KR102099516B1 - NIR responsive cross-linked micelles, drug delivery carrier targeting cancer cell and method for preparing the same - Google Patents

Abstract

The present invention relates to: a cross-linked micelle having a cancer cell target function in response to near infrared rays; a drug delivery carrier comprising the same; and a method for manufacturing the same. The drug delivery carrier having the cancer cell target function that induces drug release by using near infrared rays can be easily manufactured by a click reaction, and actively releases drugs from the outside by specifically targeting cancer cells, thereby being able to specifically kill cancer cells. Therefore, the drug delivery carrier can be usefully used in the pharmaceutical field.

Description

Cross-linked micelles that have a function of targeting cancer cells that respond to near-infrared rays, a drug delivery system comprising the same, and a method for preparing the same

The present invention relates to a cross-linked micelle having a cancer cell target function in response to near infrared rays, a drug delivery system comprising the same, and a method for manufacturing the same.

Polymer micelles based on core-shell structures in recent years have been extensively studied due to their advantages, hydrophobic drug solubility, long cycle time and specific tissue targeting. The core-shell structure of the amphiphilic block copolymer consists of an insoluble core and a water-soluble corona. Polymeric micelles are typically in the range of tens of nanometers in size and are suitable for enhanced permeability and retention (EPR) effects. The EPR effect is characterized by molecules accumulating in cancer cells larger than normal cells in sizes ranging from 40 to 200 nanometers. In other words, the side with normal cells has a tight boundary with blood vessels, whereas the side with fast-growing cancer cells has a loose boundary with blood vessels, so molecules or particles of this size cannot penetrate the normal cell side, whereas cancer The blood vessel wall on the side of the tissue passes through well and is transferred to and accumulated in cancer cells, which is effectively used for drug delivery to cancer cells.

However, the instability inherent in micelles has been a significant problem prior to many applications. That is, the polymer micelles can escape from each other as a unit at a very diluted concentration during blood circulation. It does not achieve targeted release and can be toxic to normal living tissue. Therefore, improving the stability of polymer micelles is an urgent problem that many studies are currently underway.

Cross-linking of micelles is known as the most excellent method for stabilizing the self-assembly structure. Recent advances in micelle crosslinking technology can enhance the stability of nanocarriers as well as control the drug release of micelles. Crosslinking of the hydrophilic shell affects the stealth of the drug delivery system, affects the mobility of the hydrophilic group, and unnecessary crosslinking between micelles cannot be avoided. On the other hand, core crosslinking of micelles is becoming an increasingly attractive method, which can reverse reaction or decomposition of crosslinking in a redox environment, thereby improving the release of internal active substances. Core crosslinked (crosslinked) micelles can be made by various methods such as UV irradiation of photosensitive block copolymers and metal-ligand coordination bonds. These micelles can be crosslinked according to UV / NIR light irradiation, the presence of a reducing agent or a change in pH to release the drug.

In a typical cancer cell targeted core crosslinking method, first, a hydrophobic drug is physically or chemically mounted on a core portion of a polymer micelle, and a core crosslinking reaction is performed using a crosslinking agent having a specific chemical structure. The crosslinking agent commonly used herein is a crosslinking agent that decomposes under redox reaction or weakly acidic conditions. Therefore, after the drug-mounted cross-linked micelles are maintained in the micelles during the circulation time in the blood, and after reaching and accumulating cancer cell target tissues with the EPR effect, the reduction reaction of glutathione, which is a weak acidity in the cancer cells or a large amount in cancer cells As the crosslinked structure decomposes, the drug inside the core is easily released to kill cancer cells.

However, this method has a drawback that it is impossible to artificially control drug release at a desired time at a desired site because the drug delivery agent releases the drug spontaneously from the internal environment of cancer cells. Therefore, a method of controlling drug release inside the body in a manner harmless to the body from outside the body is preferable.

For this purpose, a method of releasing the drug at a desired site in the body using a near infrared (NIR) ray, which is harmless to the living body, has been proposed to increase the drug delivery effect of the polymer cross-linked micelle, which is a biotherapeutic treatment. It has a high potential in academic applications. Using this method, the drug can be actively released from the drug delivery system. As a drug delivery system in this way, drug-loaded polymers based on diselenide bonds are more effective. Diselenide bonds are relatively stable in vivo because they have reactive oxygen sensitization, but reactive oxygen species (ROS) released by NIR irradiation by indocyanine green (ICG) loaded with drugs ) Has the property of decomposing. ICG is a tricarbocyan dye that can generate singlet oxygen, super oxide anions, hydroxyl radicals, etc. when exposed to near infrared rays. Accordingly, the cross-linked structure having a diselenide bond decomposes to release the drug around the core. Polymer drug binding through diselenide bonding includes drugs as part of polymer crosslinking or polymers with drug attached to micelles. This method can lead to improved drug delivery ability. Therefore, when a crosslinking agent including a diselenide bond is properly utilized in a crosslinking reaction to synthesize a drug and a crosslinked micelle equipped with ICG, an external stimulation-sensitive drug delivery system using near infrared rays can be effectively prepared.

On the other hand, "click chemistry (click chemistry)" reaction can form an intermolecular bond with high yield not only in reaction with a small molecule due to high reactivity, but also in a reaction with a polymer such as an oligomer or a polymer. Currently, click chemistry reactions are applied in various fields, for example, a study of cycloaddition of azide functional groups and alkynes (Rostovtsev, VV, et al., Angew. Chem. Int. Ed. 41: 2596-2599, 2002); Application of click chemistry through organic synthesis (Lee, LV, et al., J. Am. Chem. Soc. 125: 9588-9589, 2003); Easy surface functionalization using click chemistry (Fazio, F, et al., J. Am. Chem. Soc. 124: 14397-14402, 2002); Studies such as the Diels-Alder reaction (Gregoritza, M, et al., Eur. J. Pharm. Biopharm. 97, 438-453, 2015) as a powerful tool for drug delivery systems and biomaterial design are ongoing Is being made.

Republic of Korea Patent Publication No. 2009-0076856 Republic of Korea Patent Publication No. 2011-0102995

An object of the present invention is to provide a crosslinked micelle having a cancer cell target function.

Another object of the present invention is to provide a method for producing a crosslinked micelle having a cancer cell target function.

Another object of the present invention is to provide a drug carrier having a cancer cell target function.

Another object of the present invention is to provide a method for preparing a drug delivery system having a cancer cell target function.

In order to achieve the above object,

The present invention is an amphipathic copolymer comprising a hydrophilic portion and a hydrophobic portion;

A crosslinking agent comprising a diselenide bond; And

NIR dye; includes,

The hydrophilic part of the copolymer is one selected from the group consisting of poly (ethylene oxide), poly (oligo (ethylene glycol) methyl ether methacrylate) and poly (2-dimethylamino) ethyl methacrylate),

The hydrophobic portion of the copolymer is poly (furfuryl methacrylate), poly (styrene- alt -maleic anhydride), poly (α-propagyl carboxylate-ε-caprolactone), poly (α-pro Fargil-ε-caprolactone), poly (azaido ε-caprolactone-co-ε-caprolactone), poly (azaido ε-caprolactone), poly (γ-propagyl-glutamate), poly (5- (4- (prop-2-ain-1-yloxy) benzyl) -1,3-dioxolane-2,4-dione (tyrosine (alkainyl) -OCA), poly (N- (Prop-2-ain-1-yl) acrylamide), poly (N- (3-azidopropyl) acrylamide), poly (1-ethanyl-4-vinylbenzene), poly (γ-azai Do-propyl-L-glutamate) and poly (propargyl acrylate).

Here, the hydrophobic portion of the copolymer is characterized in that the furanyl (furanyl), maleimidyl (maleimidyl), alkynyl (alkynyl), or azido (azido) functional group is additionally introduced,

The crosslinking agent is a compound represented by the following formula (1),

here,

When a furanyl functional group is introduced into the hydrophobic portion of the copolymer, R ¹ and R ² of the crosslinking agent are maleimidyl,

When a maleimidyl functional group is introduced into the hydrophobic portion of the copolymer, R ¹ and R ² of the crosslinking agent are furanyl,

When an alkynyl functional group is introduced into the hydrophobic portion of the copolymer, R ¹ and R ² of the crosslinking agent are azido,

When an azido functional group is introduced into the hydrophobic portion of the copolymer, R ¹ and R ² of the crosslinking agent are alkynyl,

The functional group substituted in the hydrophobic portion of the copolymer and the functional groups substituted in R ¹ and R ² of the crosslinking agent are characterized in that they form a crosslinking by mutually clicking reaction,

The near infrared dyes include indocyanine green (ICG), methylene blue (MB), IRDye 800 CW, Cy5.5, Cy7, Cy7.5, protoporphyrin IX, IR-780, IR-783, IR-808 and MHI-148 Characterized in that at least one selected from the group consisting of,

Characterized in that the desenlenide bond of the crosslinking agent is decomposed in response to near infrared rays,

Provided is a crosslinked micelle with cancer cell target function:

[Formula 1]

(In the formula 1,

a, b, c and d are each an integer of 1-10;

R ¹ and R ² are furanyl, maleimidyl, alkynyl, or azido).

