KR20220007476A - Porous alginate-based hydrogel, preparing method thereof and drug delivery system using the same - Google Patents

Abstract

The present invention relates to a porous alginate-based hydrogel, a method for preparing the same, and a drug delivery body using the same. The porous hydrogel imparts porosity to the hydrogel as N_2 gas is generated during a click reaction at functional groups which are click-reactable with each other introduced into a polymer matrix material and a crosslinking agent, respectively. Accordingly, the porosity suppresses the initial rapid drug release and has a sustained-release drug release feature as the drug delivery body which induces the drug release. Therefore, the present invention can be usefully used in the pharmaceutical and cosmetic fields.

Description

Porous alginate-based hydrogel, manufacturing method thereof, and drug delivery system using same {Porous alginate-based hydrogel, preparing method thereof and drug delivery system using the same}

The present invention relates to a porous alginate-based hydrogel, a method for preparing the same, and a drug delivery system using the same, and a porous alginate-based hydrogel based on crosslinking including an Inverse Electron-Demand Diels-Alder click reaction. It relates to the preparation of gels and drug release.

Hydrogel - A network of hydrophilic polymer chains containing a large amount of water has been widely used as a biomaterial carrier because of its unique properties [1-3]. Because the main component is water, the hydrogel has high biocompatibility, high loading capacity, and mimics natural tissue. For drug delivery systems (DDS), hydrogels generally exhibit higher capacity compared to other DDS materials. The high moisture content is also beneficial for channel protection, especially when handling sensitive materials such as proteins or genomic materials. Moreover, the physical and chemical properties of hydrogels are easily tuned by introducing specific functional groups into the polymer backbone [8,9]. Thus, hydrogels may be the future of clinical therapy in a variety of applications.

However, most hydrogels showed fast drug release properties. The hydrophilic nature of the hydrogel enables the free diffusion of drug molecules, resulting in the release of large amounts of drug initially. The initial drug release frequently occurred even in the release stimulus response type hydrogel. Premature drug release not only reduces drug efficacy, but can also jeopardize normal tissues. Highly precise and long-term sustained-release drug release is a challenging issue in hydrogel research these days. On the other hand, the factors governing drug release behavior are not yet well understood. In particular, when dealing with natural polymers, various factors such as the nature of the polymer chain, the gelation process, the type of cross-linking agent, and the presence of a porous internal structure can influence the drug release behavior.

The use of natural carbohydrate polymers such as alginate for hydrogel preparation shows many advantages due to its good biocompatibility and low cytotoxicity. Alginate is a naturally occurring polymer that can be extracted from brown algae. The structure of alginic acid is composed of a linear acidic polysaccharide composed of β-d-mannuronate (M) and its C-5 epiisomer α-l-guluronate (G). The presence of a carboxylic acid group in each repeating unit of alginic acid provides a feasible method for adjusting its properties through functionalization and crosslinking. In the meantime, various efforts have been made to improve alginate by functionalization of polymer chains. Clara et al. reported on functionalized alginates with furan groups to demonstrate a rapid response to a pH-stimulated drug delivery system. Desai et al. reported alginic acid functionalized with norbornene and tetrazine to improve the physical properties of hydrogels for cell engineering. The functionalization of the alginate also allowed for easy crosslinking. The use of crosslinking agents can not only increase the strength of the hydrogel, but also adjust the polymer network chain entanglement, which has a high impact on diffusion kinetics. In addition, specific cross-linking has enabled smart on-demand drug release.

The structure of the crosslinking agent largely governs the properties of the hydrogel. Alginate hydrogels were generally prepared by crosslinking with divalent cations. However, the ion exchange-type alginate hydrogel is not stable under physiological conditions due to the unwanted ion exchange process, leading to rapid drug release. Therefore, today, mainly chemical crosslinking alginate hydrogels are being developed to meet the demand for sustained drug release. The chemically bonded crosslinking agent may be a polymer, a bulky material, or a small organic molecule. Depending on the size of the crosslinking agent, the mesh size, which is the open space between the polymer chains of the hydrogel, can be adjusted. Since the diffusion kinetics of the drug in the hydrogel is governed by the Stokes-Einstein equation, a smaller mesh size may increase the drug release time. In addition to the mesh size, the presence of micropores in the hydrogel influences the drug release profile according to Darcy's law of diffusion. Therefore, in order to study the drug release behavior of hydrogels, it is necessary to consider not only the presence of porosity, but also the properties of the crosslinking agent.

The present invention provides a crosslinking agent and micropore structure technology that prolongs drug release of an alginate-based hydrogel. In this invention, the crosslinking agent chemical structure and the alginate trisalt chemical structure are modified to cross-link them through an inverse electron demand Diels-Ald (IEDDA) click reaction to generate nitrogen gas. Nitrogen released during the click reaction served as a medium (porogen, porogen) for forming porous pores.

