and the polyoxazoline with a terminal maleimide group at 5° C. to 30° C. for 1 hour to 40 hours.

-Production example (b) of polyoxazoline-bound hemoglobin-
By reacting the terminal thiol group polyoxazoline with the maleimide group-introduced hemoglobin, the thiol group in the terminal thiol group polyoxazoline forms a covalent bond with the maleimide group of the maleimide group-introduced hemoglobin.
Examples of the method for introducing the polyoxazoline include stirring the maleimide group-introduced hemoglobin and the terminal thiol group polyoxazoline at 5° C. to 30° C. for 1 hour to 40 hours.

-Production example (c) of polyoxazoline-bound hemoglobin-
By reacting the terminal maleimide group polyoxazoline with the unmodified hemoglobin, the maleimide group in the terminal maleimide group polyoxazoline forms a covalent bond with the cysteine of the unmodified hemoglobin.
Examples of the method for introducing the terminal maleimide group polyoxazoline include stirring unmodified hemoglobin and the terminal maleimide group polyoxazoline at 5° C. to 30° C. for 1 hour to 40 hours.

<Characteristics of polyoxazoline-bound hemoglobin>
In the polyoxazoline-bound hemoglobin of the present embodiment, it is desirable that the binding site with the crosslinking agent in the hemoglobin be lysine, a primary amine at the protein end, or cysteine.
It is desirable that the bonding site with the terminal maleimide group polyoxazoline in the thiol group-introduced hemoglobin be the introduced thiol group. It is desirable that the bonding site with the terminal thiol group polyoxazoline in the maleimide group-introduced hemoglobin be the introduced maleimide group. It is desirable that the bonding site with the terminal maleimide group polyoxazoline in the unmodified hemoglobin be a cysteine residue.

を含むことが好ましく、以下の構造（２）、（３）又は（４）

（式中、Ｒ₁は、上述の一般式（３）、（４）又は上述の化学式（１）～（３）のいずれかを表す。）

（構造（３）、（４）中、ｍは１～１０の整数を表す。）
の構造を有することがより好ましく、以下の構造（５）又は（６）

（構造（５）、（６）中、Ｒ₁は、上述の一般式（３）、（４）又は上述の化学式（１）～（３）のいずれかを表し、ｍは１～１０の整数を表す。）
の構造を有することがさらに好ましい。 In the polyoxazoline-bound hemoglobin of this embodiment, the bond via the crosslinking agent preferably includes a structure derived from a maleimide group-introducing agent and/or a thiol group-introducing agent, and the bonding via the crosslinking agent preferably includes a structure derived from a maleimide group-introducing agent and a thiol group-introducing agent. It is more preferable that the structure is derived only from
The bond via the above crosslinking agent has the following structure (1)

It preferably contains the following structure (2), (3) or (4)

(In the formula, R ₁ represents any of the above general formulas (3) and (4) or the above chemical formulas (1) to (3).)

(In structures (3) and (4), m represents an integer from 1 to 10.)
It is more preferable to have the following structure (5) or (6)

(In structures (5) and (6), R ₁ represents any of the above general formulas (3) and (4) or the above chemical formulas (1) to (3), and m is an integer of 1 to 10. )
It is further preferable to have the structure.

Although the polyoxazoline-bound hemoglobin of this embodiment is relatively easy to prepare, it has a clear three-dimensional structure. The average particle size of the polyoxazoline-bound hemoglobin is preferably 8 to 30 nm, more preferably 8 to 20 nm.

Since the polyoxazoline-bound hemoglobin of this embodiment has an oxygen binding site in hemoglobin, it can form a stable oxygenated form (oxy form), and can efficiently supply oxygen to body tissues. The oxygen affinity (P ₅₀ ) is preferably 3 to 60 Torr, more preferably 6 to 35 Torr.

The number of bonds of polyoxazoline to core hemoglobin of the polyoxazoline-bound hemoglobin of this embodiment is preferably 1 to 10.
As a method for measuring the number of bonds of polyoxazoline to core hemoglobin in the polyoxazoline-bound hemoglobin of the present embodiment, the cyanomethemoglobin method (for example, Alfresa Pharma, Nescot Hemokit N, No. 138016-14) was used. Examples include a method of calculating based on the hemoglobin concentration calculated by quantifying heme and the thiol concentration calculated by quantifying thiol using 4,4'-dithiopyridine.

In the polyoxazoline-bound hemoglobin of this embodiment, the core hemoglobin is surrounded by polyoxazoline, so it is not immunogenic, and there is no renal excretion or leakage from vascular endothelial cells. In addition, polyoxazoline has high water solubility and excellent metabolism.

The polyoxazoline-bound hemoglobin of this embodiment does not cause precipitation or aggregation even when mixed with blood, and has high blood compatibility. In addition, it is easy to synthesize and prepare.

As described above, the polyoxazoline-bound hemoglobin of the present invention can function, for example, as an unprecedented artificial oxygen carrier that has both biocompatibility (safety) and effectiveness.

<Artificial oxygen carrier>
The artificial oxygen carrier of this embodiment includes the polyoxazoline-bound hemoglobin of the above embodiment. The artificial oxygen carrier is a substance capable of transporting oxygen molecules, and when administered to a living body, functions as a substitute for red blood cells.
The above artificial oxygen carrier can be used, for example, as a substitute for red blood cells of vertebrates such as humans, cows, pigs, horses, dogs, and cats.

Hereinafter, the present invention will be specifically explained based on Examples, but the present invention is not limited to these Examples.

