KR101974744B1 - Hydrogel including crosslinked hyaluronate graft-polymerized copolymer and method for preparing the same - Google Patents

Abstract

According to one embodiment of the present invention, a hyaluronic acid (hyaluronic acid) and an acrylate monomer (acrylate monomer) graft copolymerized (graft-polymerized copolymer); And a diacrylate cross-linker; a biocompatible hydrogel is provided.

Description

HYDROGEL INCLUDING CROSSLINKED HYALURONATE GRAFT-POLYMERIZED COPOLYMER AND METHOD FOR PREPARING THE SAME}

The present invention relates to a hydrogel comprising a crosslinked product of a hyaluronic acid graft copolymer and a preparation method thereof.

Hydrogel is a substance in which molecules interacting by chemical bonding or electrostatic attraction form a hydrophilic cross-linked polymer and absorb water corresponding to several hundred times its dry weight. Since such hydrogels exhibit excellent biocompatibility and hydrophilic properties, it is known that various applications are possible in pharmaceutical, tissue regeneration engineering, and medicine.

Hydrogels are also known as useful candidates for the preparation of tissue engineering substrates for the purpose of healing damaged human tissue. The three-dimensional structure formed by the hydrogel is also called a scaffold, and this support is used as various tissue engineering materials.

Among the materials that can be used as a material of such hydrogels, research is being conducted to apply hyaluronic acid. Hyaluronic acid is a component that exists naturally in living organisms and is distributed in various parts of the human body. Hyaluronic acid does not cause immunity problems and has excellent biocompatibility that can be used in tissue engineering and drug delivery systems, and thus may exhibit excellent efficacy as a component of hydrogel.

Despite the various advantages of hyaluronic acid, when used alone, the strength of the support skeleton is low due to insufficient viscoelastic properties or mechanical properties, and due to excessive hydrophilicity and cell adhesion ligand properties, cell adhesion, cell differentiation, etc. There is a disadvantage. Accordingly, a number of studies are underway to apply chemically modified hyaluronic acid.

However, to date, there are no results that can realize both the viscoelastic properties and the mechanical properties of the hydrogel containing hyaluronic acid.

The present invention is to solve the above-mentioned problems of the prior art, an object of the present invention is to provide a hydrogel comprising a hyaluronic acid graft polymer that can implement excellent viscoelastic properties, cell compatibility and mechanical properties and a method for preparing the same will be.

However, the problem to be solved by the present invention is not limited to the above-mentioned problem, another task that is not mentioned will be clearly understood by those skilled in the art from the following description.

According to one embodiment of the present invention, a hyaluronic acid (hyaluronic acid) and an acrylate monomer (acrylate monomer) graft copolymerized (graft-polymerized copolymer); And a diacrylate cross-linker; a biocompatible hydrogel is provided.

According to one side, the acrylate monomer may be hydroxyethyl acrylate (hydroxyethyl acrylate) or hydroxyethyl methacrylate (hydroxyethyl methacrylate).

According to one side, the diacrylate-based crosslinking agent may be poly (ethylene glycol) diacrylate (poly (ethylene glycol) diacrylate).

According to one side, the hydrogel may have a porous structure.

According to one side, the hydrogel may exhibit a pH-dependent drug release.

According to another embodiment of the present invention, (a) preparing a mixture of hyaluronic acid (hyaluronic acid) and an acrylate monomer (acrylate monomer); (b) adding a polymerization initiator to the mixture to prepare a graft-polymerized copolymer; And (c) adding a diacrylate cross-linker (diacrylate cross-linker) to the graft copolymer; provides a method for producing a biocompatible hydrogel comprising a.

According to one side, the acrylate monomer may be hydroxyethyl acrylate (HEA) or hydroxyethyl methacrylate (hydroxyethyl methacrylate).

According to one side, the polymerization initiator may be a radical polymerization initiator.

According to one side, the radical polymerization initiator may be potassium persulfate (KPS, Potassium persulfate).

According to one side, the diacrylate-based crosslinking agent may be poly (ethylene glycol) diacrylate (poly (ethylene glycol) diacrylate; PEGDA).

The hydrogel of the present invention can realize excellent viscoelastic properties and mechanical properties by using a crosslinked product of a graft copolymer in which hyaluronic acid and an acrylate monomer are graft polymerized.