Laid-open Patent Publication No. 10-2014-0013330

It is an object of the present invention to provide a porous hydrogel characterized in that it has sustained-release drug release properties.

Another object of the present invention is to provide a method for preparing the porous hydrogel.

Another object of the present invention is to provide a method for preparing a drug-supported porous hydrogel.

Another object of the present invention is to provide a sustained-release drug delivery system comprising the porous hydrogel.

Another object of the present invention is to provide an external stimulus-sensitive drug delivery system using near-infrared rays comprising the porous hydrogel.

In order to achieve the above object,

The present invention relates to an alginic acid derivative represented by the following formula (1); and

Including; a crosslinking agent comprising a bond represented by the following formula (2)

Substituent X of Formula 1 below and Substituent Y of Formula 2 are click-reactable with each other and are characterized in that they are a combination that generates _{N 2 gas during the click reaction,}

Characterized in that it has sustained-release drug release properties,

A porous hydrogel is provided.

[Formula 1]

(In Formula 1,

m is an integer from 1-100,000;

L ¹ is an ester or amide bond;

X is a click-reactive functional group of one of a norbornenyl derivative, a trans-cyclooctenyl derivative and a tetrazinyl derivative,

또는

이고,The norbornenyl derivative is

or

ego,

또는

이고,The trans-cyclooctene is

or

ego,

,

,

,

,

,

,

,

또는

이다.)The tetrazinyl derivative is

,

,

,

,

,

,

,

or

to be.)

[Formula 2]

(In Formula 2,

a, b, c and d are each an integer of 0-10, where 0 means a single bond;

Z is one of a disulfide bond, a diselenide bond, a PEG bond (polyethyleneglycol bond), and methylene;

L ² is an ester or amide bond;

Y is the same as the definition of X in Formula 1 above.)

In addition, the present invention includes a step (step 1) of adding an alginic acid derivative represented by the following formula (1) and a crosslinking agent represented by the following formula (2) to a solvent and reacting (step 1);

When X in Formula 1 is a norbornenyl derivative or a trans-cyclooctenyl derivative, Y in Formula 2 is a tetrazinyl derivative,

When X in Formula 1 is a tetrazinyl derivative, Y in Formula 2 is a norbornenyl derivative or trans-cyclooctenyl derivative,

Provided is a method for preparing a porous hydrogel.

[Formula 1]

(In Formula 1, the definition of each substituent is the same as defined in claim 1.)

[Formula 2]

(In Formula 2, the definition of each substituent is as defined in claim 1.)

Furthermore, the present invention comprises the steps of adding and mixing an alginic acid derivative represented by the following Chemical Formula 1, and a drug to a solvent (step 1); and

A step of adding and reacting a crosslinking agent represented by the following formula (2) (step 2);

When X in Formula 1 is a norbornenyl derivative or a trans-cyclooctenyl derivative, Y in Formula 2 is a tetrazinyl derivative,

When X in Formula 1 is a tetrazinyl derivative, Y in Formula 2 is a norbornenyl derivative or trans-cyclooctenyl derivative,

Provided is a method for preparing a drug-supported porous hydrogel.

[Formula 1]

(In Formula 1, the definition of each substituent is the same as defined in claim 1.)

[Formula 2]

(In Formula 2, the definition of each substituent is as defined in claim 1.)

In addition, the present invention provides a sustained-release drug delivery system comprising the porous hydrogel.

Furthermore, the present invention is the porous hydrogel; and

A near-infrared dye (NIR dye) characterized in that it forms active oxygen species upon irradiation with near-infrared rays;

In the porous hydrogel of claim 1, Z in Formula 2 is a disulfide bond or a diselenide bond,

The near-infrared dye is 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 of

Provided is an external stimulus-sensitive drug delivery system using near-infrared rays.

The porous hydrogel according to the present invention is characterized in that it imparts porosity to the hydrogel as _{N 2} gas is generated during a click reaction in the functional groups that are click-reactable with each other introduced into the polymer matrix material and the crosslinking agent, respectively, and, accordingly, the porosity As a drug delivery system that induces drug release, it can be usefully used in pharmaceutical and cosmetic fields because it suppresses rapid initial drug release and has sustained-release drug release characteristics.

1 is a view showing a reaction formula for synthesizing a porous and non-porous hydrogel according to the type of a click reaction functional group introduced into alginic acid and a crosslinking agent, and _{whether N 2 gas is generated during the click reaction.}
2 is It is a diagram showing the synthesis reaction scheme of an alginic acid derivative (Alg-Nb).
3 is a view showing a synthesis reaction scheme of a tetrazine crosslinking agent (S-Tz).
^{4 is a diagram showing 1} H-NMR spectrum of a tetrazine crosslinking agent (S-Tz).
5 is a view showing the formation of a porous hydrogel by a reverse tube test.
6 is a view showing a cross-sectional SEM photograph of a porous hydrogel.
7 is a view showing a cross-sectional SEM photograph of a non-porous hydrogel.
8 is a diagram showing a graph of the drug release rate over time of the porous hydrogel.
9 is a diagram showing a graph of the drug release rate over time of the non-porous hydrogel.
10 is a photograph confirming the degree of deterioration (degradation) of the drug-releasing porous hydrogel over time.
^{11 is 1} H NMR data of Preparation Example 1 (Alg-Nb) and Preparation Example 2 (Alg-Fu).