(Example 1)
- Preparation Example 1: Preparation of thiol group-introduced hemoglobin (HbA-SH) -
In order to introduce a thiol group into hemoglobin (Hb), the following operation was performed.
Put 13.8 mg of 2-iminothiolane hydrochloride (2-IT, Fujifilm Wako Pure Chemical Industries, Ltd.) into a microtube (1.5 mL capacity), dilute with 1 mL of phosphate buffered saline (PBS, pH 7.4), A 0.1M 2-iminothiolane solution was prepared. Next, put 1 mL of human hemoglobin (1000 μM) in a 1-neck eggplant flask (10 mL capacity), add 400 μL of 2-iminothiolane solution (2-iminothiolane/hemoglobin (2-IT/Hb) = 40 (mol/mol)), The mixture was stirred at 25°C for 3 hours. The solution was applied to a gel filtration column (GE Healthcare Japan, Sephadex G-25 Superfine) equilibrated with phosphate buffered saline (PBS, pH 7.4) to remove excess 2-iminothiolane (2IT). .
20 mL of the obtained solution was placed in a centrifugal concentrator (Merck, Amicon Ultra-15, ultramolecular weight 10 kDa), and centrifuge (BECKMAN COULTER, Allegra X-15R Centrifuge, conditions: 3,000 rpm, 10 minutes, 4 ℃) to concentrate to 2.5 mL (0.4 mM).
The number of thiol groups in thiol group-introduced hemoglobin (Hb-SH) was determined using an exchange reaction between thiol groups and disulfide bonds. 4,4'-Dithiopyridine (4,4'-DTP) reacts with free thiol (SH) groups to yield 4-Thiopyridinone (4-TP) Therefore, the amount of thiol groups can be determined by adding 4,4'-dithiopyridine (4,4'-DTP) to thiol group-introduced hemoglobin and measuring the amount of 4-thiopyridinone (4-TP) produced.
Put 2.2 mg of 4,4'-dithiopyridine (4,4'-DTP) into an Eppendorf tube (2 mL capacity), add 1 mL of phosphate buffered saline (PBS, pH 7.4), and shake well. 1 mL of 4,4'-dithiopyridine (4,4'-DTP) solution (10 mM) was prepared.
Add 2 mL of phosphate buffer aqueous solution (PB, pH 7.4) to a quartz cell for spectroscopy (light path length: 1 cm), and measure the ultraviolet-visible absorption (UV-Vis.) spectrum (190 nm-700 nm) using an ultraviolet-visible spectrophotometer (Agilent). , 8454) (blank). Next, 1500 μL of thiol group-introduced hemoglobin (5 μM) was added to a quartz cell for spectroscopy, and ultraviolet-visible absorption spectra were measured. Subsequently, 15 μL of 4,4'-dithiopyridine (4,4'-DTP) solution (10 mM) was added (4,4'-dithiopyridine/thiol group-introduced hemoglobin (4,4'-DTP/Hb-SH )=20 (mol/mol)), and was shaken well.
After standing for 30 minutes, ultraviolet-visible absorption spectra were measured. The concentration of 4-thiopyridinone (=thiol concentration) was calculated from the absorbance at 324 nm and the molar extinction coefficient of 4-thiopyridinone (4-TP) (ε ₃₂₄ =1.98×10 ⁴ M ⁻¹ cm ⁻¹ ).
On the other hand, the hemoglobin concentration in the thiol group-introduced hemoglobin solution was determined by the cyanomethemoglobin method (Alfresa Pharma, Nescort Hemokit N, No. 138016-14). The number of thiol groups in the thiol group-introduced hemoglobin (HbA-SH) was calculated by dividing the thiol concentration by the obtained hemoglobin concentration (about 6/Hb-SH).

化学反応式（１）中、ｎは反復単量体単位の数を表す。
２口ナスフラスコ（５０ｍＬ容量）に、末端がヒドロキシル基であるポリ（２－エチル－２－オキサゾリン）（平均分子量５，０００Ｄａ、ＰＯｘ（５ｋ）－ＯＨ、Ｓｉｇｍａ－ＡＬＤＲＩＣＨ社）１２０ｍｇ、及び３－ｍａｌｅｉｍｉｄｏｐｒｏｐｉｏｎｉｃ ａｃｉｄに塩化チオニルを反応させて調製した３－ｍａｌｅｉｍｉｄｏｐｒｏｐａｎｏｌｙ ｃｈｌｏｒｉｄｅ（ＭＰＣ）４５ｍｇを入れ、窒素通気を行った（３-ｍａｌｅｉｍｉｄｏｐｒｏｐａｎｏｌｙ ｃｈｌｏｒｉｄｅ／ポリオキサゾリン（ＭＰＣ／ＰＯｘ（５ｋ）－ＯＨ）＝１０（ｍｏｌ／ｍｏｌ））。
そこに、ジクロロメタン（富士フイルム和光純薬社）５ｍＬ、及びトリエチルアミン（富士フイルム和光純薬社）６６μＬを加え、２５℃で１８時間撹拌した。反応液の溶媒をロータリーエバポレーター（ＥＹＥＬＡ社）で留去し、純水３ｍLを加えよく撹拌し、遠心分離で沈殿を除去後、上清をフィルター（Ｍｅｒｃｋ Ｍｉｌｉｐｏｒｅ社、Ｍｉｌｌｅｘ－ＧＰ、０．２２μｍ、ＰＥＳ）でろ過し、純水で平衡化したゲルろ過カラム（ＧＥヘルスケア・ジャパン社、Ｓｅｐｈａｄｅｘ Ｇ-２５ Ｓｕｐｅｒｆｉｎｅ）を用いて精製した。
得られた水溶液を液体窒素で凍結した後、真空下で凍結乾燥し、¹Ｈ ＮＭＲによって構造を同定した。 - Preparation Example 2: Preparation of terminal maleimide group polyoxazoline (average molecular weight 5,000 Da, POx (5k)-eM) -
In order to synthesize polyoxazoline with a terminal maleimide group (average molecular weight 5,000 Da, POx(5k)-eM), the following operation was performed.

