KR102516738B1 - Synthesis of glycol-based compound-based crosslinking agent and manufacturing method of functional hydrogel using the same - Google Patents

Abstract

The present invention relates to a method for synthesizing a glycol-based compound-based crosslinking agent and producing a functional hydrogel including the same, and specifically, the functional hydrogel includes an azide group, and the azide group is an activator (activation group) rather than a crosslinking agent. group), it relates to a manufacturing method for a functional hydrogel capable of accurately capturing a specific protein through the azide group.

Description

Synthesis of glycol-based compound-based crosslinking agent and method for producing functional hydrogel using the same

The present invention relates to a method for synthesizing a glycol-based compound-based crosslinking agent and producing a functional hydrogel including the same, and specifically, the functional hydrogel includes an azide group, and the azide group is an activator (activation group) rather than a crosslinking agent. group), it relates to a manufacturing method for a functional hydrogel capable of accurately capturing a specific protein through the azide group.

A hydrogel is a high-molecular polymer composed of one or more monomers in the form of a cross-linked network. These hydrogels are prepared from hydrophilic polymer chains through a physical or chemical crosslinking process, and are swollen to a large extent by water, but do not dissolve in water. The mechanical properties of hydrogels are very similar to those of biological tissues, and they have excellent diffusion properties due to their high water content, so they are used in a wide range of applications such as contact lenses, drug delivery media, separators, and soft tissue replacement materials such as adhesives.

In particular, in the case of the hydrogel, unlike other materials, it contains water, so it is good for detecting organisms that can be activated only within water, such as proteins, by immobilizing antibodies, aptamers, or other recognition substances to increase the amount of the target substance. It is mainly used in biosensors to measure. Representative materials for hydrogels include synthetic polymers such as polyethylene glycol, polyacrylamide, and polyacrylic acid, or natural polymers such as hyaluronic acid, chitosan, and dextran. Signal change on the scaffold can be detected by using a detection antibody that binds to the antibody or aptamer inside the hydrogel and then shows a fluorescence signal or an electrochemical signal change.

In the case of the hydrogel used as the biosensor, it depends on the type of material to be analyzed, specifically, a biosensor for measuring protein, a biosensor for measuring nucleic acid, a biosensor for measuring glucose, a biosensor for measuring chemical substances, and an activity According to various types of biosensors, such as biosensors for sensory measurement, there are also many types of hydrogels that satisfy these requirements.

Conventionally, in order to fix the protein inside the hydrogel, a physical method of physically fixing the protein inside the hydrogel by mixing the protein with the hydrogel prepolymer and then cross-linking it is used, or A method of chelating or binding a protein to a protein using metal nanoparticle surfaces such as Ni or Au or Cu ²⁺ , Ni ²⁺ , or Zn ²⁺ metal ion particles has been used. When the protein is immobilized by the physical method, the binding force is weak, so the immobilized protein can be easily released to the outside. The method of binding the chelate or ligand may have stronger binding force than the physical method, but environmental conditions such as pH and temperature There is a problem that the binding force with the protein may be lowered due to the specific requirements.

Therefore, in order to solve the above problems, there is a need to prepare a hydrogel for a biosensor that can be used universally, and it is necessary to provide a hydrogel having excellent binding force between the hydrogel and protein.

Republic of Korea Patent Publication No. 10-2007-0013213 (2007. 02. 08)

It is intended to solve the low protein immobilization of conventional hydrogels and to provide a hydrogel capable of facilitating protein immobilization in a wide range regardless of the protein type. In addition, it is intended to provide a hydrogel capable of controlling the content of the immobilized protein. It is intended to provide a hydrogel having excellent mechanical strength and excellent water content. In addition, it is intended to provide a multipurpose hydrogel capable of binding various organic and biomaterials in addition to proteins.

In order to solve the above problems, the present invention comprises: (a) preparing a mixed solution by adding and mixing a crosslinking agent represented by Formula 1, a hydrophilic polymer monomer, and an ultraviolet crosslinking agent; (b) preparing a hydrogel by crosslinking the mixed solution; and (c) subjecting the hydrogel to azide substitution reaction (Azidation) to prepare a functional hydrogel having a structural unit represented by Formula 2 below.

[Formula 1]

[Formula 2]

(Wherein R ₁ is C ₁ -C ₆ alkylene, x and y independently satisfy 1 to 100, and n satisfies 1 to 1,000.)

According to one aspect of the present invention, Formula 1 may be prepared by synthesizing Formula 3 and acryloyl chloride.