In addition, the present invention is a step of adding and stirring an amphiphilic copolymer comprising a hydrophilic part and a hydrophobic part, a crosslinking agent comprising a diselenide bond, and a near infrared dye (NIR dye) in a solvent; And

Including the step of reacting the mixture prepared in step 1 at 50-80 ℃ for 8-16 hours, inducing cross-linking of micelle cores (step 2);

The hydrophilic part of the copolymer is one selected from the group consisting of poly (ethylene oxide), poly (oligo (ethylene glycol) methyl ether methacrylate) and poly (2-dimethylamino) ethyl methacrylate),

The hydrophobic portion of the copolymer is poly (furfuryl methacrylate), poly (styrene- alt -maleic anhydride), poly (α-propagyl carboxylate-ε-caprolactone), poly (α-pro Fargil-ε-caprolactone), poly (azaido ε-caprolactone-co-ε-caprolactone), poly (azaido ε-caprolactone), poly (γ-propagyl-glutamate), poly (5- (4- (prop-2-ain-1-yloxy) benzyl) -1,3-dioxolane-2,4-dione (tyrosine (alkainyl) -OCA), poly (N- (Prop-2-ain-1-yl) acrylamide), poly (N- (3-azidopropyl) acrylamide), poly (1-ethanyl-4-vinylbenzene), poly (γ-azai Do-propyl-L-glutamate) and poly (propargyl acrylate).

Here, the hydrophobic portion of the copolymer is characterized in that the furanyl (furanyl), maleimidyl (maleimidyl), alkynyl (alkynyl), or azido (azido) functional group is additionally introduced,

The crosslinking agent is a compound represented by the following formula (1),

here,

When a furanyl functional group is introduced into the hydrophobic portion of the copolymer, R ¹ and R ² of the crosslinking agent are maleimidyl,

When a maleimidyl functional group is introduced into the hydrophobic portion of the copolymer, R ¹ and R ² of the crosslinking agent are furanyl,

When an alkynyl functional group is introduced into the hydrophobic portion of the copolymer, R ¹ and R ² of the crosslinking agent are azido,

When an azido functional group is introduced into the hydrophobic portion of the copolymer, R ¹ and R ² of the crosslinking agent are alkynyl,

The functional group substituted in the hydrophobic portion of the copolymer and the functional groups substituted in R ¹ and R ² of the crosslinking agent are characterized in that they form a crosslinking by mutually clicking reaction,

The near infrared dyes include indocyanine green (ICG), methylene blue (MB), IRDye 800 CW, Cy5.5, Cy7, Cy7.5, protoporphyrin IX, IR-780, IR-783, IR-808 and MHI-148 Characterized in that at least one selected from the group consisting of,

Characterized in that the desenlenide bond of the crosslinking agent is decomposed in response to near infrared rays,

Provided is a method for preparing a crosslinked micelle having a cancer cell target function:

[Formula 1]

(In the formula 1,

a, b, c and d are each an integer of 1-10;

R ¹ and R ² are furanyl, maleimidyl, alkynyl, or azido).

Furthermore, the present invention is the cross-linked micelles; And it provides a drug carrier having a cancer cell target function, including a drug introduced into the cross-linked micelles.

Furthermore, the present invention comprises the steps of adding and stirring an amphiphilic copolymer comprising a hydrophilic part and a hydrophobic part, a crosslinking agent comprising a diselenide bond, a NIR dye, and a drug in a solvent (step 1); And

Including the step of reacting the mixture prepared in step 1 at 50-80 ℃ for 8-16 hours, inducing cross-linking of micelle cores (step 2);

The hydrophilic part of the copolymer is one selected from the group consisting of poly (ethylene oxide), poly (oligo (ethylene glycol) methyl ether methacrylate) and poly (2-dimethylamino) ethyl methacrylate),

The hydrophobic portion of the copolymer is poly (furfuryl methacrylate), poly (styrene- alt -maleic anhydride), poly (α-propagyl carboxylate-ε-caprolactone), poly (α-pro Fargil-ε-caprolactone), poly (azaido ε-caprolactone-co-ε-caprolactone), poly (azaido ε-caprolactone), poly (γ-propagyl-glutamate), poly (5- (4- (prop-2-ain-1-yloxy) benzyl) -1,3-dioxolane-2,4-dione (tyrosine (alkainyl) -OCA), poly (N- (Prop-2-ain-1-yl) acrylamide), poly (N- (3-azidopropyl) acrylamide), poly (1-ethanyl-4-vinylbenzene), poly (γ-azai Do-propyl-L-glutamate) and poly (propargyl acrylate).

Here, the hydrophobic portion of the copolymer is characterized in that the furanyl (furanyl), maleimidyl (maleimidyl), alkynyl (alkynyl), or azido (azido) functional group is additionally introduced,

The crosslinking agent is a compound represented by the following formula (1),

here,

When a furanyl functional group is introduced into the hydrophobic portion of the copolymer, R ¹ and R ² of the crosslinking agent are maleimidyl,

When a maleimidyl functional group is introduced into the hydrophobic portion of the copolymer, R ¹ and R ² of the crosslinking agent are furanyl,

When an alkynyl functional group is introduced into the hydrophobic portion of the copolymer, R ¹ and R ² of the crosslinking agent are azido,

When an azido functional group is introduced into the hydrophobic portion of the copolymer, R ¹ and R ² of the crosslinking agent are alkynyl,

The functional group substituted in the hydrophobic portion of the copolymer and the functional groups substituted in R ¹ and R ² of the crosslinking agent are characterized in that they form a crosslinking by mutually clicking reaction,

The drug is prednisolone 21-acetate, paclitaxel, doxorubicin, retinoic acid, cisplatin, camptothecin, Fluorouracil (5- FU), Docetaxel, Tamoxifen, Anasterozole, Carboplatin, Topotecan, Berotecan, Irinotecan, Gleevec, Vincristine, aspirin, salicylates, ibuprofen, naproxen, fenofopen, fenoprofen, indomethacin, phenyltazone ), Mesotrexate, cyclophosphamide, mechlorethamine, dexamethasone, prednisolone, celecoxib, valdecoxib, nidecyl Nimesulide, cortisone and nose Characterized in that at least one selected from the group consisting of a corticosteroid (corticosteroid), and

The near infrared dyes include indocyanine green (ICG), methylene blue (MB), IRDye 800 CW, Cy5.5, Cy7, Cy7.5, protoporphyrin IX, IR-780, IR-783, IR-808 and MHI-148 Characterized in that at least one selected from the group consisting of,

Characterized in that the desenlenide bond of the crosslinking agent is decomposed in response to near infrared rays,

Provided is a method for preparing a drug delivery system having a cancer cell target function:

[Formula 1]

(In the formula 1,

a, b, c and d are each an integer of 1-10;

R ¹ and R ² are furanyl, maleimidyl, alkynyl, or azido).

A drug delivery system having a cancer cell target function that induces drug release using near infrared rays from the outside of the present invention can be easily produced by a click reaction, specifically targets cancer cells, and actively releases drugs from the outside, thereby causing cancer cells Can be killed specifically, it can be useful in the pharmaceutical field.

1 is a diagram showing the synthesis of a diselenide crosslinking agent, PEO-b-PFMA (poly (ethylene oxide) -b-poly (furfuryl methacrylate)) and crosslinked micelles.
FIG. 2 is a diagram showing the synthesis of a diselenide crosslinking agent, PEO-b-PGMA-N ₃ (poly (ethylene oxide) -b-poly (glycidyl methacrylate) -N ₃ ) and crosslinked micelles.
3 is a diagram showing the GPC analysis of PEO-Br macroinitiator and PEO-b-PFMA.
4 is a diagram showing the GPC analysis of PEO-Br macroinitiator, PEO-b-PGMA, PEO-b-PGMA-N ₃ .
5 is a diagram showing a ¹ H NMR and ¹³ C NMR spectrum of a diselenide derivative (crosslinking agent) in which maleimide functional groups are bonded at both ends.
6 is a diagram showing a ¹ H NMR and ¹³ C NMR spectrum of a diselenide derivative (crosslinking agent) having propargyl functional groups bound at both ends.
7 is a view showing a ¹ H NMR spectrum of micelles (Production Example 5) in which the cores of (a) PEO-b-PFMA (Production Example 1) and (b) PEO-b-PFMA are crosslinked.
Figure 8 (a) PEO-b-PFMA core cross-linked micelles (non-CCL) and core cross-linked micelles (CCL) measured through the DLS changes in the mechanical diameter and (b) the core This is a diagram showing the results of confirming the size and shape of cross-linked micelles (CCL) through TEM.
9 is a diagram showing the ¹ H NMR spectrum of CCL micelles (Preparation Example 6) of (a) PEO-b-PGMA-N ₃ (Preparation Example 3) and (b) PEO-b-PGMA-N ₃ .
10 is a diagram showing the UV-Vis absorption spectrum of micelles (Example 1-1) in which the cores on which DOX and ICG were supported were crosslinked.
11 is a view showing the results of measuring the change in the dynamic diameter of micelles (Example 1-1) crosslinked with DOX and ICG-supported cores before and after irradiation with near infrared rays through DLS.
12 is (a) DOX and ICG-supported core cross-linked micelles (Example 1-1) and (b) 3 minutes after irradiation of the micelles with near-infrared rays for 5 minutes, and (c) 5 minutes of irradiation with the micelles by near-infrared rays. It is a diagram showing the result of confirming the size and shape of the state after 30 hours through TEM.
FIG. 13 is a graph showing the in vitro drug release graph from DOX / ICG / CCL micelles (Example 1-1) after irradiation with no near infrared rays or irradiation for 5 minutes.
14 is a view showing the results of measuring the change in the dynamic diameter of micelles (Example 1-2) in which DOX and ICG-supported cores were crosslinked before and after irradiation with near infrared rays through DLS.
FIG. 15 shows (a) PEO-b-PGMA-N ₃ micelles whose core is not crosslinked (b) micelles crosslinked with DOX and ICG (Example 1-2) and (c) the micelles in near infrared 5 It is a diagram showing the result of confirming the size and shape of the state elapsed 24 hours after the minute irradiation through TEM.
FIG. 16 is a graph showing the in vitro drug release from DOX / ICG / CCL micelles (Example 1-2) after irradiating for 5 minutes without irradiating near infrared rays.
FIG. 17 shows (a) HEK293 cell viability for 24 hours and (b) different concentrations of PEO-b-PFMA at different concentrations to evaluate cytotoxicity for cross-linked micelles (Example 1-1). Dop or 200 mW cm ^-2 , HepG2 for 24 hours to evaluate the cytotoxicity of micelles cross-linked with DOX (Example 1-1) with and without irradiation for 5 minutes with or without 808 nm near-infrared laser light It is a diagram showing the results of cell viability analysis.
Figure 18 is a DOX or 200 mW cm ^-2 , 808 nm NIR laser beam irradiated for 5 minutes DOX and ISG-supported PEO-b-PFMA core cross-linked micelles (Example 1-1) in the presence of HepG2 cells This is a diagram showing cell death with fluorescence photographs taken at different incubation times.