In chemical reaction formula (1), n represents the number of repeating monomer units.
In a 2-neck eggplant flask (50 mL capacity), 120 mg of poly(2-ethyl-2-oxazoline) whose terminal end is a hydroxyl group (average molecular weight 5,000 Da, POx (5k)-OH, Sigma-ALDRICH), and 3- 45 mg of 3-maleimidopropanoly chloride (MPC) prepared by reacting maleimidopropionic acid with thionyl chloride was added, and nitrogen aeration was performed. /POx(5k)-OH)=10(mol/ mol)).
5 mL of dichloromethane (Fujifilm Wako Pure Chemical Industries, Ltd.) and 66 μL of triethylamine (Fuji Film Wako Pure Chemical Industries, Ltd.) were added thereto, and the mixture was stirred at 25° C. for 18 hours. The solvent of the reaction solution was distilled off using a rotary evaporator (EYELA), 3 mL of pure water was added, stirred well, and the precipitate was removed by centrifugation, and the supernatant was filtered (Merck Millipore, Millex-GP, 0.22 μm, PES) and purified using a gel filtration column (GE Healthcare Japan, Sephadex G-25 Superfine) equilibrated with pure water.
The resulting aqueous solution was frozen with liquid nitrogen, then freeze-dried under vacuum, and the structure was identified by ¹ H NMR.

- Preparation Example 3: Preparation of polyoxazoline (average molecular weight 5,000 Da)-bound hemoglobin (POx(5k)-eMS-HbA) -
In order to prepare polyoxazoline-bound hemoglobin (POx(5k)-eMS-HbA) bound to polyoxazoline (average molecular weight 5,000 Da), the following operation was performed.
625 μL of the thiol group-introduced hemoglobin solution (HbA-SH, 0.4 mM) prepared in Preparation Example 1 was placed in a one-necked flask (5 mL volume), and 625 μL of the thiol group-introduced hemoglobin solution (HbA-SH, 0.4 mM) prepared in Preparation Example 2 was added to the polyoxazoline with a terminal maleimide group (average molecular weight 5,000 Da, 12.5 mg of POx(5k)-eM) was added and stirred for 14 hours under refrigerated light-shielding (terminated maleimide group polyoxazoline/thiol group-introduced hemoglobin (POx(5k)-eM/HbA-SH) = 10 (mol/mol) )).
The reaction solution was applied to a gel filtration column (GE Healthcare Japan, Superdex 200 p.g.) equilibrated with phosphate buffered saline (PBS, pH 7.4) to remove unreacted polyoxazoline.
The thiol concentration in a polyoxazoline-bound hemoglobin (POx(5k)-eMS-HbA) solution was determined using an exchange reaction between thiol groups and disulfide bonds. On the other hand, the hemoglobin concentration in the polyoxazoline-bound hemoglobin (POx(5k)-eMS-HbA) solution was determined by the cyanomethemoglobin method (Alfresa Pharma, Nescot Hemokit N, No. 138016-14). By dividing the thiol concentration by the obtained hemoglobin concentration, the number of thiol groups remaining in core hemoglobin in polyoxazoline-bound hemoglobin (POx(5k)-eMS-HbA) was calculated (approximately 1 group/Hb). By subtracting the number of thiol groups of the polyoxazoline-bound hemoglobin (POx(5k)-eMS-HbA) from the number of thiol groups of the thiol-group-introduced hemoglobin (HbA-SH) calculated in Preparation Example 1, the polyoxazoline-bound hemoglobin (POx( The number of polyoxazoline bonds to core hemoglobin in 5k)-eMS-HbA) was calculated (approximately 5 bonds/HbA).

(Example 2)
- Preparation Example 1: Preparation of thiol group-introduced hemoglobin (HbBv-SH) -
Thiol group-introduced hemoglobin (HbBv-SH) was prepared in the same manner as in Preparation Example 1 of Example 1, except that bovine hemoglobin was used instead of human hemoglobin.

- Preparation Example 2: Preparation of polyoxazoline (average molecular weight 5,000 Da)-bound hemoglobin (POx(5k)-eMS-HbBv) -
According to the same method as Preparation Example 3 in Example 1, except that in Preparation Example 3 in Example 1, the thiol group-introduced hemoglobin obtained in Preparation Example 1 in Example 2 was used instead of the thiol group-introduced hemoglobin. When polyoxazoline-bound hemoglobin (POx(5k)-eMS-HbBv) was prepared and the number of polyoxazoline bonds was calculated, it was approximately 6 molecules/HbBv.

化学反応式（２）中、ｎは反復単量体単位の数を表す。
２口ナスフラスコ（５０ｍＬ容量）に、末端がアミノ基であるポリ（２－エチル－２－オキサゾリン）（平均分子量５，０００Ｄａ、ＰＯｘ（５ｋ）－ＮＨ₂、Ｓｉｇｍａ－ＡＬＤＲＩＣＨ社）５０ｍｇ、及びＮ－ｓｕｃｃｉｎｉｍｉｄｙｌ ３－ｍａｌｅｉｍｉｄｏｐｒｏｐｉｏｎａｔｅ（ＳＭＰ、富士フイルム和光純薬社）２６．５ｍｇを入れ、窒素通気を行った（Ｎ－ｓｕｃｃｉｎｉｍｉｄｙｌ ３－ｍａｌｅｉｍｉｄｏｐｒｏｐｉｏｎａｔｅ／ポリオキサゾリン（ＳＭＰ／ＰＯｘ（５ｋ）－ＮＨ₂）＝１０（ｍｏｌ／ｍｏｌ））。
そこに、ジクロロメタン（富士フイルム和光純薬社）５ｍＬ、及びトリエチルアミン（富士フイルム和光純薬社）１５μＬを加え、２５℃で１８時間撹拌した。反応液の溶媒をロータリーエバポレーター（ＥＹＥＬＡ社）で留去し、純水２ｍLを加えよく撹拌し、遠心分離で沈殿を除去後、上清をフィルター（Ｍｅｒｃｋ Ｍｉｌｉｐｏｒｅ社、Ｍｉｌｌｅｘ－ＧＰ、０．２２μｍ、ＰＥＳ）でろ過し、純水で平衡化したゲルろ過カラム（ＧＥヘルスケア・ジャパン社、Ｓｅｐｈａｄｅｘ Ｇ－２５ Ｓｕｐｅｒｆｉｎｅ）にかけ、未反応のＮ－ｓｕｃｃｉｎｉｍｉｄｙｌ ３－ｍａｌｅｉｍｉｄｏｐｒｏｐｉｏｎａｔｅ（ＳＭＰ）を除去した。
得られた水溶液を液体窒素で凍結した後、真空下で凍結乾燥し、¹Ｈ ＮＭＲによって構造を同定した。 (Example 3)
- Preparation Example 1: Preparation of terminal maleimide group polyoxazoline (average molecular weight 5,000 Da, POx (5k)-aM) -
In order to synthesize polyoxazoline with a terminal maleimide group (average molecular weight 5,000 Da, POx(5k)-aM), the following operation was performed.