[Formula 3]

(Wherein R ₁ is C ₁ -C ₆ alkylene, x and y independently satisfy 1 to 100, and n satisfies 1 to 1,000.)

According to one aspect of the present invention, Formula 1 may be prepared by polymerization of a glycol-based polyol and epichlorohydrin.

According to one aspect of the present invention, the glycol-based polyol may include any one or two or more selected from ethylene glycol, propylene glycol, and isopropylene glycol.

According to one aspect of the present invention, the glycol-based polyol may have a number average molecular weight of 100 to 1,000 g/mol.

According to one aspect of the present invention, Formula 1 may have a weight average molecular weight of 1,000 to 10,000 g/mol.

According to one aspect of the present invention, the crosslinking in step (C) may include an ultraviolet crosslinking method.

According to one aspect of the present invention, the hydrophilic polymer monomer of step (B) may include at least one monomer selected from methacrylic acid, acrylic acid, and N-isopropylacrylamide.

According to one aspect of the present invention, a functional hydrogel prepared by the manufacturing method of the functional hydrogel can be provided.

The manufacturing method for the functional hydrogel of the present invention contains a highly functional azide group, so it can covalently bind with alkyne group-linked proteins, antibodies, and organic substances through a click reaction, and the click reaction As a covalent bond, it may have a very strong binding force compared to physical or ligand binding of conventional hydrogels.

The manufacturing method for the functional hydrogel of the present invention can control the content of the azide group, so that the content of the protein, antibody and organic matter to be bound can also be controlled.

In addition, the functional hydrogel of the present invention is prepared with an azide-containing crosslinking agent and a hydrophilic polymer monomer, so it has a high water content and excellent mechanical strength, and the binding with proteins, antibodies, and organic substances is covalent, so that it can be changed at pH and temperature. It also has the effect of not being easily detached.

1 is a 1H-NMR image of poly(epichlorohydrin-co-tetrahydrofuran)diol.
2 is an FT-IR image of a fabficated hydrogel, an Azide functionalized hydrogel, and an after click reaction hydrogel.
Figure 3 is a photograph comparing the shape of the hydrogel before azide treatment and the hydrogel after azide treatment.
Figure 4 is a fluorescence laser optical microscope image before and after sending a click reaction to the azide-treated hydrogel with alkyne spiropyran.

Hereinafter, the present invention will be described in more detail through specific examples or examples including the accompanying drawings. However, the following specific examples or examples are only one reference for explaining the present invention in detail, but the present invention is not limited thereto, and may be implemented in various forms.

Also, unless defined otherwise, all technical and scientific terms have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Terms used in the description in the present invention are merely to effectively describe specific embodiments and are not intended to limit the present invention.

Also, the singular forms used in the specification and appended claims may be intended to include the plural forms as well, unless the context dictates otherwise.

In addition, when a certain component is said to "include", this means that it may further include other components without excluding other components unless otherwise stated.

In the case of a hydrogel used as a conventional biosensor, there is a problem in that the binding with organisms such as proteins is weak and the binding is broken under the influence of pH and temperature, so it is not easy to detect proteins smoothly.

Accordingly, the present invention provides (a) preparing a mixed solution by adding and mixing a crosslinking agent represented by Formula 1, a hydrophilic polymeric monomer, and an ultraviolet crosslinking agent; (b) preparing a hydrogel by crosslinking the mixed solution; And (c) subjecting the hydrogel to azide substitution reaction (Azidation) to prepare a functional hydrogel having a structural unit of Formula 2; did

[Formula 1]

[Formula 2]

(Wherein R ₁ is C ₁ -C ₆ alkylene, x and y independently satisfy 1 to 100, and n satisfies 1 to 1,000.)

In the case of the functional hydrogel of the special structure, the hydrophilic polymer is composed of a main chain, so the water content is high, and the crosslinking agent having a vinyl group at the end contains an azide group, so that a click reaction is performed through the azide group. It can covalently bind proteins, antibodies, and organic substances containing an alkyne group.

The azide group has selectivity to bind only to alkynes, so it is very good to use as a biosensor for detecting a specific protein. It has the advantage of not breaking or breaking. In addition, in the case of the functional hydrogel, the content of the azide group included in the crosslinking agent can be controlled.

In addition, in the case of the functional hydrogel, there is an advantage in that a specific protein into which an alkyne group has been introduced can be selectively immobilized, so that only a desired specific protein can be selected from a protein library. In addition, the specific protein immobilized on the functional hydrogel is strongly covalently bonded within the hydrogel, but does not affect the structure of the protein, so that the protein's functionality can be sufficiently exhibited without deterioration.