Hereinafter, the present invention will be described in detail.

porous hydrogel

The present invention relates to an alginic acid derivative represented by the following formula (1); and

Including; a crosslinking agent comprising a bond represented by the following formula (2)

Substituent X of Formula 1 below and Substituent Y of Formula 2 are click-reactable with each other and are characterized in that they are a combination that generates _{N 2 gas during the click reaction,}

Characterized in that it has sustained-release drug release properties,

A porous hydrogel is provided.

[Formula 1]

(In Formula 1,

m is an integer from 1-100,000;

L ¹ is an ester or amide bond;

X is a click-reactive functional group of one of a norbornenyl derivative, a trans-cyclooctenyl derivative and a tetrazinyl derivative,

또는

이고,The norbornenyl derivative is

or

ego,

또는

이고,The trans-cyclooctene is

or

ego,

,

,

,

,

,

,

,

또는

이다.)The tetrazinyl derivative is

,

,

,

,

,

,

,

or

to be.)

[Formula 2]

(In Formula 2,

a, b, c and d are each an integer of 0-10, where 0 means a single bond;

Z is one of a disulfide bond, a diselenide bond, a PEG bond (polyethyleneglycol bond), and methylene;

L ² is an ester or amide bond;

Y is the same as the definition of X in Formula 1 above.)

When X in Formula 1 is a norbornenyl derivative or a trans-cyclooctenyl derivative, Y in Formula 2 is a tetrazinyl derivative,

When X in Formula 1 is a tetrazinyl derivative, Y in Formula 2 is a norbornenyl derivative or a trans-cyclooctenyl derivative.

And, the number of tetrazine (Tz) functional groups of the tetrazinyl derivative of X or Y in Formula 1,

The ratio of the number of norbornene (Nb) functional groups of the norborneneyl derivative or cyclooctene (Co) functional groups of the trans-cyclooctenyl derivative that click-react with the tetrazine functional group in X or Y;

Nb or Co: Tz may be 1-20: 1-20, preferably 10:4-10.

The porous hydrogel is characterized in that the rapid initial drug release is suppressed and has a constant sustained-release property (see Experimental Example 4).

The porous hydrogel may support a drug,

The drug is, for example, insulin, anti-rheumatic agents, prednisolone 21-acetate, paclitaxel, doxorubicin, retinoic acid, cis-platin, camptocecin (camptothecin), Fluorouracil (5-FU), docetaxel (Docetaxel), tamoxifen (Tamoxifen), anasterozole, topotecan (topotecan), gleevec (gleevec), vincristine (vincristine), aspirin (aspirin) , salicylates, ibuprofen, fenoprofen, indomethacin, phenyltazone, methotrexate, cyclophosphamide, Dexamethasone, nimesulide, cortisone, corticosteroid, mother and child placenta extract, protein from animal placenta, α-lipoic acid, α-Tocopherol , retinoids, glutathione, etc. can be used, and all commercially available drugs can be used.

In a preferred embodiment, the alginic acid derivative may be one compound of Formulas 3 to 6 below.

[Formula 3]

(In Formula 3,

m is an integer from 1-100,000.)

[Formula 4]

(In Formula 4,

m is an integer from 1-100,000.)

[Formula 5]

(In Formula 5,

m is an integer from 1-100,000.)

[Formula 6]

(In Formula 6,

m is an integer from 1-100,000.)

In a preferred embodiment, the crosslinking agent may be one compound of Formulas 7 to 21 below.

[Formula 7]

[Formula 8]

[Formula 9]

[Formula 10]

[Formula 11]

[Formula 12]

(In Formula 12,

n is an integer of 1-20.)

[Formula 13]

(In the formula 13,

n is an integer of 1-20.)

[Formula 14]

(In Formula 14,

n is an integer of 1-20.)

[Formula 15]

(In Formula 15,

n is an integer of 1-20.)

[Formula 16]

(In Formula 16,

n is an integer of 1-20.)

[Formula 17]

[Formula 18]

[Formula 19]

[Formula 20]

[Formula 21]

Method for preparing porous hydrogel

The present invention includes a step (step 1) of adding an alginic acid derivative represented by the following formula (1) and a crosslinking agent represented by the following formula (2) to a solvent and reacting (step 1);

When X in Formula 1 is a norbornenyl derivative or a trans-cyclooctenyl derivative, Y in Formula 2 is a tetrazinyl derivative,

When X in Formula 1 is a tetrazinyl derivative, Y in Formula 2 is a norbornenyl derivative or trans-cyclooctenyl derivative,

Provided is a method for preparing a porous hydrogel.