In chemical reaction formula (2), n represents the number of repeating monomer units.
In a 2-neck eggplant flask (50 mL capacity), 50 mg of poly(2-ethyl-2-oxazoline) whose terminal end is an amino group (average molecular weight 5,000 Da, POx (5k)-NH ₂ , Sigma-ALDRICH), and N - 26.5 mg of succinimidyl 3-maleimidopropionate (SMP, Fuji Film Wako Pure Chemical Industries, Ltd.) was added and nitrogen aeration was performed (N-succinimidyl 3-maleimidopropionate/polyoxazoline (SMP/POx (5k) -NH ₂ )=10( mol/mol)).
5 mL of dichloromethane (Fuji Film Wako Pure Chemical Industries, Ltd.) and 15 μL of triethylamine (Fuji Film Wako Pure Chemical Industries, Ltd.) were added thereto, and the mixture was stirred at 25° C. for 18 hours. The solvent of the reaction solution was distilled off using a rotary evaporator (EYELA), 2 mL of pure water was added, stirred well, and the precipitate was removed by centrifugation, and the supernatant was filtered (Merck Millipore, Millex-GP, 0.22 μm, PES) and applied to a gel filtration column (GE Healthcare Japan, Sephadex G-25 Superfine) equilibrated with pure water to remove unreacted N-succinimidyl 3-maleimidopropionate (SMP).
The resulting aqueous solution was frozen with liquid nitrogen, then freeze-dried under vacuum, and the structure was identified by ¹ H NMR.

- Preparation Example 2: Preparation of polyoxazoline (average molecular weight 5,000 Da)-bound hemoglobin (POx(5k)-aMS-HbBv) -
In Preparation Example 3 of Example 1, instead of the terminal maleimide group polyoxazoline (average molecular weight 5,000 Da, POx(5k)-eM), the terminal maleimide group polyoxazoline (average molecular weight 5,000Da, POx(5k)-aM) was used, and the thiol group-introduced hemoglobin (HbBv-SH) obtained in Preparation Example 1 of Example 2 was used. Polyoxazoline-conjugated hemoglobin (POx(5k)-aMS-HbBv) was prepared according to the method. When the number of polyoxazoline bonds was calculated, it was approximately 5 bonds/HbBv.

化学反応式（３）中、ｎは反復単量体単位の数を表す。
２口ナスフラスコ（５０ｍＬ容量）に、末端がヒドロキシル基であるポリ（２－エチル－２－オキサゾリン）（平均分子量２，０００Ｄａ、ＰＯｘ（２ｋ）－ＮＨＯＨ、Ｓｉｇｍａ－ＡＬＤＲＩＣＨ社）４８ｍｇ、及び３－ｍａｌｅｉｍｉｄｏｐｒｏｐｉｏｎｉｃ ａｃｉｄに塩化チオニルを反応させて調製した３－ｍａｌｅｉｍｉｄｏｐｒｏｐａｎｏｌｙ ｃｈｌｏｒｉｄｅ（ＭＰＣ）４５ｍｇを入れ、窒素通気を行った（３－ｍａｌｅｉｍｉｄｏｐｒｏｐａｎｏｌｙ ｃｈｌｏｒｉｄｅ／ポリオキサゾリン（ＭＰＣ／ＰＯｘ（２ｋ）－ＮＨＯＨ）＝１０（ｍｏｌ／ｍｏｌ））。
そこに、ジクロロメタン（富士フイルム和光純薬社）５ｍＬ、及びトリエチルアミン（富士フイルム和光純薬社）６６μＬを加え、２５℃で１８時間撹拌した。反応液の溶媒をロータリーエバポレーター（ＥＹＥＬＡ社）で留去し、純水３ｍLを加えよく撹拌し、遠心分離で沈殿を除去後、上清をフィルター（Ｍｅｒｃｋ Ｍｉｌｉｐｏｒｅ社、Ｍｉｌｌｅｘ－ＧＰ、０．２２μｍ、ＰＥＳ）でろ過し、純水で平衡化したゲルろ過カラム（ＧＥヘルスケア・ジャパン社、Ｓｅｐｈａｄｅｘ Ｇ－２５ Ｓｕｐｅｒｆｉｎｅ）を用いて精製した。
得られた水溶液を液体窒素で凍結した後、真空下で凍結乾燥し、¹Ｈ ＮＭＲによって構造を同定した。 (Example 4)
- Preparation Example 1: Preparation of terminal maleimide group polyoxazoline (average molecular weight 2,000 Da, POx(2k)-aeM)) -
In order to synthesize polyoxazoline with a terminal maleimide group (average molecular weight 2,000 Da, POx(2k)-aeM), the following operation was performed.

In chemical reaction formula (3), n represents the number of repeating monomer units.
In a 2-neck eggplant flask (50 mL capacity), 48 mg of poly(2-ethyl-2-oxazoline) whose terminal end is a hydroxyl group (average molecular weight 2,000 Da, POx (2k)-NHOH, Sigma-ALDRICH), and 3- 45 mg of 3-maleimidopropanoly chloride (MPC) prepared by reacting maleimidopropionic acid with thionyl chloride was added, and nitrogen aeration was performed. /POx(2k)-NHOH)=10(mol/ mol)).
5 mL of dichloromethane (Fujifilm Wako Pure Chemical Industries, Ltd.) and 66 μL of triethylamine (Fuji Film Wako Pure Chemical Industries, Ltd.) were added thereto, and the mixture was stirred at 25° C. for 18 hours. The solvent of the reaction solution was distilled off using a rotary evaporator (EYELA), 3 mL of pure water was added, stirred well, and the precipitate was removed by centrifugation, and the supernatant was filtered (Merck Millipore, Millex-GP, 0.22 μm, PES) and purified using a gel filtration column (GE Healthcare Japan, Sephadex G-25 Superfine) equilibrated with pure water.
The resulting aqueous solution was frozen with liquid nitrogen, then freeze-dried under vacuum, and the structure was identified by ¹ H NMR.