According to one aspect of the present invention, Formula 1 may be prepared by synthesizing Formula 3 and acryloyl chloride.

[Formula 3]

(Wherein R ₁ is C ₁ -C ₆ alkylene, x and y independently satisfy 1 to 100, and n satisfies 1 to 1,000.)

Chemical Formula 3 provides a block-copolymer by combining epichlorohydrin with glycol-based polyol. Specifically, epichlorohydrin is continuously bonded to both ends of the glycol-based polyol to obtain Block-copolymers of the same structure can be prepared.

The glycol-based polyol is not limited, but may be a glycol-based polyol containing C ₁ -C ₆ alkylene, preferably ethylene glycol, propylene glycol, or isopropylene glycol.

The number average molecular weight of the glycol-based polyol may be 100 to 1,000 g/mol, preferably 300 to 1,000 g/mol, and more preferably 600 to 1,000 g/mol, but is not limited thereto.

When a glycol-based polyol that satisfies the above range is used, it is well soluble in a solvent such as water or alcohol and has excellent flexibility.

The block-copolymer including a chlorine group bonded to the glycol-based polyol and epichlorohydrin may have a weight average molecular weight of 1,000 to 10,000 g/mol, preferably 1,500 to 8,000 g/mol, and more preferably It may be 2,000 to 5,000 g / mol, but is not limited thereto.

As the block-copolymer containing the chlorine group satisfies the weight average molecular weight, it is possible to provide a hydrogel having excellent flexibility and excellent hardness.

A crosslinking agent in which the vinyl-based functional group includes two or more chlorine groups can be prepared by binding a vinyl compound to both ends of the block-copolymer containing a chlorine group of Formula 3, preferably by adding acryloyl chloride. Thus, a crosslinking agent having two or more vinyl-based functional groups at both ends may be provided, but is not limited thereto.

A hydrogel containing a chlorine group may be prepared by mixing and crosslinking the crosslinking agent containing the chlorine group and the hydrophilic polymer monomer.

The hydrophilic polymeric monomer is not limited as long as it is a hydrophilic polymeric monomer containing a vinyl group, and may preferably include one or more monomers selected from methacrylic acid, acrylic acid, and N-isopropylacrylamide, and more preferably It may contain N-isopropylacrylamide.

A hydrogel containing a chlorine group may be prepared by carrying out chain polymerization of the hydrophilic polymer monomer with the crosslinking agent containing a chlorine group.

The hydrogel containing the chlorine group may be polymerized with 1 to 100 parts by weight of the crosslinking agent containing the chlorine group, preferably 10 to 80 parts by weight, more preferably 30 to 50 parts by weight, based on 100 parts by weight of the hydrophilic polymer monomer. It may include parts by weight, but is not limited thereto.

The content of the crosslinking agent is not limited depending on desired physical properties, but as the crosslinking agent in the above range is included, flexibility and swelling properties may be excellent, and at the same time, strength may be excellent.

In order to dissolve the hydrophilic polymer monomer and the crosslinking agent containing the chlorine group, water, an alcohol solvent, an organic solvent, etc. may be used, preferably an alcohol solvent, and more preferably ethanol, but limited thereto. it is not going to be

A radical initiator may be added to prepare the hydrogel containing the chlorine group. The radical initiator may include a thermal initiator and a photoinitiator, preferably a photoinitiator.

When using the thermal initiator, the radical initiation temperature is relatively high, so depending on the type of hydrophilic polymer, the hydrogel itself may not be produced. However, in the case of the photoinitiator, the hydrogel is well prepared regardless of the type of hydrophilic polymer. And, the added crosslinking agent also has the advantage of not being deformed.

The amount of the photoinitiator may be the amount used in conventionally prepared hydrogels, and preferably may include 0.0001 to 0.1 parts by weight based on 100 parts by weight of the hydrophilic polymer monomer, but is not limited thereto.

The photoinitiator is not limited as long as it is a generally used photoinitiator, preferably darocur, but is not limited thereto.

In order to prepare a functional hydrogel to be produced, a step of substituting the chlorine group of the hydrogel containing a chlorine group with an azide group is required. As a method of substituting the chlorine group, the hydrogel containing the chlorine group and sodium azide may be added to an organic solvent and then stirred at 30 to 50 ° C. for 12 hours or more, and the substitution may be performed through the substitution reaction. A functional hydrogel containing an azide group can be prepared.