[Formula 1]

(In Formula 1, the definition of each substituent is as defined in Formula 1 above.)

[Formula 2]

(In Formula 2, the definition of each substituent is as defined in Formula 2 above.)

The solvent is phosphate buffer, dimethyl sulfoxide (DMSO), ethanol, anhydrous tetrahydrofuran (THF), benzene, KOH / MeOH, MeOH, toluene, CH ₂ Cl ₂ , hexane, dimethylformamide (DMF), diisopropyl Ether, diethyl ether, dioxane, dimethylacetamide (DMA), acetone, chlorobenzene, etc. may be used alone or in combination, and a phosphate buffer may be preferably used.

Method for preparing drug-supported porous hydrogel

The present invention comprises the steps of adding and mixing an alginic acid derivative represented by the following Chemical Formula 1, and a drug in a solvent (step 1); and

A step of adding and reacting a crosslinking agent represented by the following formula (2) (step 2);

When X in Formula 1 is a norbornenyl derivative or a trans-cyclooctenyl derivative, Y in Formula 2 is a tetrazinyl derivative,

When X in Formula 1 is a tetrazinyl derivative, Y in Formula 2 is a norbornenyl derivative or trans-cyclooctenyl derivative,

Provided is a method for preparing a drug-supported porous hydrogel.

[Formula 1]

(In Formula 1, the definition of each substituent is as defined in Formula 1 above.)

[Formula 2]

(In Formula 2, the definition of each substituent is as defined in Formula 2 above.)

The solvent is phosphate buffer, dimethyl sulfoxide (DMSO), ethanol, anhydrous tetrahydrofuran (THF), benzene, KOH / MeOH, MeOH, toluene, CH ₂ Cl ₂ , hexane, dimethylformamide (DMF), diisopropyl Ether, diethyl ether, dioxane, dimethylacetamide (DMA), acetone, chlorobenzene, etc. may be used alone or in combination, and a phosphate buffer may be preferably used.

The drug is, for example, insulin, anti-rheumatic agents, prednisolone 21-acetate, paclitaxel, doxorubicin, retinoic acid, cis-platin, camptocecin (camptothecin), Fluorouracil (5-FU), docetaxel (Docetaxel), tamoxifen (Tamoxifen), anasterozole, topotecan (topotecan), gleevec (gleevec), vincristine (vincristine), aspirin (aspirin) , salicylates, ibuprofen, fenoprofen, indomethacin, phenyltazone, methotrexate, cyclophosphamide, Dexamethasone, nimesulide, cortisone, corticosteroid, mother and child placenta extract, protein from animal placenta, α-lipoic acid, α-Tocopherol , retinoids, glutathione, etc. can be used, and all commercially available drugs can be used.

sustained release drug delivery system

The present invention provides a sustained-release drug delivery system comprising the porous hydrogel.

The drug is, for example, insulin, anti-rheumatic agents, prednisolone 21-acetate, paclitaxel, doxorubicin, retinoic acid, cis-platin, camptocecin (camptothecin), Fluorouracil (5-FU), docetaxel (Docetaxel), tamoxifen (Tamoxifen), anasterozole, topotecan (topotecan), gleevec (gleevec), vincristine (vincristine), aspirin (aspirin) , salicylates, ibuprofen, fenoprofen, indomethacin, phenyltazone, methotrexate, cyclophosphamide, Dexamethasone, nimesulide, cortisone, corticosteroid, mother and child placenta extract, protein from animal placenta, α-lipoic acid, α-Tocopherol , retinoids, glutathione, etc. can be used, and all commercially available drugs can be used.

External stimulus-sensitive drug delivery system using near-infrared rays

The present invention is the porous hydrogel of claim 1; and

A near-infrared dye (NIR dye) characterized in that it forms active oxygen species upon irradiation with near-infrared rays;

In the porous hydrogel of claim 1, Z in Formula 2 is a disulfide bond or a diselenide bond,

The near-infrared dye is 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 of

Provided is an external stimulus-sensitive drug delivery system using near-infrared rays.

The external stimulus-sensitive drug delivery system using the near infrared rays is characterized in that a disulfide bond or a diselenide bond included in the crosslinking agent is disintegrated in response to the near infrared rays.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples only illustrate the present invention, and the content of the present invention is not limited by the following examples.

<Preparation Example 1> Synthesis of alginic acid-norbornene (Alg-Nb)

1 g of sodium alginate (5.32 mmol relative to COOH functional groups) was dissolved in a buffer solution (pH 5.8) at a concentration of 0.5% w/v. Next, 0.36 g (3.16 mmol) of N-hydroxysuccinimide (NHS) and 1.11 g (5.8 mmol) of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDCI) were added to the alginate solution. After stirring for 10 minutes, 5-norbornene-2-methylamine (Nb-NH ₂ ) 0.32 g (2.63 mmol) was added to the mixture. After stirring at room temperature for 24 hours, the mixture was precipitated in acetone solution, and the filtered solid was dried in a vacuum oven, rehydrated with distilled water, and then dialyzed in a 13 kDa MWCO dialysis bag for 4 days. Finally, by freeze-drying, functionalized 25.53% of purified Alg-Nb was obtained in 77% yield.