- Preparation Example 2: Preparation of polyoxazoline (average molecular weight 2,000 Da)-bound hemoglobin (POx(2k)-aeMS-HbBv) -
In Preparation Example 3 of Example 1, instead of the terminal maleimide group polyoxazoline (average molecular weight 5,000 Da, POx(5k)-eM), the terminal maleimide group polyoxazoline obtained in Preparation Example 1 of Example 4 (average molecular weight Same as Preparation Example 3 in Example 1, except that 2,000Da, POx(2k)-aeM)) was used, and the thiol group-introduced hemoglobin (HbBv-SH) obtained in Preparation Example 1 in Example 2 was used. Polyoxazoline-conjugated hemoglobin (POx(2k)-aeMS-HbBv) was prepared according to the method described above. When the number of polyoxazoline bonds was calculated, it was approximately 7 bonds/HbBv.

(Example 5)
- Preparation Example 1: Preparation of polyoxazoline (average molecular weight 5,000 Da)-bound hemoglobin (POx(5k)-eM-HbBv) -
In order to prepare polyoxazoline-bound hemoglobin (POx(5k)-eM-HbBv) bound to polyoxazoline (average molecular weight 5,000 Da), the following operation was performed.
Put 500 μL of bovine hemoglobin solution (HbBv, 500 μM) into a one-necked flask (5 mL volume), and add the terminal maleimide group polyoxazoline (average molecular weight 5,000 Da, POx (5k)-eM) obtained in Preparation Example 2 in Example 1. .8 mg was added thereto, and the mixture was stirred for 18 hours under a refrigerator and shielded from light (terminated maleimide group polyoxazoline/hemoglobin (POx(5k)-eM/HbBv) = 5 (mol/mol)).
The reaction solution was applied to a gel filtration column (GE Healthcare Japan, Superdex 200 p.g.) equilibrated with phosphate buffered saline (PBS, pH 7.4) to remove unreacted polyoxazoline.
The thiol concentration in a polyoxazoline-bound hemoglobin (POx(5k)-eM-HbBv) solution was determined using an exchange reaction between thiol groups and disulfide bonds. On the other hand, the hemoglobin concentration in the polyoxazoline-bound hemoglobin (POx(5k)-eM-HbBv) solution was determined by the cyanomethemoglobin method (Alfresa Pharma, Nescot Hemokit N, No. 138016-14). By dividing the thiol concentration by the obtained hemoglobin concentration, the number of thiol groups in polyoxazoline-bound hemoglobin (POx(5k)-eM-HbBv) was calculated (0 groups/HbBv). By subtracting the number of thiol groups in the polyoxazoline-bound hemoglobin (POx(5k)-eM-HbBv) from the number of thiol groups in HbBv (2/HbBv), The number of polyoxazolines bound to core hemoglobin was calculated (2/HbBv).

(Example 6)
- Preparation Example 1: Preparation of maleimide group-introduced hemoglobin (HbBv-M) -
In order to introduce a maleimide group into bovine hemoglobin (HbBv), the following operation was performed.
39.9 mg of N-succinimidyl 3-Maleimidopropionate (SMP, Fuji Film Wako Pure Chemical Industries, Ltd.) was placed in a sample bottle (5 mL capacity) and dissolved in 1 mL of dimethyl sulfoxide to prepare a 0.15 M SMP solution. Next, put 1 mL of bovine hemoglobin (1 mM) in a 1-neck eggplant flask (5 mL capacity), and add 0.1 mL of SMP solution (N-succinimidyl 3-Maleimidopropionate/hemoglobin (SMP/HbBv) = 15 (mol/mol)). The mixture was stirred for 30 minutes under refrigeration and shielded from light. After filtration with a filter (Merck Millipore, Millex-GP, 0.22 μm, PES), the solution was equilibrated with phosphate buffered saline (PBS, pH 7.4) using a gel filtration column (GE Healthcare Japan). Excess N-succinimidyl 3-Maleimidopropionate was removed.
20 mL of the obtained solution was placed in a centrifugal concentrator (Merck, Amicon Ultra-15, ultramolecular weight 10 kDa), and centrifuge (BECKMAN COULTER, Allegra X-15R Centrifuge, conditions: 3,000 rpm, 10 minutes, 4 ℃) to concentrate to 2.5 mL (0.4 mM).