Accordingly, the hydrogel can be applied to various fields such as tissue engineering scaffolds, injectable gels, 3D printing materials, drug carriers, and the like. In particular, the hydrogel may be made of a porous structure of a uniform shape may exhibit an excellent effect in controlling drug release and mechanical properties.

It is to be understood that the effects of the present invention are not limited to the above effects, and include all effects deduced from the configuration of the invention described in the detailed description or claims of the present invention.

1 illustrates a method for preparing a biocompatible hydrogel according to an embodiment of the present invention according to a reaction step.
Figure 2 shows the IR analysis of the biocompatible hydrogel according to an embodiment of the present invention.
Figure 3 shows the H ¹ NMR analysis of the biocompatible hydrogel according to an embodiment of the present invention.
Figure 4 shows an SEM image of the biocompatible hydrogel according to an embodiment of the present invention.
Figure 5 shows the results of TGA analysis of the biocompatible hydrogel according to an embodiment of the present invention.
Figure 6 shows the DMOG and TCN drug release behavior of the biocompatible hydrogel according to an embodiment of the present invention.
Figure 7 shows the swelling degree according to the pH conditions of the biocompatible hydrogel according to an embodiment of the present invention.
Figure 8 shows the rheology (Rheology) analysis of the biocompatible hydrogel according to an embodiment of the present invention.
9 to 12 show the cell suitability measurement results of the biocompatible hydrogel according to an embodiment of the present invention.

Hereinafter, exemplary embodiments will be described in detail with reference to the accompanying drawings. Like reference numerals in the drawings denote like elements.

Various modifications may be made to the embodiments described below. The examples described below are not intended to be limited to the embodiments and should be understood to include all modifications, equivalents, and substitutes for them.

The terminology used herein is for the purpose of describing particular example embodiments only and is not intended to be limiting of examples. Singular expressions include plural expressions unless the context clearly indicates otherwise. In this specification, terms such as "comprise" or "have" are intended to indicate that there is a feature, number, step, action, component, part, or combination thereof described on the specification, and one or more other features. It is to be understood that the present invention does not exclude the possibility of the presence or the addition of numbers, steps, operations, components, components, or a combination thereof.

Unless defined otherwise, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by one of ordinary skill in the art. Terms such as those defined in the commonly used dictionaries should be construed as having meanings consistent with the meanings in the context of the related art and shall not be construed in ideal or excessively formal meanings unless expressly defined in this application. Do not.

In addition, in the description with reference to the accompanying drawings, the same components regardless of reference numerals will be given the same reference numerals and redundant description thereof will be omitted. In the following description of the embodiment, when it is determined that the detailed description of the related known technology may unnecessarily obscure the gist of the embodiment, the detailed description thereof will be omitted.

According to one embodiment of the present invention, a hyaluronic acid (hyaluronic acid) and an acrylate monomer (acrylate monomer) graft copolymerized (graft-polymerized copolymer); And a diacrylate cross-linker; a biocompatible hydrogel is provided.

The hyaluronic acid itself exhibits excellent biocompatibility, but because of its weak viscoelastic properties and mechanical properties, such hyaluronic acid may be supplemented by using an acrylate-based monomer and a diacrylate-based crosslinking agent.

Specifically, the acrylate monomer may be hydroxyethyl acrylate (hydroxyethyl acrylate) or hydroxyethyl methacrylate (hydroxyethyl methacrylate), preferably 2-hydroxyethyl acrylate (2-HEA) However, the present invention is not limited thereto.

The hyaluronic acid and the acrylate monomer may form a graft copolymer. The graft copolymer can effectively improve the viscoelastic properties and mechanical properties of hyaluronic acid by forming a dense molecular structure compared to the linear copolymer.

In addition, the graft copolymer may be crosslinked by a diacrylate-based crosslinking agent containing two or more unsaturated functional groups. Specifically, the diacrylate crab crosslinking agent is poly (ethylene glycol) diacrylate, poly (ethylene glycol) tetraacrylate, poly (ethylene glycol) ) It may be a polyacrylate (poly (ethylene glycol) multiacrylate), but is not limited thereto.