The chemical structure of Alg-Nb is shown in FIGS. 1 and 2 .

¹ H NMR data of Alg-Nb of Preparation Example 1 is shown in FIG. 11(A).

<Preparation Example 2> Synthesis of alginic acid-furan (Alg-Fu)

1.25 g of sodium alginate (6.6452 mmol relative to COOH functional groups) was dissolved in a buffer solution (pH 5.8) at a concentration of 0.5% w/v. Next, 0.45 g (3.95 mmol) of NHS and 1.39 g (7.25 mmol) of EDCI were added to the alginate solution. After stirring for 10 minutes, furfuryl amine (Fu-NH ₂ ) 0.32 g (3.3 mmol) was added to the mixture. The reaction was stirred at room temperature for 24 hours, after which the reaction was stopped by adding hydroxylamine. The mixture was precipitated in excess acetone, which was dissolved in distilled water as a 1% w/v solution, and then dialyzed with distilled water in a 13 kDa MWCO dialysis bag for 4 days. The product was freeze-dried to yield purified Alg-Fu in 80% yield.

The Alg-Fu chemical structural formula is shown in FIG. 1 .

¹ H NMR data of Alg-Fu of Preparation Example 2 is shown in FIG. 11(B).

<Preparation Example 3> Preparation of tetrazine disulfide crosslinking agent (S-Tz)

The acid group of 5-(4-(1,2,4,5-tetrazin-3-yl)benzylamino)-5-oxopentanoic acid (Tz-COOH) was reacted with cystamine dihydrochloride (CYS.HCl) as follows. Tz-COOH powder (200 mg, 0.66 mmol) in a mixed solvent of DMF and DCM contained in a two-neck flask was stirred under _{N 2 atmosphere.} And CYS.HCl (67.9 mg, 0.3 mmol) was dissolved in acetonitrile and then added to the Tz-COOH solution. Then, 16.4 mg of N,N'-dicyclohexylcarbodiimide (DCC) dissolved in dichloromethane (DCM) was added to the mixture solution and reacted at room temperature for 12 hours. Then, the solid white impurities in the solution were filtered off and 1N HCl was added. Then the solution was extracted with DCM, the organic solvent was evaporated, and the residue was separated by chromatography to obtain the product.

The chemical structure of S-Tz is shown in FIGS. 1 and 3 .

¹ H NMR, δ = 10.58 (s), 8.49-8.42 (m, 3H), 8.27-8.20 (d, 1H), 7.56-7.49 (d, 2H), 4.43-4.34 (d, 2H), 4.02-3.88 (t, 2H), 3.54-3.39 (m, 2H), 2.32-2.25 (t, 2H), 2.23-2.16 (t, 2H), 1.81 (m, 2H).

<Preparation Example 4> Preparation of maleimide disulfide crosslinking agent (S-Ma)

<Preparation Example 4-1> Synthesis of 5-(2-(2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)-5-oxopentanoic acid (HEMI-GA)

1-(2-hydroxyethyl)-1H-pyrrole-2,5-dione (HEMI, 62 g, 4.39 mmol) was added to 50 ml acetonitrile under _{N 2 .} 50 mg (4.39 mmol) of Glutaric anhydrided was added to the HEMI solution, and the mixture was refluxed at 50 °C for 1 h. The reaction was then stirred at room temperature for 12 hours and then the solvent was removed by evaporation. The remaining mixture was dissolved in water, extracted with ethyl acetate, and washed with brine. The organic solution was evaporated to give a yellow liquid product (HEMI-GA).

<Preparation Example 4-2> Bis(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl) 5,5'-((disulfanediylbis(ethane-2,1-diyl) Synthesis of ))bis(azanediyl))bis(5-oxopentanoate) (S-Ma)

HEMI-GA 200mg (0,78mmol) was dissolved in DCM under _{N 2 atmosphere and stirred.} In another vessel, CYS.HCl (67.9 mg, 0.3 mmol) was dissolved in acetonitrile and then added to the HEMI-GA solution. Then, 16.4 mg of DCC was added to the mixture solution, followed by stirring at room temperature for 12 hours. The solid white impurities in the solution were filtered off and 1 N HCl was added. The solution was then extracted with DCM and water. After the organic solution was washed with brine and evaporated, the product was separated by chromatography.

The chemical structure of S-Ma is shown in FIG. 1 .