化学反応式（４）中、ｎは反復単量体単位の数を表す。
２口ナスフラスコ（５０ｍＬ容量）に、末端がアミノ基であるポリ（２－エチル－２－オキサゾリン）（平均分子量５，０００Ｄａ、ＰＯｘ（５ｋ）－ＮＨ₂、Ｓｉｇｍａ－ＡＬＤＲＩＣＨ社）５０ｍｇ、及びＮ－ｓｕｃｃｉｎｉｍｉｄｙｌ ３－（２－ｐｙｒｉｄｙｌｄｉｔｈｉｏ）ｐｒｏｐｉｏｎａｔｅ（ＳＰＤＰ、東京化成工業社）３１ｍｇを入れ、窒素通気を行った（Ｎ－ｓｕｃｃｉｎｉｍｉｄｙｌ ３－（２－ｐｙｒｉｄｙｌｄｉｔｈｉｏ）ｐｒｏｐｉｏｎａｔｅ／ポリオキサゾリン（ＳＰＤＰ／ＰＯｘ（５ｋ）－ＮＨ₂）＝１０（ｍｏｌ／ｍｏｌ））。
そこに、ジクロロメタン（富士フイルム和光純薬社）５ｍＬを加え、室温で１５時間撹拌した。反応液の溶媒をロータリーエバポレーター（ＥＹＥＬＡ社）で留去し、純水３ｍＬ、ジチオトレイトール（ＤＴＴ、富士フイルム和光純薬社）３１ｍｇを加え、２５℃で２時間撹拌した（ジチオトレイトール／ポリオキサゾリン（ＤＴＴ／ＰＯｘ（５ｋ）－ＮＨ₂）＝２０（ｍｏｌ）／ｍｏｌ））。遠心分離で沈殿を除去後、上清をフィルター（Ｍｅｒｃｋ Ｍｉｌｉｐｏｒｅ社、Ｍｉｌｌｅｘ－ＧＰ、０．２２μｍ、ＰＥＳ）でろ過し、純水で平衡化したゲルろ過カラム（ＧＥヘルスケア・ジャパン社、ＰＤ－１０）にかけ、未反応物を除去した。
得られた水溶液を液体窒素で凍結した後、真空下で凍結乾燥し、¹Ｈ ＮＭＲによって構造を同定した。チオール基とジスルフィド結合の交換反応を利用して、得られたポリオキサゾリン溶液中のチオール濃度を定量し、末端チオール基ポリオキサゾリン（ＰＯｘ（５ｋ）－ａＳＨ）のチオール基の導入率を算出した。 - Preparation Example 2: Preparation of terminal thiol group polyoxazoline (average molecular weight 5,000 Da, POx(5k)-aSH) -
In order to synthesize a terminal thiol group polyoxazoline (average molecular weight 5,000 Da, POx(5k)-aSH), the following operation was performed.

In chemical reaction formula (4), n represents the number of repeating monomer units.
In a 2-neck eggplant flask (50 mL capacity), 50 mg of poly(2-ethyl-2-oxazoline) whose terminal end is an amino group (average molecular weight 5,000 Da, POx (5k)-NH ₂ , Sigma-ALDRICH), and N 31 mg of -succinimidyl 3-(2-pyridyldithio) propionate (SPDP, Tokyo Kasei Kogyo Co., Ltd.) was added, and nitrogen aeration was performed. PDP/POx (5k) - NH ₂ )=10 (mol/mol)).
5 mL of dichloromethane (Fuji Film Wako Pure Chemical Industries, Ltd.) was added thereto, and the mixture was stirred at room temperature for 15 hours. The solvent of the reaction solution was distilled off using a rotary evaporator (EYELA), 3 mL of pure water and 31 mg of dithiothreitol (DTT, Fujifilm Wako Pure Chemical Industries, Ltd.) were added, and the mixture was stirred at 25°C for 2 hours (dithiothreitol/polymer). Oxazoline (DTT/POx(5k)-NH ₂ )=20(mol)/mol)). After removing the precipitate by centrifugation, the supernatant was filtered with a filter (Merck Millipore, Millex-GP, 0.22 μm, PES), and a gel filtration column (GE Healthcare Japan, PD-) equilibrated with pure water was used. 10) to remove unreacted substances.
The resulting aqueous solution was frozen with liquid nitrogen, then freeze-dried under vacuum, and the structure was identified by ¹ H NMR. Using the exchange reaction between thiol groups and disulfide bonds, the thiol concentration in the obtained polyoxazoline solution was determined, and the introduction rate of thiol groups in the polyoxazoline with terminal thiol groups (POx (5k)-aSH) was calculated.

- Preparation Example 3: Preparation of polyoxazoline (average molecular weight 5,000 Da)-bound hemoglobin (POx(5k)-aSM-HbBv) -
In order to prepare polyoxazoline-bound hemoglobin (POx(5k)-aSM-HbBv) bound to polyoxazoline (average molecular weight 5,000 Da), the following operation was performed.
Pour 0.5 mL of the maleimide group-introduced hemoglobin solution (HbBv-M, 400 μM) obtained in Preparation Example 1 in Example 6 into a one-necked flask (5 mL capacity), and add the terminal thiol group-introduced hemoglobin solution obtained in Preparation Example 2 in Example 6. 17.5 mg of oxazoline (average molecular weight 5,000 Da, POx(5k)-aSH) was added and stirred for 18 hours under refrigerated light protection (thiol-terminated polyoxazoline/maleimide group-introduced hemoglobin (POx(5k)-aSH)/HbBv -M=10 (mol/mol)).
The reaction solution was applied to a gel filtration column (GE Healthcare Japan, Superdex 200 p.g.) equilibrated with phosphate buffered saline (PBS, pH 7.4) to remove unreacted polyoxazoline. When the number of polyoxazoline bonds was calculated, it was approximately 6 bonds/HbBv.