The crosslinking agent is connected to form a more dense structure between the graft copolymers, through which the hydrogel including the crosslinked product of the graft copolymer as a final product may have a porous structure, the porous structure to the degree of crosslinking It can have a variety of forms. According to the degree of cross-linking (degree x-linking) and the porous structure, the hydrogel can be applied as a drug carrier, a tissue engineering support that can support the drug therein, in addition to the 3D printing material, injectable gel, etc. It can also be applied.

On the other hand, the hydrogel may carry a drug delivery function in vivo by supporting the drug therein. At this time, the hydrogel may exhibit a pH-dependent drug release.

Specifically, the hydrogel may exhibit sustained release drug release in an acidic condition having a low pH, while it may exhibit rapid release drug release in a basic condition having a high pH. That is, since the drug release properties can be adjusted differently according to the pH environment to which the hydrogel is applied, the hydrogel may be selected and applied according to the pH of the application site.

1 illustrates a method for preparing a biocompatible hydrogel according to an embodiment of the present invention according to a reaction step.

According to another embodiment of the present invention as shown in Figure 1, (a) preparing a mixture of hyaluronic acid (hyaluronic acid) and an acrylate monomer (acrylate monomer); (b) adding a polymerization initiator to the mixture to prepare a graft-polymerized copolymer; And (c) adding a diacrylate cross-linker (diacrylate cross-linker) to the graft copolymer; provides a method for producing a biocompatible hydrogel comprising a.

In the step (a), after preparing hyaluronic acid and an acrylate monomer which are monomers required for graft polymerization, these may be mixed. In this case, the hyaluronic acid may be used not only by itself, but also in the form of a salt, for example, sodium hyaluronate, but is not limited thereto.

The acrylate monomer may be hydroxyethyl acrylate (hydroxyethyl acrylate) or hydroxyethyl methacrylate (hydroxyethyl methacrylate), preferably 2-hydroxyethyl acrylate (2-HEA), but It is not limited.

In step (b), a graft polymerization reaction between the mixed monomers may be started by adding a polymerization initiator. At this time, the polymerization initiator may be a radical polymerization initiator so that the polymerization reaction can proceed to a radical reaction. Specifically, the radical polymerization initiator may be potassium persulfate (KPS, Potassium persulfate), but is not limited thereto. Any compound capable of initiating a radical polymerization reaction such as benzoyl peroxide may be used.

The polymerization initiator exhibits radical activity to generate hyaluronic acid radicals by separating protons of hyaluronic acid or hyaluronate. The generated hyaluronic acid radical may react with the acrylate monomer to perform a graft polymerization reaction between the hyaluronic acid and the acrylate monomer.

In step (c), a biocompatible hydrogel may be prepared by adding a diacrylate cross-linker to a graft copolymer between hyaluronic acid and an acrylate monomer produced by a graft polymerization reaction. .

In this case, the diacrylate-based crosslinking agent may be a poly (ethylene glycol) diacrylate containing two unsaturated functional groups in order to proceed with an appropriate crosslinking reaction, but is not limited thereto.

That is, the biocompatible hydrogel as a final product may be prepared in a form in which a graft copolymer between hyaluronic acid-acrylate monomers is crosslinked by a diacrylate crosslinking agent.

The biocompatible hydrogel may be made of a porous structure, and thus may function as a drug carrier for carrying a drug therein. In addition, the hydrogel may be applied as a 3D printing material, an injectable gel, a tissue engineering support, etc., because it has appropriate viscoelastic properties and mechanical properties, but is not limited thereto.

Hereinafter, the present invention will be described in more detail with reference to Examples. The following examples are described for the purpose of illustrating the present invention, but the scope of the present invention is not limited thereto.

Example

Hydrogels were synthesized at high temperature using 0.25 g of sodium hyaluronate and 2 ml of 2-hydroxyethyl acrylate (2-HEA) as monomers, and 0.0025 g of potassium persulfate (KPS) as a polymerization initiator. . At this time, in order to control the porous structure of the hydrogel, the polymerization reaction was carried out while controlling the poly (ethylene glycol) diacrylate (PEGDA) crosslinking agent to 0.25 ml, 0.50 ml, and 0.75 ml to form HA-p (2-HEA)-. PEG hydrogels were synthesized. Specific contents of the compound used are shown in Table 1 below.

division Temperature
(℃) Sodium hyaluronate
(g) KPS
(g) 2-HEA
(Ml) PEGDA
(Ml) Example 1 75 0.25 0.0025 2.0 0.25 Example 2 75 0.25 0.0025 2.0 0.50 Example 3 75 0.25 0.0025 2.0 0.75

Experimental Example  1: IR results analysis

In order to confirm the chemical structure of the hydrogel prepared according to Example 2, the spectrum was analyzed by using an ATR-FTIR spectrometer (Travel IR, Smiths Detection, USA), and the results of hyaluronic acid (HA) and 2-hydro It is shown in Figure 2 together with the ATR-FTIR spectra of oxyethyl acrylate (2-HEA), poly (ethylene glycol) diacrylate (PEGDA).