¹ H NMR, δ = 12.03 (s, 3H), 7.02-6.99 (s, 2H), 4.20-4.08 (t, 2H), 3.71-3.59 (t, 2H), 2.32-2.16 (m, 4H), 1.73 - 1.31 (m, 2H).

<Examples 1-1 to 1-4> Preparation of porous hydrogel

In a typical porous hydrogel preparation method, Alg-Nb (Preparation Example 1) solution was mixed with S-Tz (Preparation Example 3) solution at room temperature until a hydrogel was formed within a gelation time of several minutes, and the effect of crosslinking rate 4 different precursor ratios were prepared to examine A hydrogel was formed by mixing 2 w / v % of Alg-Nb and S-Tz in PBS buffer so that the ratio of functional group Nb / Tz was 10/10, 10/8, 10/6, and 10/4.

The appearance of the porous hydrogel formed by the reverse tube test prepared in Examples 1-1 to 1-4 is shown in FIG. 5 .

<Comparative Examples 1-1 to 1-2> Preparation of non-porous hydrogel

Using the same procedure as in Example, Alg-Fu (Preparation Example 2) and S-Ma (Preparation Example 4) crosslinking agent were mixed so that the Fu / Ma functional group ratio was 10/10 and 10/4 to form a non-porous hydrogel did

<Example 2> DOX loading on porous hydrogel

To load doxorubicin (DOX) on the porous hydrogel, 1 mg/mL of DOX dissolved in PBS was added to the Alg-Nb solution. Thereafter, S-Tz was mixed with the DOX/Alg-Nb solution to generate A1/DOX to A4/DOX hydrogels.

At this time, the Nb/Tz ratio was carried out in the same manner as in Examples 1-1 (A1) to 1-4 (A4).

<Comparative Example 2> DOX loading on non-porous hydrogel

To load doxorubicin (DOX) on the non-porous hydrogel, 1 mg/mL of DOX dissolved in PBS was added to the Alg-Fu solution. Then, S-Ma was mixed with the DOX/Alg-Fu solution to generate B1/DOX to B4/DOX hydrogels.

In this case, the Fu/Ma ratio was carried out in the same manner as in Comparative Examples 1-1 (B1) to 1-2 (B4).

<Experimental Example 1> Gelation time and hydrogel content (hydrogel content)

The gelation time was determined using a reverse tube test and a digital time meter, and the results are shown in Table 1 below.

The content of the hydrogel is, after freeze-drying the hydrogel, the dried hydrogel is washed with PBS to remove unreacted reactants. Then, the hydrogel was dried and weighed, and the results are shown in Table 1 below.

The hydrogel content is calculated by the following (1) formula.

Hydrogel content (%) = W ₁ /W ₀ Х 100 (1)

where W ₀ is the primary weight of the dry hydrogel and W ₁ is the dry weight of the hydrogel after swelling and washing.

In Table 1, 'Nb/Tz' represents the ratio of the number of norbornene functional groups/the number of tetrazine functional groups, and 'Fu/Ma' represents the ratio of the number of furan functional groups/the number of maleimide functional groups.

Hydrogel Feed ratio ^a Gelation time (min) ^b Hydrogel Content (%) ^c A1 (Example 1-1) 10/10 (Nb/Tz) 2.51 ± 0.36 96 A2 (Example 1-2) 10/8 (Nb/Tz) 3.59 ± 0.15 94 A3 (Example 1-3) 10/6 (Nb/Tz) 8.93 ± 0.25 94 A4 (Example 1-4) 10/4 (Nb/Tz) 15.72 ± 0.51 92 B1 (Comparative Example 1-1) 10/10 (Fu/Ma) 20.25 ± 0.72 95 B4 (Comparative Example 1-2) 10/4 (Fu/Ma) 32.18 ± 0.77 92

^a ionic equivalent ratio of click functional groups

^b Gelation time measured at 37°C

^c Measured by gravimetric method

<Experimental Example 2> Swelling ratio of the hydrogel

The swelling properties of the hydrogels were measured by a simple gravity method. The dried hydrogel discs are immersed in PBS (0.01 M) at pH 7.4 at physiological temperature. The swollen hydrogel is weighed at a given time, and the measurement is continued until no increase in the hydrogel weight is observed. The swelling ratio (SR) was calculated by the formula (2).

SR(100%) = (Ws - Wd)/Wd (2)

where Ws is the weight of the swollen hydrogel and Wd is the weight of the final dried hydrogel.

Swelling ratio test results are requested

<Experimental Example 3> Observation of the morphological microstructure of the hydrogel

The cross-section of the hydrogel was observed using SEM to investigate the internal morphological microstructure of the alginate hydrogel, and the results are shown in FIG. 6 .

Specifically, after the swelling state reaches the parallel condition, the hydrogel is freeze-dried in vacuum until all water is removed, the freeze-dried hydrogel is cut vertically, and the specimen is coated with gold and observed with an SEM instrument. did

SEM images of A1 (Example 1-1), A2 (Example 1-2), A3 (Example 1-3), and A4 (Example 1-4) porous hydrogels are shown in FIG. 6 ,

SEM images of the non-porous hydrogels B1 (Comparative Example 1-1) and B4 (Comparative Example 1-2) are shown in FIG. 7 .