化学反応式（５）中、ｎは反復単量体単位の数を表す。
２口ナスフラスコ（５０ｍＬ容量）に、３，３'－Ｄｉｔｈｉｏｄｉｐｒｏｐｉｏｎｉｃ Ａｃｉｄ（ＤＴＤＰＡ、東京化成工業社）５．１ｍｇ、Ｎ，Ｎ'―Ｄｉｃｙｃｌｏｈｅｘｙｌｃａｒｂｏｄｉｉｍｉｄｅ（ＤＣＣ、東京化成工業社）１０ｍｇ及び４－Ｄｉｍｅｔｈｙｌａｍｉｎｏｐｙｒｉｄｉｎｅ（ＤＭＡＰ、東京化成工業社）３ｍｇを入れ、窒素通気を行った後、ジクロロメタン（富士フイルム和光純薬社）５ｍＬを加え、室温で５分間撹拌した。そこに、末端がヒドロキシル基であるポリ（２－エチル－２－オキサゾリン）（平均分子量５，０００Ｄａ、ＰＯｘ（５ｋ）－ＯＨ、Ｓｉｇｍａ－ＡＬＤＲＩＣＨ社）６０．６ｍｇ、ジクロロメタン（富士フイルム和光純薬社）５ｍＬを加え、室温で２４間撹拌した。反応液の溶媒をロータリーエバポレーター（ＥＹＥＬＡ社）で留去し、純水２ｍＬ、ジチオトレイトール（ＤＴＴ、富士フイルム和光純薬社）７．５ｍｇを加え、２５℃で２時間撹拌した（ジチオトレイトール／ポリオキサゾリン（ＤＴＴ／ＰＯｘ（５ｋ）－ＮＨ₂）＝４（ｍｏｌ）／ｍｏｌ））。遠心分離で沈殿を除去後、上清をフィルター（Ｍｅｒｃｋ Ｍｉｌｉｐｏｒｅ社、Ｍｉｌｌｅｘ－ＧＰ、０．２２μｍ、ＰＥＳ）でろ過し、純水で平衡化したゲルろ過カラム（ＧＥヘルスケア・ジャパン社、ＰＤ－１０）にかけ、未反応物を除去した。
得られた水溶液を液体窒素で凍結した後、真空下で凍結乾燥し、¹Ｈ ＮＭＲによって構造を同定した。チオール基とジスルフィド結合の交換反応を利用して、得られたポリオキサゾリン溶液中のチオール濃度を定量し、末端チオール基ポリオキサゾリン（ＰＯｘ（５ｋ）－ｅＳＨ）のチオール基の導入率を算出した。 (Example 7)
- Preparation Example 1: Preparation of terminal thiol group polyoxazoline (average molecular weight 5,000 Da, POx(5k)-eSH) -
In order to synthesize a terminal thiol group polyoxazoline (average molecular weight 5,000 Da, POx(5k)-eSH), the following operation was performed.

In chemical reaction formula (5), n represents the number of repeating monomer units.
In a 2-neck eggplant flask (50 mL capacity), 5.1 mg of 3,3'-Dithiodipropionic Acid (DTDPA, Tokyo Kasei Kogyo Co., Ltd.), 10 mg of N,N'-Dicyclohexylcarbodiimide (DCC, Tokyo Kasei Kogyo Co., Ltd.), and 4-Dimethylaminop. yridine (DMAP After bubbling with nitrogen, 5 mL of dichloromethane (Fuji Film Wako Pure Chemical Industries, Ltd.) was added, and the mixture was stirred at room temperature for 5 minutes. There, 60.6 mg of poly(2-ethyl-2-oxazoline) with a hydroxyl group at the end (average molecular weight 5,000 Da, POx (5k)-OH, Sigma-ALDRICH), dichloromethane (Fujifilm Wako Pure Chemical Industries, Ltd.) ) and stirred at room temperature for 24 hours. The solvent of the reaction solution was distilled off using a rotary evaporator (EYELA), 2 mL of pure water and 7.5 mg of dithiothreitol (DTT, Fujifilm Wako Pure Chemical Industries, Ltd.) were added, and the mixture was stirred at 25°C for 2 hours. /polyoxazoline (DTT/POx(5k)-NH ₂ )=4(mol)/mol)). After removing the precipitate by centrifugation, the supernatant was filtered with a filter (Merck Millipore, Millex-GP, 0.22 μm, PES), and a gel filtration column (GE Healthcare Japan, PD-) equilibrated with pure water was used. 10) to remove unreacted substances.
The resulting aqueous solution was frozen with liquid nitrogen, then freeze-dried under vacuum, and the structure was identified by ¹ H NMR. Using the exchange reaction between thiol groups and disulfide bonds, the thiol concentration in the obtained polyoxazoline solution was determined, and the introduction rate of thiol groups in the polyoxazoline with terminal thiol groups (POx (5k)-eSH) was calculated.

- Preparation Example 2: Preparation of polyoxazoline (average molecular weight 5,000 Da)-bound hemoglobin (POx(5k)-eSM-HbBv) -
In order to prepare polyoxazoline-bound hemoglobin (POx(5k)-eSM-HbBv) bound to polyoxazoline (average molecular weight 5,000 Da), the following operation was performed.
Add 0.5 mL of the maleimide group-introduced hemoglobin solution (HbBv-M, 400 μM) obtained in Preparation Example 1 in Example 6 to a one-necked flask (5 mL capacity), and add 28 mg of oxazoline (average molecular weight 5,000 Da, POx(5k)-eSH) was added and stirred for 20 hours under refrigerated light protection (thiol-terminated polyoxazoline/maleimide group-introduced hemoglobin (POx(5k)-eSH)/HbBv-M = 10 (mol/mol)).
The reaction solution was applied to a gel filtration column (GE Healthcare Japan, Superdex 200 p.g.) equilibrated with phosphate buffered saline (PBS, pH 7.4) to remove unreacted polyoxazoline. When the number of polyoxazoline bonds was calculated, it was approximately 6 bonds/HbBv.

(Dynamic light scattering (DLS) measurement)
Dynamic light scattering (DLS) measurement of a phosphate buffered saline solution (PBS, pH 7.4) of polyoxazoline-bound hemoglobin (POx(5k)-eMS-Hb) obtained in Preparation Example 3 in Example 1 was performed using zeta potential. - Particle size/molecular weight measurement system (Otsuka Electronics, ELSZ-2000) was used. The average particle size of unmodified human hemoglobin (Hb) was 6.3 nm. The average particle size of polyoxazoline-conjugated hemoglobin (POx(5k)-eMS-HbA) was 12.0 nm. It was found that binding of polyoxazoline to hemoglobin increases the molecular size. Note that the average particle size of unmodified bovine hemoglobin was 6.4 nm.