Referring to Figure 2, the hydrogel (d) of Example 2 is 3425 cm ^-1 (HA and 2-HEA), 2398 cm ^-1 (2-HEA), 2878 cm ^-1 (PEGDA) and 1723 cm ^-1 All of the peaks (PEGDA) in (PEGDA) can be confirmed that it is a compound containing both HA, 2-HEA and PEGDA.

Experimental Example  2: H ^One  NMR results analysis

In order to confirm the chemical structure of the hydrogel prepared according to Example 2, spectral analysis was performed using a H ¹ NMR spectrometer (DD2 700, Agilent Technologies-Korea, USA), and the result was hyaluronic acid (HA), It is shown in FIG. 3 together with the H ¹ NMR spectrum of 2-hydroxyethyl acrylate (2-HEA), poly (ethylene glycol) diacrylate (PEGDA).

Figure 3 shows the H ¹ NMR spectrum of HA, 2-HEA, PEGDA and the hydrogel prepared according to Example 2. Referring to FIG. 3, it can be seen that the hydrogel according to Example 2 is composed of a compound having a structure including all of HA, 2-HEA, and PEGDA.

Experimental Example  3: Hydrogel  Structure observation

In order to confirm the microstructure of each of the hydrogels prepared according to Examples 1 to 3, a scanning electron microscope (SEM, Joel, Korea) image of each hydrogel was observed and the results are shown in FIG. 4.

(A) and (b) of FIG. 4 show the surface and cross-sectional images of the hydrogel according to Example 1, respectively, and (c) and (d) show the surface and the cross-sectional images of the hydrogel according to Example 2, respectively. (E) and (f) show surface and cross-sectional images of the hydrogel according to Example 3, respectively.

Referring to Figure 4, each of the hydrogel has a porous structure, it can be seen that the structure can be adjusted according to the content of the crosslinking agent. When the concentration of the crosslinking agent was low, the porous structure was not uniform (a), and even when the concentration of the crosslinking agent was too high, an uneven porous structure was produced (c). Such a hydrogel having a porous structure may be applied to drug carriers, etc., suggesting that the release property of the drug may also be controlled by controlling the structure.

Experimental Example  4: TGA (thermos- gravimetric  analysis

In order to confirm the pyrolysis characteristics of the hydrogel prepared according to Example 2, TGA analysis was performed at a scan rate of 5 ° C./min using a thermogravimetric analyzer (DTG-60, Shimadzu, Japan) under a nitrogen atmosphere. The results are shown in Figure 5 together with the TGA analysis of hyaluronic acid (HA).

Referring to FIG. 5, the hydrogel of Example 2 is more firmly formed in the molecule due to crosslinking, so that pyrolysis occurs at a relatively high temperature compared to hyaluronic acid.

Experimental Example  5: drug release evaluation

In order to evaluate drug release properties of the hydrogels prepared according to Examples 1 to 3, 27.9 μM and 13.9 μM of DMOG (dimethyloxalyglycine) and TCN (tetracycline) drugs, respectively, 7 mm in height and 10 mm in diameter HA-p The (2-HEA) -PEG gel sample was placed in a 48 well polystyrene well and then induced to release the drug at 37 ° C. for 4 days in 100 ml buffer solution (pH 7.0 and 7.4), Drug amounts were analyzed using a UV-Vis spectrometer (BioMATE 3, Thermo Scientific, USA) and the results are shown in FIG. 6.

Experimental Example  6: swelling degree evaluation

In order to confirm the water properties of the hydrogels prepared according to Examples 1 to 3, swelling was evaluated. Depending on the swelling characteristics of the hydrogel may be used as an indirect indicator for the release properties of the material, for example, the drug contained therein may vary.