As shown in FIGS. 6 and 7, the morphologies of A1 to A4 hydrogels produced by the reverse electron demand Diels-Alder reaction between norbornene and tetrazine functional groups were porous, and the higher the Nb/Tz ratio, the more A structure with small pores was formed. And the B1 and B4 hydrogels produced by the general Diels-Alder reaction between furan and maleimide functional groups showed a dense non-porous sheet structure.

<Experimental Example 4> Drug release (in vitro test)

For drug release studies, Example 2 (A1/DOX; A4/DOX) and Comparative Example 2 (B1/DOX; B4/DOX) were immersed in 15 mL of PBS (pH 7.4) and shaken at room temperature. At designated time intervals, 3 mL aliquots were extracted for UV-vis analysis at a wavelength of 485 nm. The concentration of released DOX was determined using a calibration curve. After every sampling of the 3 mL solution, it was replenished with 3 mL fresh PBS to maintain a constant volume. Each experiment was performed in triplicate.

8 is a graph of the drug release rate over time of the porous hydrogel (Example 2).

9 is a graph of the drug release rate over time of the non-porous hydrogel (Comparative Example 2).

As shown in FIGS. 8 and 9 , it was confirmed that the porous hydrogel of Example 2 (A1/DOX; A4/DOX) exhibited a uniform DOX release rate over time, whereas Comparative Example 2 (B1/DOX; B4 / DOX) of the non-porous hydrogel showed a rapid initial release rate within about 30 minutes, and then the DOX release rate was very low until 15 hours. could have been expected

So far, the present invention has been looked at with respect to preferred embodiments thereof. Those of ordinary skill 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 are to be considered in an illustrative rather than a restrictive sense. The scope of the present invention is particularly indicated in the claims rather than the foregoing description, and all differences within the scope equivalent thereto should be construed as being included in the present invention.

Claims (14)

an alginic acid derivative represented by the following formula (1); and
Including; a crosslinking agent comprising a bond represented by the following formula (2)
Substituent X of Formula 1 below and Substituent Y of Formula 2 are click-reactable with each other and are characterized in that they are a combination that generates _{N 2 gas during the click reaction,}
Characterized in that it has sustained-release drug release properties,
Porous hydrogels:
[Formula 1]

(In Formula 1,
m is an integer from 1-100,000;
L ¹ is an ester or amide bond;
X is a click-reactive functional group of one of a norbornenyl derivative, a trans-cyclooctenyl derivative and a tetrazinyl derivative,
The norbornenyl derivative is

or

ego,
The trans-cyclooctene is

or

ego,
The tetrazinyl derivative is

,

,

,

,

,

,

,

or

to be.)

[Formula 2]

(In Formula 2,
a, b, c and d are each an integer of 0-10, where 0 means a single bond;
Z is one of a disulfide bond, a diselenide bond, a PEG bond (polyethyleneglycol bond), and methylene;
L ² is an ester or amide bond;
Y is the same as the definition of X in Formula 1 above.)
According to claim 1,
When X in Formula 1 is a norbornenyl derivative or a trans-cyclooctenyl derivative, Y in Formula 2 is a tetrazinyl derivative,
When X in Formula 1 is a tetrazinyl derivative, Y in Formula 2 is a norbornenyl derivative or trans-cyclooctenyl derivative, characterized in that
Porous hydrogels.
According to claim 1,
The number of tetrazine (Tz) functional groups in the tetrazinyl derivative of X or Y in Formula 1,
The ratio of the number of norbornene (Nb) functional groups of the norborneneyl derivative or cyclooctene (Co) functional groups of the trans-cyclooctenyl derivative that click-react with the tetrazine functional group in X or Y;
Nb or Co: Tz is 1-20: 1-20, characterized in that the porous hydrogel.
According to claim 1,
The porous hydrogel may support a drug,
The drugs include insulin, anti-rheumatic agents, prednisolone 21-acetate, paclitaxel, doxorubicin, retinoic acid, cis-platin, camptothecin ), Fluorouracil (5-FU), Docetaxel, Tamoxifen, Anasterozole, Topotecan, Gleevec, Vincristine, Aspirin, Salali Sylates, ibuprofen, fenoprofen, indomethacin, phenyltazone, methotrexate, cyclophosphamide, dexamethasone ( dexamethasone), nimesulide, cortisone, corticosteroid, mother and child placenta extract, protein from animal placenta, α-lipoic acid, α-tocopherol, retinoy A porous hydrogel, characterized in that at least one of Retinoids and Glutathione.
According to claim 1,
The alginic acid derivative is a porous hydrogel, characterized in that it is one compound of the following Chemical Formulas 3 to 6.
[Formula 3]

(In Formula 3,
m is an integer from 1-100,000.)