Hereinafter, the present invention will be described in detail.

Cross-linked micelle with cancer cell target function

The present invention is an amphipathic copolymer comprising a hydrophilic portion and a hydrophobic portion;

A crosslinking agent comprising a diselenide bond; And

NIR dye; includes,

The hydrophilic part of the copolymer is one selected from the group consisting of poly (ethylene oxide), poly (oligo (ethylene glycol) methyl ether methacrylate) and poly (2-dimethylamino) ethyl methacrylate),

The hydrophobic portion of the copolymer is poly (furfuryl methacrylate), poly (styrene- alt -maleic anhydride), poly (α-propagyl carboxylate-ε-caprolactone), poly (α-pro Fargil-ε-caprolactone), poly (azaido ε-caprolactone-co-ε-caprolactone), poly (azaido ε-caprolactone), poly (γ-propagyl-glutamate), poly (5- (4- (prop-2-ain-1-yloxy) benzyl) -1,3-dioxolane-2,4-dione (tyrosine (alkainyl) -OCA), poly (N- (Prop-2-ain-1-yl) acrylamide), poly (N- (3-azidopropyl) acrylamide), poly (1-ethanyl-4-vinylbenzene), poly (γ-azai Do-propyl-L-glutamate) and poly (propargyl acrylate).

Here, the hydrophobic portion of the copolymer is characterized in that the furanyl (furanyl), maleimidyl (maleimidyl), alkynyl (alkynyl), or azido (azido) functional group is additionally introduced,

The crosslinking agent is a compound represented by the following formula (1),

Here, when a furanyl functional group is introduced into the hydrophobic portion of the copolymer, R ¹ and R ² of the crosslinking agent are maleimidyl,

When a maleimidyl functional group is introduced into the hydrophobic portion of the copolymer, R ¹ and R ² of the crosslinking agent are furanyl,

When an alkynyl functional group is introduced into the hydrophobic portion of the copolymer, R ¹ and R ² of the crosslinking agent are azido,

When an azido functional group is introduced into the hydrophobic portion of the copolymer, R ¹ and R ² of the crosslinking agent are alkynyl,

The functional group substituted in the hydrophobic portion of the copolymer and the functional groups substituted in R ¹ and R ² of the crosslinking agent are characterized in that they form a crosslinking by mutually clicking reaction,

The near infrared dyes include indocyanine green (ICG), methylene blue (MB), IRDye 800 CW, Cy5.5, Cy7, Cy7.5, protoporphyrin IX, IR-780, IR-783, IR-808 and MHI-148 Characterized in that at least one selected from the group consisting of,

Characterized in that the desenlenide bond of the crosslinking agent is decomposed in response to near infrared rays,

Provided is a crosslinked micelle with cancer cell target function:

[Formula 1]

(In the formula 1,

a, b, c and d are each an integer of 1-10;

R ¹ and R ² are furanyl, maleimidyl, alkynyl, or azido).

In one embodiment of the present invention, the micelle is a hydrophilic shell; And a hydrophobic core.

In one embodiment of the present invention, the cross-linked micelles may introduce drugs into the cross-linked micelles,

The drug is prednisolone 21-acetate, paclitaxel, doxorubicin, retinoic acid, cisplatin, camptothecin, Fluorouracil (5- FU), Docetaxel, Tamoxifen, Anasterozole, Carboplatin, Topotecan, Berotecan, Irinotecan, Gleevec, Vincristine, aspirin, salicylates, ibuprofen, naproxen, fenofopen, fenoprofen, indomethacin, phenyltazone ), Mesotrexate, cyclophosphamide, mechlorethamine, dexamethasone, prednisolone, celecoxib, valdecoxib, nidecyl Nimesulide, cortisone and nose It may be at least one selected from the group consisting of a corticosteroid (corticosteroid), and may preferably be from doxorubicin (doxorubicin).

In one embodiment of the present invention, the crosslinking agent may be a compound represented by Formula 2 below:

[Formula 2]

.

.

In one embodiment of the present invention, the crosslinking agent may be a compound represented by Formula 3 below:

[Formula 3]

.

.

In one embodiment of the present invention, the copolymer comprises: poly (ethylene oxide) -b-poly (furfuryl methacrylate); Or poly (ethylene oxide) -b-poly (furfuryl methacrylate) in which azido functional group is introduced; poly (ethylene oxide) -b-poly (in which furanyl functional group is introduced) Glycidyl methacrylate); Or poly (ethylene oxide) -b-poly (glycidyl methacrylate) into which an azido functional group has been introduced.

In one embodiment of the present invention, the near-infrared dye, indocyanine green (ICG), methylene blue (MB), IRDye 800 CW, Cy5.5, Cy7, Cy7.5, protoporphyrin IX, IR-780, IR It may be one selected from the group consisting of -783, IR-808 and MHI-148, preferably indocyanine green.

Manufacturing method of crosslinked micelle

The present invention comprises the steps of adding and stirring an amphiphilic copolymer comprising a hydrophilic part and a hydrophobic part, a crosslinking agent comprising a diselenide bond, and a NIR dye in a solvent (step 1); And

Including the step of reacting the mixture prepared in step 1 at 50-80 ℃ for 8-16 hours, inducing cross-linking of micelle cores (step 2);

The hydrophilic part of the copolymer is one selected from the group consisting of poly (ethylene oxide), poly (oligo (ethylene glycol) methyl ether methacrylate) and poly (2-dimethylamino) ethyl methacrylate),

The hydrophobic portion of the copolymer is poly (furfuryl methacrylate), poly (styrene- alt -maleic anhydride), poly (α-propagyl carboxylate-ε-caprolactone), poly (α-pro Fargil-ε-caprolactone), poly (azaido ε-caprolactone-co-ε-caprolactone), poly (azaido ε-caprolactone), poly (γ-propagyl-glutamate), poly (5- (4- (prop-2-ain-1-yloxy) benzyl) -1,3-dioxolane-2,4-dione (tyrosine (alkainyl) -OCA), poly (N- (Prop-2-ain-1-yl) acrylamide), poly (N- (3-azidopropyl) acrylamide), poly (1-ethanyl-4-vinylbenzene), poly (γ-azai Do-propyl-L-glutamate) and poly (propargyl acrylate).

Here, the hydrophobic portion of the copolymer is characterized in that the furanyl (furanyl), maleimidyl (maleimidyl), alkynyl (alkynyl), or azido (azido) functional group is additionally introduced,

The crosslinking agent is a compound represented by the following formula (1),

here,

When a furanyl functional group is introduced into the hydrophobic portion of the copolymer, R ¹ and R ² of the crosslinking agent are maleimidyl,

When a maleimidyl functional group is introduced into the hydrophobic portion of the copolymer, R ¹ and R ² of the crosslinking agent are furanyl,

When an alkynyl functional group is introduced into the hydrophobic portion of the copolymer, R ¹ and R ² of the crosslinking agent are azido,

When an azido functional group is introduced into the hydrophobic portion of the copolymer, R ¹ and R ² of the crosslinking agent are alkynyl,

The functional group substituted in the hydrophobic portion of the copolymer and the functional groups substituted in R ¹ and R ² of the crosslinking agent are characterized in that they form a crosslinking by mutually clicking reaction,

The near infrared dyes include indocyanine green (ICG), methylene blue (MB), IRDye 800 CW, Cy5.5, Cy7, Cy7.5, protoporphyrin IX, IR-780, IR-783, IR-808 and MHI-148 Characterized in that at least one selected from the group consisting of,

Characterized in that the desenlenide bond of the crosslinking agent is decomposed in response to near infrared rays,

Provided is a method for preparing a crosslinked micelle having a cancer cell target function:

[Formula 1]

(In the formula 1,

a, b, c and d are each an integer of 1-10;

R ¹ and R ² are furanyl, maleimidyl, alkynyl, or azido).

Drug carrier with cancer cell target function

The present invention is the cross-linked micelles; And a drug introduced into the crosslinked micelle,

The drug is prednisolone 21-acetate, paclitaxel, doxorubicin, retinoic acid, cisplatin, camptothecin, Fluorouracil (5- FU), Docetaxel, Tamoxifen, Anasterozole, Carboplatin, Topotecan, Berotecan, Irinotecan, Gleevec, Vincristine, aspirin, salicylates, ibuprofen, naproxen, fenofopen, fenoprofen, indomethacin, phenyltazone ), Mesotrexate, cyclophosphamide, mechlorethamine, dexamethasone, prednisolone, celecoxib, valdecoxib, nidecyl Nimesulide, cortisone and nose It provides a drug delivery system with a target cancer cell function, characterized in that at least one selected from the group consisting of a corticosteroid (corticosteroid).