(Oxygen affinity (P ₅₀ ) measurement)
The ultraviolet-visible absorption spectrum of the polyoxazoline-bound hemoglobin (POx(5k)-aMS-HbBv) obtained in Preparation Example 2 in Example 3 in a phosphate buffered saline solution (PBS, pH 7.4) in a carbon monoxide atmosphere is , λ _max : 420 nm, 538 nm, 569 nm, which matched the spectral pattern of carbon monoxide form (carbonyl form) of bovine hemoglobin. The site was found to bind carbon monoxide.
A phosphate buffered saline solution (PBS, pH 7.4) of this polyoxazoline-bound hemoglobin (POx(5k)-aMS-HbBv) was placed in a one-necked eggplant flask, and the cap was sealed with septum rubber. Oxygen was aerated while irradiated with light. Then, the UV-visible absorption spectrum showed λ _max : 414 nm, 541 nm, and 577 nm, which matched the spectral pattern of the oxygenated form (oxy form) of bovine hemoglobin. It was found that the hemoglobin site of HbBv) binds oxygen.
Subsequently, when nitrogen was bubbled through the phosphate buffered saline solution (PBS, pH 7.4) of this polyoxazoline-bound hemoglobin (POx(5k)-aMS-HbBv), the ultraviolet-visible absorption spectrum changed to λ _max : 430 nm, 555 nm. , which matched the spectral pattern of deoxygenated bovine hemoglobin, indicating that the hemoglobin site of polyoxazoline-bound hemoglobin (POx(5k)-aMS-HbBv) was deoxygenated. Ta.
When oxygen was aerated again, the spectrum pattern was that of an oxy form, indicating that polyoxazoline-bound hemoglobin (POx(5k)-aMS-HbBv) reversibly adsorbed and desorbed oxygen.
Using an oxygen dissociation and bonding curve recorder (TSC, Hemox Analyzer), we determined the oxygen affinity (P ₅₀ ) (when the oxygen bonding rate was 50% in the oxygen dissociation curve graph) from the changes in the ultraviolet-visible absorption spectrum for different oxygen partial pressures. The P ₅₀ of polyoxazoline-bound hemoglobin (POx(5k)-aMS-HbBv) was 10 Torr (37°C). The P ₅₀ of unmodified human hemoglobin was 12 Torr, and the P ₅₀ of unmodified bovine hemoglobin was 23 Torr, and the polyoxazoline-bound hemoglobin of the present Example had an oxygen affinity equal to or higher than that of unmodified hemoglobin.

The artificial oxygen carrier of the present invention containing polyoxazoline-bound hemoglobin as an active ingredient can be used as a highly safe red blood cell substitute even when administered in vivo. The target is not limited to humans, but the artificial oxygen carrier can also be administered to animals (pets such as dogs and cats, livestock, etc.). In addition, the artificial oxygen carrier containing the polyoxazoline-bound hemoglobin of the present invention as an active ingredient can be used as a preservation solution or perfusion solution for transplanted organs or tissues, a culture solution for regenerated tissue, a sensitizer for anticancer treatment of tumors, and preoperative blood. It is also expected to be used as a diluent, a replenishing fluid for extracorporeal circulation circuits such as heart-lung machines, an oxygen supply fluid for ischemic areas (myocardial infarction, cerebral infarction, respiratory failure, etc.), a chronic anemia treatment, and a perfusion fluid for liquid ventilation.

100 Polyoxazoline-bound hemoglobin 10 Hemoglobin 20 Polyoxazoline

Claims (10)

comprising hemoglobin as a core and polyoxazoline as a shell covalently bonded to the hemoglobin via a crosslinking agent,
The covalent bond via the crosslinking agent includes the following structure (2),
(In the formula, R ₁ represents any of the following general formulas (3), (4) or the following chemical formulas (1) to (3).)
(In general formula (3), n represents an integer from 1 to 10.)
(In general formula (4), n represents an integer of 2, 4, 6, 8, 10 or 12.)
A polyoxazoline-bound hemoglobin characterized by: The polyoxazoline-bound hemoglobin according to claim 1, wherein the binding site with the crosslinking agent in the hemoglobin is lysine, a primary amine at a protein end, or cysteine. The covalent bond via the crosslinking agent includes a structure derived from a maleimide group-introducing agent,
The polyoxazoline-bound hemoglobin according to claim 1 or 2, wherein the maleimide group-introducing agent contains at least one selected from the group consisting of compounds represented by the following general formulas (1) and (2).
(In general formula (1), R ₂ represents a hydrogen atom or SO ₃ ⁻ Na ⁺ , and R ₁ represents any of the following general formulas (3), (4) or the following chemical formulas (1) to (3). (In addition, in general formula (2), R ₃ represents the following general formula (3), and R ₄ represents OH or Cl.)
(In general formula (3), n represents an integer from 1 to 10.)
(In general formula (4), n represents an integer of 2, 4, 6, 8, 10 or 12.)
The polyoxazoline-bound hemoglobin according to any one of claims 1 to 3 , wherein the covalent bond via the crosslinking agent includes the following structure (3) or structure (4).
(In structures (3) and (4), m represents an integer from 1 to 10.) The covalent bond via the crosslinking agent further includes a structure derived from a thiol group-introducing agent,
Claims 1 to 3, wherein the thiol group-introducing agent is at least one compound selected from the group consisting of compounds represented by the following chemical formula (4), the following general formula (5), and the following general formula (6). 4. The polyoxazoline-bound hemoglobin according to any one of 4.
(In general formula (5), n represents an integer from 1 to 10.)
(In general formula (6), R ₁ represents OH or Cl, and n and m represent integers of 1 to 10. ) The polyoxazoline-bound hemoglobin according to any one of claims 1 to 5, which has hemoglobin on the left side of the structure (2) and polyoxazoline on the right side of the structure (2). The polyoxazoline-bound hemoglobin according to any one of claims 1 to 5, which has a polyoxazoline on the left side of the structure (2) and has hemoglobin on the right side of the structure (2). The polyoxazoline-bound hemoglobin according to any one of claims 1 to 7, wherein the covalent bond via the crosslinking agent includes the following structure (5) or structure (6).
(In the formula, R ₁ represents any of the following general formulas (3), (4) or the following chemical formulas (1) to (3), and m represents an integer from 1 to 10. )
(In general formula (3), n represents an integer from 1 to 10.)
(In general formula (4), n represents an integer of 2, 4, 6, 8, 10 or 12.)
The polyoxazoline-bound hemoglobin according to any one of claims 1 to 8, having an oxygen affinity (Pa) of 6 to 35 Torr. An artificial oxygen carrier comprising the polyoxazoline-bound hemoglobin according to any one of claims 1 to 9 .

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