[Formula 4]

(In Formula 4,
m is an integer from 1-100,000.)

[Formula 5]

(In Formula 5,
m is an integer from 1-100,000.)

[Formula 6]

(In Formula 6,
m is an integer from 1-100,000.)
According to claim 1,
The crosslinking agent is a porous hydrogel, characterized in that the compound of one of the following formulas 7 to 21.
[Formula 7]

[Formula 8]

[Formula 9]

[Formula 10]

[Formula 11]

[Formula 12]

(In Formula 12,
n is an integer of 1-20.)
[Formula 13]

(In the formula 13,
n is an integer of 1-20.)
[Formula 14]

(In Formula 14,
n is an integer of 1-20.)
[Formula 15]

(In Formula 15,
n is an integer of 1-20.)
[Formula 16]

(In Formula 16,
n is an integer of 1-20.)
[Formula 17]

[Formula 18]

[Formula 19]

[Formula 20]

[Formula 21]

and adding an alginic acid derivative represented by the following formula (1) and a crosslinking agent represented by the following formula (2) to a solvent and reacting (step 1); and
When X in Formula 1 is a norbornenyl derivative or a trans-cyclooctenyl derivative, Y in Formula 2 is a tetrazinyl derivative,
When X in Formula 1 is a tetrazinyl derivative, Y in Formula 2 is a norbornenyl derivative or trans-cyclooctenyl derivative,
A method for preparing a porous hydrogel.
[Formula 1]

(In Formula 1, the definition of each substituent is the same as defined in claim 1.)

[Formula 2]

(In Formula 2, the definition of each substituent is as defined in claim 1.)
8. The method of claim 7,
The solvent is phosphate buffer, dimethyl sulfoxide (DMSO), ethanol, anhydrous tetrahydrofuran (THF), benzene, KOH / MeOH, MeOH, toluene, CH ₂ Cl ₂ , hexane, dimethylformamide (DMF), diisopropyl A method for producing a porous hydrogel, characterized in that at least one selected from the group consisting of ether, diethyl ether, dioxane, dimethylacetamide (DMA), acetone and chlorobenzene.
adding and mixing the alginic acid derivative represented by the following Chemical Formula 1, and a drug in a solvent (step 1); and
A step of adding and reacting a crosslinking agent represented by the following formula (2) (step 2);
When X in Formula 1 is a norbornenyl derivative or a trans-cyclooctenyl derivative, Y in Formula 2 is a tetrazinyl derivative,
When X in Formula 1 is a tetrazinyl derivative, Y in Formula 2 is a norbornenyl derivative or trans-cyclooctenyl derivative,
A method for preparing a drug-supported porous hydrogel.
[Formula 1]

(In Formula 1, the definition of each substituent is the same as defined in claim 1.)

[Formula 2]

(In Formula 2, the definition of each substituent is as defined in claim 1.)
10. The method of claim 9,
The solvent is phosphate buffer, dimethyl sulfoxide (DMSO), ethanol, anhydrous tetrahydrofuran (THF), benzene, KOH / MeOH, MeOH, toluene, CH ₂ Cl ₂ , hexane, dimethylformamide (DMF), diisopropyl A method for producing a porous hydrogel, characterized in that at least one selected from the group consisting of ether, diethyl ether, dioxane, dimethylacetamide (DMA), acetone and chlorobenzene.
10. The method of claim 9,
The drugs include insulin, anti-rheumatic agents, prednisolone 21-acetate, paclitaxel, doxorubicin, retinoic acid, cis-platin, camptothecin ), Fluorouracil (5-FU), Docetaxel, Tamoxifen, Anasterozole, Topotecan, Gleevec, Vincristine, Aspirin, Salali Sylates, ibuprofen, fenoprofen, indomethacin, phenyltazone, methotrexate, cyclophosphamide, dexamethasone ( dexamethasone), nimesulide, cortisone, corticosteroid, mother and child placenta extract, protein from animal placenta, α-lipoic acid, α-tocopherol, retinoy To (Retinoids), and glutathione (Glutathione) characterized in that at least one of, a method for producing a drug-supported porous hydrogel.
A sustained-release drug delivery system comprising the porous hydrogel of claim 1.
The porous hydrogel of claim 1; and
A near-infrared dye (NIR dye) characterized in that it forms active oxygen species upon irradiation with near-infrared rays;
In the porous hydrogel of claim 1, Z in Formula 2 is a disulfide bond or a diselenide bond,
The near-infrared dye is 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 of
An external stimulus-sensitive drug delivery system using near-infrared rays.
14. The method of claim 13,
The external stimulus-sensitive drug delivery system using the near-infrared light is characterized in that a disulfide bond or a diselenide bond contained in the cross-linking agent is disintegrated in response to the near-infrared light, an external stimulus-sensitive drug using near-infrared rays carrier.

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