Method for manufacturing drug delivery system having cancer cell target function

The present invention comprises the steps of adding and stirring an amphiphilic copolymer comprising a hydrophilic part and a hydrophobic part, a crosslinking agent containing a diselenide bond, a NIR dye, and a drug in a solvent (step 1); And

Including the step of reacting the mixture prepared in step 1 at 50-80 ℃ for 8-16 hours, inducing cross-linking of micelle cores (step 2);

The hydrophilic part of the copolymer is one selected from the group consisting of poly (ethylene oxide), poly (oligo (ethylene glycol) methyl ether methacrylate) and poly (2-dimethylamino) ethyl methacrylate),

The hydrophobic portion of the copolymer is poly (furfuryl methacrylate), poly (styrene- alt -maleic anhydride), poly (α-propagyl carboxylate-ε-caprolactone), poly (α-pro Fargil-ε-caprolactone), poly (azaido ε-caprolactone-co-ε-caprolactone), poly (azaido ε-caprolactone), poly (γ-propagyl-glutamate), poly (5- (4- (prop-2-ain-1-yloxy) benzyl) -1,3-dioxolane-2,4-dione (tyrosine (alkainyl) -OCA), poly (N- (Prop-2-ain-1-yl) acrylamide), poly (N- (3-azidopropyl) acrylamide), poly (1-ethanyl-4-vinylbenzene), poly (γ-azai Do-propyl-L-glutamate) and poly (propargyl acrylate).

Here, the hydrophobic portion of the copolymer is characterized in that the furanyl (furanyl), maleimidyl (maleimidyl), alkynyl (alkynyl), or azido (azido) functional group is additionally introduced,

The crosslinking agent is a compound represented by the following formula (1),

here,

When a furanyl functional group is introduced into the hydrophobic portion of the copolymer, R ¹ and R ² of the crosslinking agent are maleimidyl,

When a maleimidyl functional group is introduced into the hydrophobic portion of the copolymer, R ¹ and R ² of the crosslinking agent are furanyl,

When an alkynyl functional group is introduced into the hydrophobic portion of the copolymer, R ¹ and R ² of the crosslinking agent are azido,

When an azido functional group is introduced into the hydrophobic portion of the copolymer, R ¹ and R ² of the crosslinking agent are alkynyl,

The functional group substituted in the hydrophobic portion of the copolymer and the functional groups substituted in R ¹ and R ² of the crosslinking agent are characterized in that they form a crosslinking by mutually clicking reaction,

The drug is prednisolone 21-acetate, paclitaxel, doxorubicin, retinoic acid, cisplatin, camptothecin, Fluorouracil (5- FU), Docetaxel, Tamoxifen, Anasterozole, Carboplatin, Topotecan, Berotecan, Irinotecan, Gleevec, Vincristine, aspirin, salicylates, ibuprofen, naproxen, fenofopen, fenoprofen, indomethacin, phenyltazone ), Mesotrexate, cyclophosphamide, mechlorethamine, dexamethasone, prednisolone, celecoxib, valdecoxib, nidecyl Nimesulide, cortisone and nose Characterized in that at least one selected from the group consisting of a corticosteroid (corticosteroid), and

The near infrared dyes include indocyanine green (ICG), methylene blue (MB), IRDye 800 CW, Cy5.5, Cy7, Cy7.5, protoporphyrin IX, IR-780, IR-783, IR-808 and MHI-148 Characterized in that at least one selected from the group consisting of,

Characterized in that the desenlenide bond of the crosslinking agent is decomposed in response to near infrared rays,

Provided is a method for preparing a drug delivery system having a cancer cell target function:

[Formula 1]

(In the formula 1,

a, b, c and d are each an integer of 1-10;

R ¹ and R ² are furanyl, maleimidyl, alkynyl, or azido).

In the manufacturing method of the present invention, the amphipathic copolymer may be prepared through a Reversable Addition Fragmentation Chain Trasfer (RAFT) polymerization reaction or an atomic transfer radical polymerization (ATRP) molecular controlled polymerization reaction.

In the preparation method of the present invention, the crosslinking agent may be a compound represented by the following Chemical Formula 2:

[Formula 2]

.

.

In the preparation method of the present invention, the crosslinking agent may be a compound represented by the following Chemical Formula 3:

[Formula 3]

.

.

Hereinafter, the present invention will be described in more detail by the following examples. However, the following examples are only illustrative of the present invention, and the contents of the present invention are not limited by the following examples.

Manufacturing example  One. PEO -b- PFMA  Synthesis of block copolymer

PEO-b-PFMA (poly (ethylene oxide) -b-poly (furfuryl methacrylate)) is prepared by SET-LRP of FMA (furfuryl methacrylate) using a PEO-Br macro initiator. It was prepared (top right in Figure 1). In a typical procedure for PEO-b-PFMA synthesis, the ratio of reactants to [monomer]: [macro initiator]: [copper wire]: [Me ₆ TREN] is 30: 1: 8: 0.36 (molar equivalents). Round bottom flask with FMA (1.881g, 11.320mmol), PEO-Br (2g, 0.377mmol), copper wire (12.5cm, d = 0.5mm, 0.205g), Me ₆ TREN (0.0312g, 0.1258mmol) and DMF Put in The solution was purged with N ₂ and the reaction was allowed to proceed at room temperature for 18 hours. The resulting mixture was diluted in dichloromethane and passed through a neutral alumina column to remove copper from the solution. The product was concentrated on a rotary evaporator and precipitated with cold diethyl ether. The final product was collected and dried under vacuum at room temperature (2.91 g, yield 75%). The results of GPC analysis of the PEO-b-PFMA block copolymer are shown in FIG. 3.

Preparation Example 2 Synthesis of PEO-b-PGMA block copolymer

Block copolymers of PEO-b-PGMA (poly (ethylene oxide) -b-poly (glycidyl methacrylate)) were synthesized via ATRP using PEO-Br macro initiator (Figure 2 top right). ). In general, PEO-Br (0.25 g, 0.11 mmol), GMA (0.711 g, 5 mmol), AIBN (0.008 g, 0.05 mmol) and DMF (3 mL) were placed in a round flask, followed by nitrogen replacement for 30 minutes. The mixture was stirred for 7 hours in an oil bath at 75 ° C. Thereafter, the product was dissolved in THF and purified by precipitation in excess ethyl ether. The polymer was dried in a vacuum oven at 40 ° C. for 12 hours (yield: 90%).

Manufacturing example  3. PEO -b- PGMA -N ₃  Synthesis of block copolymer

PEO-b-PGMA-N ₃ (poly (ethylene oxide) -b-poly (glycidyl methacrylate) via epoxide ring-opening reaction of PGMA segments of PEO-b-PGMA ) -N ₃ ) was prepared (Fig. 2 top right). Rounded mixture of PEO-b-PGMA (0.40 g, 1.4 mmol of epoxide moieties), NaN ₃ (0.406 g, 6.3 mmol), NH ₄ Cl (0.334 g, 6.3 mmol) and DMF (5 mL) The flask was stirred at 50 ° C for 24 hours. After removing the insoluble salt through the filter, the filtrate was dialyzed in distilled water for 24 hours (MW cutoff, 1 kDa). Distilled water was changed about every 4 hours. The final product was obtained by freeze-drying (0.39 g, 97%). The results of GPC analysis of the PEO-b-PGMA-N ₃ block copolymer are shown in FIG. 4.

Preparation Example 4 Synthesis of crosslinking agent containing diselenide functional group

<4-1> 3,3'-D Selan Diyl  Dipropionic acid ( DSeDPA Synthesis of)

In a flask under a nitrogen atmosphere, 20 mL of deionized water and selenium powder (4.73 g, 60 mmol) were added. And NaBH ₄ (4.54 g, 120 mmol) was dissolved in 50 mL of deionized water and then added dropwise to the selenium suspension. Stir at 0 ° C. until the mixture is a colorless solution showing complete dissolution. After 30 minutes another amount of selenium (4.73 g, 60 mmol) was added to the solution. The mixture was heated to 105 ° C. for 30 minutes until a brown solution formed. 3-Bromo propionic acid (18.36 g, 120 mmol) was dissolved in 30 mL of deionized water, the pH was adjusted to 8 with sodium carbonate, then added to the brown solution, and the mixture was stirred at room temperature for 12 hours under a nitrogen atmosphere. Then the mixture was filtered and the resulting yellow filtrate was acidified with 1M HCl solution and extracted twice with ethyl acetate. The extract was dried by recrystallization twice in ethyl acetate, and dried to obtain 11.3 g of DSeDPA (62% yield).

<4-2> Synthesis of diselenide-containing dimaleimide crosslinking agent

To synthesize a dimaleimide crosslinking agent containing a diselenide bond, first 10 mL of hydroxy ethyl maleimide (HEMI, 5 g, 0.035 mol), DCC (8.04 g, 0.039 mol) and DMAP (47.61 mg, 0.0039 mol) It was dissolved in anhydrous dichloromethane and cooled to 0 ° C for 30 minutes. Then, DSeDPA (5.17 g, 0.017 mol) of Preparation Example 2 dissolved in 5 mL of dichloromethane was added dropwise to the solution, and the mixture was further stirred at 0 ° C. for 30 minutes (upper left in FIG. 1). Subsequently, the reaction was left at room temperature and further stirred for 24 hours. The by-product was filtered off and the product solution was concentrated. The product was finally separated by silica flash column chromatography to obtain 2.52 g (75% yield) of a diselenide-containing dimaleimide crosslinker. The ¹ H-NMR and ¹³ C-NMR spectra of the dimaleimide crosslinking agent containing the synthesized diselenide bond are shown in FIG. 5.