The organic solvent may be a polar organic solvent, preferably dimethylformamide (DMF) or dimethylacetamide (DMAC), but is not limited thereto.

By using a method in which a hydrogel containing a chlorine group is first prepared and then substituted with an azide group, hydrogel formation is smoother than when a crosslinking agent substituted with an azide group is used in advance, and the azide group having particularly excellent reactivity is used as a chain Damage during polymerization or crosslinking can be prevented.

The functional hydrogel containing the azide group can be purified by alternately swelling an organic solvent and water to remove unreacted sodium azide.

The azide group of the prepared functional hydrogel can bind only the desired protein due to the selectivity of binding only to alkyne, which is a specific functional group, and since the bond is a covalent bond, it can maintain a stronger bond than conventional physical and ligand bonds.

In addition, the covalent bond has an advantage in that it is easy to maintain the activation condition of the protein because it can maintain binding property even in a change in pH or temperature.

In the functional hydrogel, when an enzyme is introduced into the hydrogel through a double bond, a column tube capable of continuously enzymatic reaction can be made by manufacturing a column using the hydrogel, and using the hydrogel characteristics, which is a biocompatible material, It has the advantage of being able to be used in various ways, such as being able to directly load biomaterials such as proteins.

The present invention will be described in more detail based on the following Examples and Comparative Examples. However, the following Examples and Comparative Examples are only one example for explaining the present invention in more detail, and the present invention is not limited by the following Examples and Comparative Examples.

[Production Example 1]

[Preparation of glycol-based oligomer containing chlorine group]

Using a cannula transfer to a jacketed reactor, 50 parts by weight of polyethylene glycol (Mn 500, Aldrich) and 50 parts by weight of methylene chloride are added to the reactor.

The temperature of the jacketed reactor was controlled at 25°C using a circulator. Boron trifluoride tetrahydrofuran complex (BF ₃ -THF), which is a cationic catalyst, was added to the reactor with a 0.25 equivalent syringe compared to polyethylene glycol. Then, it was stirred for 30 minutes.

Epichlorohydrin (Aldrich) was added drop by drop for 3 hours using a dropping funnel. Thereafter, the reaction was further performed at room temperature at 25° C. for 30 minutes. Thereafter, distilled water was added to terminate the reaction, and an extraction process was performed 2 to 3 times with n-hexane. The solvent was removed from the extracted methylene chloride layer solution using a rotary evaporator. Thereafter, the mixture was dried in a vacuum oven at 60° C. for 12 hours to obtain a glycol-based oligomer containing a chlorine group, which is the compound of Formula 3.

[Preparation of a glycol-based oligomer crosslinking agent containing a chlorine group]

100 parts by weight of the glycol-based oligomer having a chlorine group and 2,000 parts by weight of methylene chloride were added to the jacketed reactor using cannula transfer. Thereafter, each reactor was controlled using a circulator at a temperature of 5 °C. Triethylamine (Aldrich), a catalyst, was added in 2 units compared to the glycol-based oligomer. Thereafter, acryloyl chloride was added slowly for 30 minutes while dropping dropwise into the reactor by adjusting the stop cock of the dropping funnel. After that, the reaction was carried out at room temperature for 12 hours. After the reaction, the extraction process was repeated twice with a 1.5MK ₂ CO ₃ solution, and the extraction process was performed twice or more with distilled water. After the extraction, the remaining moisture was captured with sodium sulfate, and then the sodium sulfate was removed using a Buchner funnel. Then, the methylene chloride solution was removed using a rotary evaporator. Thereafter, the mixture was dried in a vacuum oven at 60° C. and 760 mmHg under reduced pressure for 12 hours to obtain a glycol-based oligomer crosslinking agent containing a chlorine group.

[Production Example 2]

[Preparation of a glycol-based oligomer crosslinking agent containing an azide group]

After dissolving 100 parts by weight of the glycol-based oligomer crosslinking agent containing a chlorine group prepared in Preparation Example in 1,000 parts by weight of dimethylformamide, 400 parts by weight of sodium azide was added to replace the chlorine group with azide to include an azide group An oligomer crosslinking agent was prepared. The prepared glycol-based oligomer crosslinking agent containing an azide group was purified by removing unreacted sodium azide through a swelling process using dimethylformamide and distilled water.