<4-3> Dipropargill  3,3- Diselenium dipropionate ( Contains selenide Dipropargill  Synthesis of crosslinking agent)

Propargyl alcohol (1.11g, 20mmol), DCC (4.13g, 20mol) and DMAP (0.24) to prepare dipropagyl 3,3-diselenium dipropionate, a crosslinking agent containing a diselenide bond mg, 2 mmol) was dissolved in 10 mL of THF and cooled in an icebath for 30 minutes. DSeDPA (3.05 g, 10 mmol) of Example 1-2 was dissolved in 5 mL THF and added dropwise to the mixture, followed by stirring in an icebath for 30 minutes (Fig. 2, top left). Subsequently, the reaction was left at room temperature and further stirred for 24 hours. The precipitated white by-product was filtered and the product was concentrated to dissolve in dichloromethane. The product solution was washed with water, dried over MgSO ₄ and concentrated on a rotary evaporator. The product was separated by silica flash column chromatography (ethyl acetate: hexane = 1: 1) to obtain 2.60 g (68.5% yield) of dipropagyl 3,3-diselenium dipropionate. The ¹ H-NMR and ¹³ C-NMR spectra of the synthesized dipropargyl 3,3-diselenium dipropionate are shown in FIG. 6.

Manufacturing example  5. Diels Alder  By click response PEO -b- PFMA  CCL Mycell  Produce

Figure 1 shows the synthetic route used for the preparation of CCL micelles (CCL PEO-b-PFMA) of diselenide-containing crosslinker, PEO-b-PFMA and PEO-b-PFMA. Did.

The cross-linking reaction of PEO-b-PFMA by the Diels-Alder reaction is a PEO-b-PFMA block copolymer (50 mg, furfuryl residue 0.013 mmol) and acelenide in 1 mL of acetonitrile. After dissolving the maleimide crosslinking agent (38.5 mg, 0.07 mmol), excess water was added dropwise to the solution while stirring vigorously for 1 hour. Subsequently, the solution was heated to 60 ° C. for 6 hours to induce cross-linking of the micelle core (bottom in FIG. 1).

Manufacturing example  6. Alkanes - Azide  By click response PEO -b- PGMA -N ₃ CCL Mycell  Produce

Figure 2 shows the synthetic route used for the preparation of CCL micelles of dipropagyl 3,3-diselenium dipropionate crosslinker, PEO-b-PGMA-N ₃ and PEO-b-PGMA-N ₃ .

The crosslinking reaction of PEO-b-PGMA-N ₃ by Alkyne-Azide Click reaction is a copolymer (50mg, azide residue 0.013mmol) and dipropargyl 3 in 5mL of acetonitrile, After dissolving the 3-diselenium dipropionate crosslinking agent (26.6 mg, 0.07 mmol), 50 mL of water was added dropwise to the solution for 24 hours with vigorous stirring. Then, CuSO ₄ · 5H ₂ O (2.5mg, 10μmol) and sodium ascorbate (Sodium ascorbate, 2.0mg, 10μmol) aqueous solution was added and stirred at room temperature for 24 hours to induce crosslinking of the micelle core (FIG. 2) lower).

Example  1. Copolymer, Crosslinker Indocyanine green and Doxorubicin (drug)  Preparation of crosslinked micelles containing

<1-1> PEO -b- PFMA  CCL Mycell  Indo cyanine green ( indocyanine  green, ICG) Doxorubicin (DOX) Loading

For de-neutralization of amino groups in DOX.HCl, DOX.HCl and triethylamine (TEA) having a 1: 3 molar ratio were dissolved in DMF, and then the DOX solution was stirred overnight at room temperature. ICG was dissolved in phosphate buffer (pH 7.4) at a concentration of 1 mg / mL. To encapsulate the DOX and ICG in CCL micelles of PEO-b-PFMA PEO-b -PFMA (20mg), di-selenide crosslinking agent ^{(2mg, 3.8 × 10 -3 mmol} ), DOX (350μL solution, 3.5mg) and ICG (100 μL solution, 0.18 mg) was uniformly dissolved in 2 mL of acetonitrile. PBS (pH 7.4, 5.0 mL, 10 mM) was added dropwise to the solution with vigorous stirring for 1 hour. Subsequently, the solution was heated at 60 ° C. for 12 hours to induce crosslinking of the micelle core. The CCL solution was transferred to a dialysis membrane (MW cutoff, 3 kDa) and dialyzed against 1 L of distilled water. Distilled water was replaced every 24 hours for 24 hours. Then, the CCL nanoparticles were centrifuged at 2500 rpm for 20 minutes to remove unsupported DOX and ICG to cross-link micelles with cores carrying DOX and ICG inside the CCL micelles of PEO-b-PFMA (Example 1- 1, CCL / ICG / DOX).

<1-2> PEO -b- PGMA -N ₃ CCL Mycell  Inside ICG and DOX Loading

For de-neutralization of amino groups in DOX.HCl, DOX.HCl and triethylamine (TEA) having a 1: 3 molar ratio were dissolved in DMF, and then the DOX solution was stirred overnight at room temperature. ICG was dissolved in phosphate buffer (pH 7.4) at a concentration of 1 mg / mL. PEO-b-PGMA-N ₃ in order to encapsulate the DOX and ICG in CCL micelles of _{PEO-b-PGMA-N 3} (20mg), di-selenide crosslinking agent _{(5mg), CuSO 4 · 5H} 2 O (1mg) and sodium Ascorbate (0.8 mg), DOX (350 μL solution, 3.5 mg) and ICG (100 μL solution, 0.18 mg) were uniformly dissolved in 2 mL of acetonitrile. PBS (pH 7.4, 5.0 mL, 10 mM) was added dropwise to the solution with vigorous stirring for 1 hour. Subsequently, the solution was heated at 60 ° C. for 12 hours to induce crosslinking of the micelle core. The CCL solution was transferred to a dialysis membrane (MW cutoff, 3 kDa) and dialyzed against 1 L of distilled water. Distilled water was replaced every 24 hours for 24 hours. Then, the CCL nanoparticles were centrifuged at 2500 rpm for 20 minutes to remove unsupported DOX and ICG to cross-link micelles with cores carrying DOX and ICG inside the CCL micelles of PEO-b-PGMA-N ₃ (Example 1-2) was prepared.

Example  2. Copolymer, Crosslinker  And indocyanine green Micelle  Produce

<2-1> PEO -b- PFMA  CCL Mycell  Indo cyanine green ( indocyanine  green, ICG) supported

ICG was dissolved in phosphate buffer (pH 7.4) at a concentration of 1 mg / mL. To encapsulate ICG into CCL micelles of PEO-b-PFMA, PEO-b-PFMA (20 mg), diselenide crosslinking agent (2 mg, 3.8 x 10 ^-3 mmol), and ICG (100 μL solution, 0.18 mg) in 2 mL Dissolved uniformly in acetonitrile. PBS (pH 7.4, 5.0 mL, 10 mM) was added dropwise to the solution with vigorous stirring for 1 hour. Subsequently, the solution was heated at 60 ° C. for 12 hours to induce crosslinking of the micelle core. The CCL solution was transferred to a dialysis membrane (MW cutoff, 3 kDa) and dialyzed against 1 L of distilled water. Distilled water was replaced every 24 hours for 24 hours. Then, CCL nanoparticles were centrifuged at 2500 rpm for 20 minutes to remove unsupported ICG to prepare micelles crosslinked with ICG-supported cores in CCL micelles of PEO-b-PFMA (Example 2-1). Did.

<2-2> PEO-b-PGMA-N ₃ ICG loaded inside CCL micelle

ICG was dissolved in phosphate buffer (pH 7.4) at a concentration of 1 mg / mL. _{PEO-b-PGMA-N 3} (20mg), di-selenide crosslinking agent to encapsulate the ICG CCL micelles of _{PEO-b-PGMA-N 3} (2mg, 3.8 × 10 -3 mmol), Cu, and ICG (100μL Solution, 0.18 mg) was uniformly dissolved in 2 mL of acetonitrile. PBS (pH 7.4, 5.0 mL, 10 mM) was added dropwise to the solution with vigorous stirring for 1 hour. Subsequently, the solution was heated at 60 ° C. for 12 hours to induce crosslinking of the micelle core. The CCL solution was transferred to a dialysis membrane (MW cutoff, 3 kDa) and dialyzed against 1 L of distilled water. Distilled water was replaced every 24 hours for 24 hours. Then, the CCL nanoparticles were centrifuged at 2500 rpm for 20 minutes to remove unsupported ICG to cross-link micelles with an ICG-supported core inside the CCL micelle of PEO-b-PGMA-N ₃ (Example 2-2). ).

Example  3. Copolymer, Crosslinker  And Doxorubicin (drug)  Containing crosslink Micelle  Produce

<3-1> PEO -b- PFMA  CCL Mycell  Inside Doxorubicin (DOX) Loading

For de-neutralization of amino groups in DOX.HCl, DOX.HCl and triethylamine (TEA) having a 1: 3 molar ratio were dissolved in DMF, and then the DOX solution was stirred overnight at room temperature. To encapsulate DOX into CCL micelles of PEO-b-PFMA, PEO-b-PFMA (20 mg), diselenide crosslinker (2 mg, 3.8 × 10 ^-3 mmol) and DOX (350 μL solution, 3.5 mg) in 2 mL of aceto Dissolve uniformly in nitrile. PBS (pH 7.4, 5.0 mL, 10 mM) was added dropwise to the solution with vigorous stirring for 1 hour. Subsequently, the solution was heated at 60 ° C. for 12 hours to induce crosslinking of the micelle core. The CCL solution was transferred to a dialysis membrane (MW cutoff, 3 kDa) and dialyzed against 1 L of distilled water. Distilled water was replaced every 24 hours for 24 hours. Then, the CCL nanoparticles were centrifuged at 2500 rpm for 20 minutes to remove unsupported DOX, and the micelles crosslinked with DOX cores in the CCL micelles of PEO-b-PFMA (Example 3-1, CCL / DOX).

<3-2> PEO -b- PGMA -N ₃ CCL Mycell  Inside DOX Loading

For de-neutralization of amino groups in DOX.HCl, DOX.HCl and triethylamine (TEA) having a 1: 3 molar ratio were dissolved in DMF, and then the DOX solution was stirred overnight at room temperature. PEO-b-PGMA-N ₃ in order to encapsulate DOX in CCL micelles of _{PEO-b-PGMA-N 3} (20mg), di-selenide crosslinking agent _{(5mg), CuSO 4 · 5H} 2 O (1mg) and sodium ascorbyl bait (0.8 mg) and DOX (350 μL solution, 3.5 mg) were uniformly dissolved in 2 mL of acetonitrile. PBS (pH 7.4, 5.0 mL, 10 mM) was added dropwise to the solution with vigorous stirring for 1 hour. Subsequently, the solution was heated at 60 ° C. for 12 hours to induce crosslinking of the micelle core. The CCL solution was transferred to a dialysis membrane (MW cutoff, 3 kDa) and dialyzed against 1 L of distilled water. Distilled water was replaced every 24 hours for 24 hours. Then, CCL nanoparticles were centrifuged at 2500 rpm for 20 minutes to remove unsupported DOX, and micelles cross-linked with DOX cores in CCL micelles of PEO-b-PGMA-N ₃ (Example 3-2) Was prepared.

Experimental example  One. Diels Alder  By click response PEO -b- PFMA  CCL Of micelle (Production Example 5)  Analysis and characteristics

7 shows ¹ H-NMR of PEO-b-PFMA (Preparation Example 1) and CCL micelles of PEO-b-PFMA (Preparation Example 5). In the spectrum of PEO-b-PFMA copolymer (Preparation Example 1) (Fig. 7 (a)), the proton of the methylene group of PEO was 3.55 ppm and the furfuryl methylene proton of PFMA was found at 4.92 ppm. However, in the spectrum of PEO-b-PFMA CCL micelle (Preparation Example 5) (Fig. 7 (b)), only protons of the methylene group of PEO, a hydrophilic cell region, were shown.

From the DLS analysis, the hydraulic diameters of PEO-b-PFMA micelles and PEO-b-PFMA CCL micelles were compared. As shown in Fig. 8 (a), the diameter of the PEO-b-PFMA micelle was 112 nm, but the diameter of the core-crosslinked PEO-b-PFMA CCL micelle was about 91 nm. This is due to the shrinkage of the core part due to crosslinking. The morphology of CCL micelles was observed by TEM (FIG. 8 (b)), and the spherical shape showed an average size of 100 nm, which was in good agreement with the DLS results.

Experimental example  2. Alkanes - Azide  By click response PEO -b- PGMA -N ₃ CCL Of micelle (Production Example 6)  Analysis and characteristics

8 shows ¹ H-NMR of CCL micelles (Preparation Example 6) of PEO-b-PGMA-N ₃ (Preparation Example 3) and PEO-b-PGMA-N ₃ . In the spectrum of the PEO-b-PGMA-N ₃ copolymer (Preparation Example 3) (FIG. 9 (a)), the proton of the methylene group of PEO was 3.55 ppm and the methyl proton of PGMA-N ₃ was 0.7-1.0 ppm. . However, in the spectrum of CCL micelles of PEO-b-PGMA-N ₃ (Preparation Example 6) (FIG. 9 (b)), only protons of the methylene group of PEO, a hydrophilic cell region, were shown.

Experimental example  3. PEO -b- PFMA  CCL Micelle ICG  And DOX  Load efficiency analysis

ICG and / or DOX carried on CCL micelles (CCL / ICG / DOX, CCL / ICG and CCL / DOX) of PEO-b-PFMA of Examples 1-1, 2-1 and 3-1 It was analyzed by UV-vis spectrophotometer, it is shown in Figure 10. The mass of DOX and ICG supported in the micelle was determined using ultraviolet-visible spectroscopy with standard calibration curves at 485 nm and 780 nm. The drug loading (DLC) and drug loading (DLE) were calculated by the following equations, and the loading properties of ICG and DOX for PEO-b-PFMA CCL micelles are shown in Table 1 below.

DLC (%) = (weight of drug supported on nanoparticles / total weight of nanoparticles supported on drug) × 100 and

DLE (%) = (weight of drug detected in nanoparticles / weight of drug added) × 100.

Samples DLE of DOX (%) DLC of DOX (%) DLE of ICG (%) DLC of ICG (%) CCL / DOX 62.38 9.92 - - CCL / ICG - - 50.91 0.41 CCL / ICG / DOX 76.11 11.83 60.56 0.48

10, the characteristic absorption peaks of DOX and ICG were found at 490 nm and 780 nm, respectively. When DOX was mounted on the CCL (CCL / DOX), a red transition from 490 nm to 505 nm indicates that the DOX was successfully mounted on the CCL micelle. In addition, a red shift was also observed in the CCL / ICG peak signal shifted from 780 nm to 803 nm. In particular, the simultaneous mounting of ICG and DOX in the CCL micelle structure (CCL / ICG / DOX, Example 1-1) is the separate loading of ICG and DOX summarized in Table 1 above (CCL / ICG (Example 2-1) or Compared to CCL / DOX (Example 3-1)), the loading capacity was significantly improved. For example, after co-loading, DOX's DLE increased 14.97% and ICG's DLE increased 11.46%.

Experimental example  4. Near infrared  By investigation PEO -b- PFMA / ICG / DOX Micelle  Drug release pattern analysis

The in vitro drug release of DOX from PEO-b-PFMA / ICG / DOX micelles (CCL / ICG / DOX) of Example 1-1 was investigated by dialysis. A solution of 3 mL of micelle (1 mg mL ^-1 ) loaded with DOX is transferred to a dialysis membrane (MW cutoff, 13 kDa) and 5 min for 15 mL of PBS (0.01 M, pH 7.4) or acetate buffer (0.01 M, pH 5.0) Dialysis was performed at 37 ° C. with near infrared (NIR) irradiation (200 mW cm ⁻² , 808 nm). The dialysate (3 mL) was withdrawn at a predetermined time interval and calibrated with an equal amount of fresh buffer solution. The drug release was determined by the UV-Vis absorbance at 485 nm, which was described as the percentage of cumulative DOX outside the dialysis membrane relative to the total DOX of the nanocarriers.

The size distribution graph and TEM photographs measured by DLS for the size change of CCL / ICG / DOX micelles irradiated with near infrared rays are shown in FIGS. 11 and 12, respectively.

Figure 13 shows the DOX drug release according to the near infrared irradiation at pH 5 and pH 7.4. As shown in FIG. 13, CCL / ICG / DOX micelles (Example 1-1) significantly increased the release of DOX by near infrared irradiation (+ NIR). In the absence of irradiation (-NIR), the cumulative emission of DOX was less than 25% from the initial loading value. In contrast, DOX released about 65% at pH 5 and about 42% at pH 7.4 after 30 hours of incubation by near infrared irradiation (+ NIR). The higher release performance at pH 5 is due to the good solubility of DOX in acidic environments. By near-infrared, ICG generates ROS, which cleaves the deselenide bonds of the core of the micelle, resulting in the release of DOX mounted therein. In the absence of near infrared irradiation under the same conditions, drug release is suppressed due to the compactness and robustness of the CCL structure. These results suggest that while CCL micelles do not release DOX in the blood (pH close to 7.4), DOX can be released from CCL micelles after entering target cancer cells (pH 5-5.5).

Experimental example  5. Near infrared  By investigation PEO -b- PGMA / ICG / DOX Micelle  Drug release pattern analysis

The in vitro drug release of DOX from PEO-b-PGMA / ICG / DOX micelles (CCL / ICG / DOX) of Example 1-2 was investigated by dialysis. A solution of 3 mL of micelle (1 mg mL ^-1 ) loaded with DOX is transferred to a dialysis membrane (MW cutoff, 13 kDa) and 5 min for 15 mL of PBS (0.01 M, pH 7.4) or acetate buffer (0.01 M, pH 5.0) Dialysis was performed at 37 ° C. with near infrared (NIR) irradiation (200 mW cm ⁻² , 808 nm). The dialysate (3 mL) was withdrawn at a predetermined time interval and calibrated with an equal amount of fresh buffer solution. The drug release was determined by the UV-Vis absorbance at 485 nm, which was described as the percentage of cumulative DOX outside the dialysis membrane relative to the total DOX of the nanocarriers.