[Example 1]

44 parts by weight of the glycol-based oligomer crosslinking agent containing the chlorine group and 0.004 parts by weight of a photoinitiator (dorcur 1173, Ciba®) and 80 parts by weight of ethanol were mixed with respect to 100 parts by weight of N-isopropylacrylamide (NIPAM, Aldrich). and dissolved. Thereafter, a hydrogel was prepared by performing a radical reaction for 10 minutes through UV irradiation. After swelling the prepared hydrogel in dimethylformamide, 100 parts by weight of sodium azide was added and stirred at 40 °C for 12 hours. The hydrogel was alternately swollen using dimethylformamide and water, and unreacted sodium azide was removed to prepare a hydrogel containing an azide group.

Figure 3 compares the hydrogel before substituting the azide group and the hydrogel substituted with the azide group, and it was confirmed that the color changed as the azide group was substituted.

[Example 2]

Spiropyran containing an alkyne group and a copper catalyst were added to the functional hydrogel containing an azide group prepared in Example 1, and a click reaction was performed to prepare a functional hydrogel to which a fluorescent substance was covalently bonded.

As described in FIG. 4, the difference in fluorescence between the functional hydrogel containing the azide and the functional hydrogel to which the fluorescent material was covalently bonded was observed using a fluorescence laser optical microscope image, and the functional hydrogel to which the fluorescent material was covalently bonded. It was confirmed that the fluorescence of the gel was clearly visible.

In addition, the FT-IR graph shown in FIG. 2 shows the FT-IR of a hydrogel containing a chlorine group, a hydrogel containing an azide group, and a hydrogel containing an azide group after a click reaction. It was confirmed that the azide (N ₃ ) peak at 2,250 cm ^-1 was lowered. Through this, it was confirmed that the azide group had a click reaction.

[Comparative Example 1]

The same procedure was performed except that the oligomer crosslinking agent containing an azide group prepared in Preparation Example 2 was used instead of the glycol-based oligomer crosslinking agent containing a chlorine group in Example 1. When using the above method, the hydrogel was not properly formed. It is believed that this is because the highly reactive azide group bound to the crosslinking agent caused a side reaction when preparing the hydrogel of Comparative Example 1 and hindered the hydrogel production.

As described above, the present invention has been described by specific details and limited embodiments and drawings, but this is only provided to help a more general understanding of the present invention, the present invention is not limited to the above embodiments, and the present invention Those skilled in the art can make various modifications and variations from these descriptions.

Therefore, the spirit of the present invention should not be limited to the described embodiments, and it will be said that not only the claims to be described later, but also all modifications equivalent or equivalent to these claims belong to the scope of the present invention. .

Claims (9)

(a) preparing a mixed solution by adding and mixing a crosslinking agent represented by Formula 1, a hydrophilic polymer monomer, and an ultraviolet crosslinking agent;
(b) preparing a hydrogel by crosslinking the mixed solution; and
(c) subjecting the hydrogel to azide substitution reaction (Azidation) to prepare a functional hydrogel having a structural unit represented by Formula 2 below;
Including,
Formula 1 below is a method for producing a functional hydrogel comprising a weight average molecular weight of 1,000 to 10,000 g / mol.
[Formula 1]

[Formula 2]

(Wherein R ₁ is C ₁ -C ₆ alkylene, x and y independently satisfy 1 to 100, and n satisfies 1 to 1,000.) According to claim 1,
Formula 1 is a method for producing a functional hydrogel, which is prepared by synthesizing the following formula 3 and acryloyl chloride.
[Formula 3]

(Wherein R ₁ is C ₁ -C ₆ alkylene, x and y independently satisfy 1 to 100, and n satisfies 1 to 1,000.) According to claim 1,
Formula 1 is a method for producing a functional hydrogel, which is prepared by polymerization of a glycol-based polyol and epichlorohydrin. According to claim 3,
The glycol-based polyol is a method for producing a functional hydrogel comprising any one or two or more selected from ethylene glycol, propylene glycol, and isopropylene glycol. According to claim 4,
The glycol-based polyol is a method for producing a functional hydrogel comprising a number average molecular weight of 100 to 1,000 g / mol. delete According to claim 1,
The method of producing a functional hydrogel, wherein the crosslinking in step (C) includes an ultraviolet crosslinking method. According to claim 1,
The hydrophilic polymer monomer of step (B) comprises at least one monomer selected from methacrylic acid, acrylic acid and N-isopropylacrylamide, a method for producing a functional hydrogel. A functional hydrogel prepared from any one selected from claims 1 to 5 and 7 to 8.

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