14 and 15 show TEM images and size distribution graphs measured by DLS for changes in the size of CCL / ICG / DOX micelles irradiated with near infrared rays.

Figure 16 shows the DOX drug release according to the near infrared irradiation at pH 5 and pH 7.4. As shown in FIG. 16, CCL / ICG / DOX micelles (Example 1-2) significantly increased the release of DOX by near infrared irradiation (+ NIR). In the absence of irradiation (-NIR), the cumulative emission of DOX was less than 25% from the initial loading value. In contrast, DOX released about 60% at pH 5 and about 40% at pH 7.4 after 30 hours of incubation by near infrared irradiation (+ NIR). The higher release performance at pH 5 is due to the good solubility of DOX in acidic environments. By near-infrared, ICG generates ROS, which cleaves the deselenide bonds of the core of the micelle, resulting in the release of DOX mounted therein. In the absence of near infrared irradiation under the same conditions, drug release is suppressed due to the compactness and robustness of the CCL structure. These results suggest that while CCL micelles do not release DOX in the blood (pH close to 7.4), DOX can be released from CCL micelles after entering target cancer cells (pH 5-5.5).

Experimental example  6. PEO -b- PFMA / ICG / DOX Micelle  Cytotoxicity analysis

Generic cells HEK293 (human embryo kidney) and cancer cells HepG2 (human hepatocyte carcinoma) were provided by the American Type Culture Collection (ATCC; Manassas, VA, USA). Cells were cultured and maintained in MEM medium. The medium was supplemented with 10% FBS and 1% antibiotic (penicillin-streptomycin). The cultured cells were cultured in a humidified atmosphere of 5% CO ₂ at 37 ° C. Cell viability assays were performed to determine the toxic effects of PEO-b-PFMA CCL micelles (Preparation Example 5) on HEK293 and the cytotoxic effects of CCL / ICG / DOX micelles (Example 1-1) on HepG2 cells. . HEK293 and HepG2 cells were inoculated into each well of a 96-well plate at a density of 1.0 × 10 ⁴ cells and cultured in a humidified atmosphere of 5% CO ₂ at 37 ° C. for 24 hours. Next, cells were subjected to different concentrations of CCL micelles (5.0, 10, 50, 100, 150, 250 and 500 μg mL ⁻¹ ) and CCL / ICG / DOX and DOX (0.1, 0.5, 1.0, 5.0, 10 and 15 μg) / mL ^{- 1} ). For drug release, HepG2 cells were treated with drug-bearing crosslinked micelles (CCL / ICG / DOX) exposed to NIR laser (200mW cm ^-2 at 808 nm NIR) for 5 minutes. Absorbance was measured at 460 nm using an ELISA plate reader (Molecular Devices, Sunnyvale, CA, USA). Cytotoxicity is expressed as a percentage of cell viability relative to the blank control.

17 (a) shows that CCL micelles are not toxic to HEK293 cells after 24 hours of incubation at a concentration of 0 to 100 μg / mL ⁻¹ . And at 150, 250, 500 μg / mL ^-1 concentrations, it showed slightly reduced cell viability to 91.5, 84.3 and 76.1%. These results show that CCL micelles up to 150 μg / mL ^-1 concentration can be safely used as nanocarriers for drug delivery and have biocompatibility. Figure 17 (b) is a graph assessing the cytotoxic effect on human hepatocellular carcinoma (HepG2) cells after a near-infrared irradiation (5 minutes) of CCL micelles equipped with DOX. Cell survival was reduced in a concentration-dependent manner in CCL / ICG / DOX micelles subjected to 5-minute near-infrared irradiation at the same concentration as free DOX without near-infrared irradiation at concentrations of 0.1, 0.5, 1, 5, 10 and 15 μg / mL ⁻¹ . In contrast, CCL / ICG / DOX micelles not subjected to near-infrared irradiation showed minimal toxic effects on HepG2 cells. That is, the case of apoptosis CCL / ICG / DOX micelles the near infrared is irradiated There is no significant cell death to 0.1μg / mL ^-1 1, 5, 10 and 15 μg / mL ^-1 to 84.16, 79.14, 67.4 and 62.56% The rate was reached. On the other hand, CCL / ICG / DOX micelles without NIR laser irradiation showed negligible cytotoxic effects with 97.57, 96.6, and 95.08% mortality at 5, 10, and 15 μg / mL ^-1 concentrations. These results show that CCL / ICG / DOX micelles induce cell death to HepG2 cells by near infrared irradiation.

Experimental example  7. DAPI  By dyeing PEO -b- PFMA / ICG / DOX On micelle  Cell death assay

The nuclear morphology of HepG2 cells was monitored by fluorescence microscopy using DAPI staining. HepG2 cells (500 μl) with a density of 1.0 × 10 ⁴ cells mL ^-1 were placed in a coverslip bottom plate and incubated at 37 ° C. in a humidified atmosphere of 5% CO ₂ for 24 hours. The next day, cells were treated with 1 mL of MEM medium containing CCL / ICG / DOX of Example 1-1 irradiated with NIR laser for 5 minutes, and cultured at 37 ° C and 5% CO ₂ . After incubation, cells were washed with cold PBS and 1 mL of DAPI solution (1 μg / mL in methanol) was added. The plate was wrapped in aluminum foil to prevent light exposure and incubated for 20 minutes in an incubator. Finally, cells were washed twice with cold PBS and fixed to coverslip using ProLong® Gold Antifade reagent (10 μL). The nuclear morphology of the cells was observed with a Laser Scanning Confocal Microscope (Carl Zeiss LSM700, Jane, Germany).

After DAPI staining, typical characteristics of chromatin condensation and apoptosis were observed in HepG2 cells treated with free DOX and CCL / ICG / DOX micelles (Example 1-1) (dose: 15 μg / mL ⁻¹ ). 18 shows DAPI stained nuclei of HepG2 cells. Free DOX and DOX loaded CCL micelles induced typical apoptosis. Cells treated with free DOX showed strong DOX fluorescence in HepG2 cells after incubation for 8 and 16 hours. On the other hand, in the case of HepG2 cells treated with CCL / ICG / DOX micelles irradiated with near-infrared light, DOX fluorescence of the nucleus was slightly lower, but it was higher in the cytoplasm. This indicates that the drug-loaded micelle enters the cell slowly compared to free DOX through endocytosis and releases the drug of the core. These results show that CCL / ICG / DOX micelles release DOX by near-infrared irradiation and cause the death of HepG2 cells.

So far, the present invention has been focused on the preferred embodiments. Those skilled in the art to which the present invention pertains will understand that the present invention can be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments should be considered in terms of explanation, not limitation. The scope of the present invention is shown in the claims rather than the foregoing description, and all differences within the equivalent range should be interpreted as being included in the present invention.

Claims (13)

Amphiphilic copolymers comprising a hydrophilic portion and a hydrophobic portion;
A crosslinking agent comprising a diselenide bond; And
NIR dye; includes,
The hydrophilic part of the copolymer is one selected from the group consisting of poly (ethylene oxide), poly (oligo (ethylene glycol) methyl ether methacrylate) and poly (2-dimethylamino) ethyl methacrylate),
The hydrophobic portion of the copolymer is poly (furfuryl methacrylate), poly (styrene- alt -maleic anhydride), poly (α-propagyl carboxylate-ε-caprolactone), poly (α-pro Fargil-ε-caprolactone), poly (azaido ε-caprolactone-co-ε-caprolactone), poly (azaido ε-caprolactone), poly (γ-propagyl-glutamate), poly (5- (4- (prop-2-ain-1-yloxy) benzyl) -1,3-dioxolane-2,4-dione (tyrosine (alkainyl) -OCA), poly (N- (Prop-2-ain-1-yl) acrylamide), poly (N- (3-azidopropyl) acrylamide), poly (1-ethanyl-4-vinylbenzene), poly (γ-azai Do-propyl-L-glutamate) and poly (propargyl acrylate).
Here, the hydrophobic portion of the copolymer is characterized in that the furanyl (furanyl), maleimidyl (maleimidyl), alkynyl (alkynyl), or azido (azido) functional group is additionally introduced,
The crosslinking agent is a compound represented by the following formula (1),
here,
When a furanyl functional group is introduced into the hydrophobic portion of the copolymer, R ¹ and R ² of the crosslinking agent are maleimidyl,
When a maleimidyl functional group is introduced into the hydrophobic portion of the copolymer, R ¹ and R ² of the crosslinking agent are furanyl,
When an alkynyl functional group is introduced into the hydrophobic portion of the copolymer, R ¹ and R ² of the crosslinking agent are azido,
When an azido functional group is introduced into the hydrophobic portion of the copolymer, R ¹ and R ² of the crosslinking agent are alkynyl,
The functional group substituted in the hydrophobic portion of the copolymer and the functional groups substituted in R ¹ and R ² of the crosslinking agent are characterized in that they form a crosslinking by mutually clicking reaction,
The near infrared dyes include indocyanine green (ICG), methylene blue (MB), IRDye 800 CW, Cy5.5, Cy7, Cy7.5, protoporphyrin IX, IR-780, IR-783, IR-808 and MHI-148 Characterized in that at least one selected from the group consisting of,
Characterized in that the diselenide bond of the crosslinking agent disintegrates in response to near infrared rays,
Cross-linked micelles with cancer cell target function:
[Formula 1]

(In the formula 1,
a, b, c and d are each an integer of 1-10;
R ¹ and R ² are furanyl, maleimidyl, alkynyl, or azido).
According to claim 1,
The micelle is a hydrophilic shell; And a hydrophobic core; a crosslinked micelle having a cancer cell target function.
According to claim 1,
The cross-linked micelle may introduce a drug into the cross-linked micelle,
The drug is prednisolone 21-acetate, paclitaxel, doxorubicin, retinoic acid, cisplatin, camptothecin, Fluorouracil (5- FU), Docetaxel, Tamoxifen, Anasterozole, Carboplatin, Topotecan, Berotecan, Irinotecan, Gleevec, Vincristine, aspirin, salicylates, ibuprofen, naproxen, fenofopen, fenoprofen, indomethacin, phenyltazone ), Mesotrexate, cyclophosphamide, mechlorethamine, dexamethasone, prednisolone, celecoxib, valdecoxib, nidecyl Nimesulide, cortisone and nose Cross-linked micelle, characterized in that at least one selected from the group consisting of a corticosteroid (corticosteroid).
According to claim 1,
The cross-linking agent is a cross-linked micelle characterized in that the compound represented by the formula (2):
[Formula 2]

.
According to claim 1,
The cross-linking agent is a cross-linked micelle characterized in that the compound represented by the formula (3):
[Formula 3]

.
According to claim 1,
The copolymer,
Poly (ethylene oxide) -b-poly (furfuryl methacrylate); Or poly (ethylene oxide) -b-poly (furfuryl methacrylate) having an azido functional group introduced therein;
According to claim 1,
The copolymer,
Poly (ethylene oxide) -b-poly (glycidyl methacrylate) having a furanyl functional group introduced therein; Or poly (ethylene oxide) -b-poly (glycidyl methacrylate) into which an azido functional group is introduced; Cross-linked micelle, characterized in that.
Adding and stirring an amphiphilic copolymer comprising a hydrophilic part and a hydrophobic part, a crosslinking agent comprising a diselenide bond, and a NIR dye (step 1); And
Including the step of reacting the mixture prepared in step 1 at 50-80 ℃ for 8-16 hours, inducing cross-linking of micelle cores (step 2);
The hydrophilic part of the copolymer is one selected from the group consisting of poly (ethylene oxide), poly (oligo (ethylene glycol) methyl ether methacrylate) and poly (2-dimethylamino) ethyl methacrylate),
The hydrophobic portion of the copolymer is poly (furfuryl methacrylate), poly (styrene- alt -maleic anhydride), poly (α-propagyl carboxylate-ε-caprolactone), poly (α-pro Fargil-ε-caprolactone), poly (azaido ε-caprolactone-co-ε-caprolactone), poly (azaido ε-caprolactone), poly (γ-propagyl-glutamate), poly (5- (4- (prop-2-ain-1-yloxy) benzyl) -1,3-dioxolane-2,4-dione (tyrosine (alkainyl) -OCA), poly (N- (Prop-2-ain-1-yl) acrylamide), poly (N- (3-azidopropyl) acrylamide), poly (1-ethanyl-4-vinylbenzene), poly (γ-azai Do-propyl-L-glutamate) and poly (propargyl acrylate).
Here, the hydrophobic portion of the copolymer is characterized in that the furanyl (furanyl), maleimidyl (maleimidyl), alkynyl (alkynyl), or azido (azido) functional group is additionally introduced,
The crosslinking agent is a compound represented by the following formula (1),
here,
When a furanyl functional group is introduced into the hydrophobic portion of the copolymer, R ¹ and R ² of the crosslinking agent are maleimidyl,
When a maleimidyl functional group is introduced into the hydrophobic portion of the copolymer, R ¹ and R ² of the crosslinking agent are furanyl,
When an alkynyl functional group is introduced into the hydrophobic portion of the copolymer, R ¹ and R ² of the crosslinking agent are azido,
When an azido functional group is introduced into the hydrophobic portion of the copolymer, R ¹ and R ² of the crosslinking agent are alkynyl,
The functional group substituted in the hydrophobic portion of the copolymer and the functional groups substituted in R ¹ and R ² of the crosslinking agent are characterized in that they form a crosslinking by mutually clicking reaction,
The near infrared dyes include indocyanine green (ICG), methylene blue (MB), IRDye 800 CW, Cy5.5, Cy7, Cy7.5, protoporphyrin IX, IR-780, IR-783, IR-808 and MHI-148 Characterized in that at least one selected from the group consisting of,
Characterized in that the desenlenide bond of the crosslinking agent is decomposed in response to near infrared rays,
Manufacturing method of cross-linked micelles with cancer cell target function:
[Formula 1]

(In the formula 1,
a, b, c and d are each an integer of 1-10;
R ¹ and R ² are furanyl, maleimidyl, alkynyl, or azido).
The cross-linked micelle of claim 1; And a drug introduced into the crosslinked micelle,
The drug is prednisolone 21-acetate, paclitaxel, doxorubicin, retinoic acid, cisplatin, camptothecin, Fluorouracil (5- FU), Docetaxel, Tamoxifen, Anasterozole, Carboplatin, Topotecan, Berotecan, Irinotecan, Gleevec, Vincristine, aspirin, salicylates, ibuprofen, naproxen, fenofopen, fenoprofen, indomethacin, phenyltazone ), Mesotrexate, cyclophosphamide, mechlorethamine, dexamethasone, prednisolone, celecoxib, valdecoxib, nidecyl Nimesulide, cortisone and nose A drug delivery system with a target cancer cell function, characterized in that at least one selected from the group consisting of a corticosteroid (corticosteroid).
Adding and stirring an amphiphilic copolymer comprising a hydrophilic part and a hydrophobic part, a crosslinking agent comprising a diselenide bond, a NIR dye, and a drug in a solvent (step 1); And
Including the step of reacting the mixture prepared in step 1 at 50-80 ℃ for 8-16 hours, inducing cross-linking of micelle cores (step 2);
The hydrophilic part of the copolymer is one selected from the group consisting of poly (ethylene oxide), poly (oligo (ethylene glycol) methyl ether methacrylate) and poly (2-dimethylamino) ethyl methacrylate),
The hydrophobic portion of the copolymer is poly (furfuryl methacrylate), poly (styrene- alt -maleic anhydride), poly (α-propagyl carboxylate-ε-caprolactone), poly (α-pro Fargil-ε-caprolactone), poly (azaido ε-caprolactone-co-ε-caprolactone), poly (azaido ε-caprolactone), poly (γ-propagyl-glutamate), poly (5- (4- (prop-2-ain-1-yloxy) benzyl) -1,3-dioxolane-2,4-dione (tyrosine (alkainyl) -OCA), poly (N- (Prop-2-ain-1-yl) acrylamide), poly (N- (3-azidopropyl) acrylamide), poly (1-ethanyl-4-vinylbenzene), poly (γ-azai Do-propyl-L-glutamate) and poly (propargyl acrylate).
Here, the hydrophobic portion of the copolymer is characterized in that the furanyl (furanyl), maleimidyl (maleimidyl), alkynyl (alkynyl), or azido (azido) functional group is additionally introduced,
The crosslinking agent is a compound represented by the following formula (1),
here,
When a furanyl functional group is introduced into the hydrophobic portion of the copolymer, R ¹ and R ² of the crosslinking agent are maleimidyl,
When a maleimidyl functional group is introduced into the hydrophobic portion of the copolymer, R ¹ and R ² of the crosslinking agent are furanyl,
When an alkynyl functional group is introduced into the hydrophobic portion of the copolymer, R ¹ and R ² of the crosslinking agent are azido,
When an azido functional group is introduced into the hydrophobic portion of the copolymer, R ¹ and R ² of the crosslinking agent are alkynyl,
The functional group substituted in the hydrophobic portion of the copolymer and the functional groups substituted in R ¹ and R ² of the crosslinking agent are characterized in that they form a crosslinking by mutually clicking reaction,
The near infrared dyes include indocyanine green (ICG), methylene blue (MB), IRDye 800 CW, Cy5.5, Cy7, Cy7.5, protoporphyrin IX, IR-780, IR-783, IR-808 and MHI-148 Characterized in that at least one selected from the group consisting of,
The drug is prednisolone 21-acetate, paclitaxel, doxorubicin, retinoic acid, cisplatin, camptothecin, Fluorouracil (5- FU), Docetaxel, Tamoxifen, Anasterozole, Carboplatin, Topotecan, Berotecan, Irinotecan, Gleevec, Vincristine, aspirin, salicylates, ibuprofen, naproxen, fenofopen, fenoprofen, indomethacin, phenyltazone ), Mesotrexate, cyclophosphamide, mechlorethamine, dexamethasone, prednisolone, celecoxib, valdecoxib, nidecyl Nimesulide, cortisone and nose Characterized in that at least one selected from the group consisting of a corticosteroid (corticosteroid), and
Characterized in that the desenlenide bond of the crosslinking agent is decomposed in response to near infrared rays,
Preparation method of drug delivery system having cancer cell target function:
[Formula 1]

(In the formula 1,
a, b, c and d are each an integer of 1-10;
R ¹ and R ² are furanyl, maleimidyl, alkynyl, or azido).
The method of claim 10,
The amphipathic copolymer is a manufacturing method characterized in that it is prepared through a RAFT (Reversable Addition Fragmentation chain Trasfer) polymerization reaction or an atomic transfer radical polymerization (ATRP) molecular controlled polymerization reaction.
The method of claim 10,
The crosslinking agent is a production method characterized in that the compound represented by the following formula (2):
[Formula 2]

.
The method of claim 10,
The crosslinking agent is a production method characterized in that the compound represented by the following formula (3):
[Formula 3]

.

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