Pre-weighed 0.25 g of dried hydrogel was supported in 50 ml of diluted buffer solution (pH 2.5, 7.0 and 7.4) at 37 ° C. for 16 hours. After 1 hour, the hydrogel was removed from the medium and washed, and the hydrogel was weighed until the weight reached equilibrium. Swelling degree (%) was calculated according to the following equation (1), the results are shown in FIG.

[Equation 1]

Swelling degree (%) = [(weight of specimen after immersion-weight of specimen before immersion) / weight of specimen before immersion] × 100 (%)

Experimental Example  7: Rheology (Rheology) analysis

In order to confirm the rheological properties of the hydrogel prepared according to Example 2, shear storage modulus (G ′) and 37 ° C. under a Rotational rheometer (TA Instrument Ltd., DHR-1) were used. Shear loss modulus (G ") characterization was performed.

Measurement gap and specimen dimensions were measured by adding HA-p (2-HEA) -PEG hydrogel samples synthesized to 1.0 mm and 25 mm plates, respectively, to provide gel oscillation of the sample while providing torque oscillation. Analyzed. Minimum torque oscillation was set at 10 nM-m and shear rate was set at a range of 0.1-1,300 / sec. For stress sweep measurements, frequency and oscillation stress were set to 1 Hz and 1 to 1,000 Pa, respectively. The minimum and maximum torques used were 10 nN-m and 150 mN-m. In the case of frequency sweep measurement, the torque resolution and strain were 0.1 nN-m and 1%, respectively, and the frequency was set at 0.1 to 10 Hz. The analysis results are shown in FIG. 8.

Referring to FIG. 8, it can be seen that the shear storage modulus (G ′) and shear loss modulus (G ″) are increased in the frequency range (a), and the shear storage modulus (G ′) and shear loss modulus (G ″) are increased. ), The gel formation can be confirmed by overlapping (b), and the shear viscosity decreases according to the shear rate (c), thereby observing the properties of the hydrogel.

Experimental Example  8: Cell suitability measurement

The suitability for the cells of the hydrogels prepared according to Examples 1 to 3 was measured.

Cell culture was performed as follows. Mouse-derived osteoblastic cells (MC3T3) were cultured in 10% Fetal Bovine Serum (FBS) and 100unit / ㎖ penicillin-streptomycin alpha- MEM medium put in a polystyrene culture dish 100㎜ × 20㎜ containing, 5% CO _2, 37 In vitro cell culture was carried out in a ℃ incubator.

The live & dead assay was performed as follows. 6 × (600 μl PBS, 1.2 μl EthD-1, 0.3 μl Calcein AM) was added to a 10 ml conical tube, and mixed. After removing the medium from 6 samples, each of 10: 1, 2: 1, and 1:10 samples at pH 7.0 and pH 7.4, add 600 µl of each prepared live & dead solution to each well, and block the light. It was left to stand in a 5% CO ₂ , 37 ° C. incubator for 30 minutes. After 30 minutes, the live & dead solution was removed, washed once with PBS, and observed under a fluorescence microscope. All the lights were blocked and progressed during the live & dead assay.

Cell proliferation evaluation was performed using cell counting kit-8 (CCK-8) to observe the cell number and cell proliferation rate. 2 ml of CCK-8 solution was mixed with an alpha-MEM 18 ml medium solution containing 10% FBS to prepare 20 ml. 3 ml of each sample was added 1ml of cck mixed solution to each sample and then incubated for 2 hours in a 5% CO ₂ , 37 ℃ incubator. After 2 hours, 100 μl of sample was taken from each well and transferred to each well three times in a 96well plate. For each sample the absorbance of the solution was measured at 450 nm wavelength using a Microplate reader (Tecan).

The cell suitability measurement results are shown in FIGS. 9 to 12. 9 to 12, it can be seen that the hydrogels according to Examples 1 to 3 exhibit excellent biocompatibility.

Although the embodiments have been described by the limited embodiments and the drawings as described above, various modifications and variations are possible to those skilled in the art from the above description. For example, the techniques described may be performed in a different order than the described method, and / or the components described may be combined or combined in a different form than the described method, or replaced or substituted by other components or equivalents. Appropriate results can be achieved.

Therefore, other implementations, other embodiments, and equivalents to the claims are within the scope of the following claims.

Claims (10)

Graft copolymers in which hyaluronic acid and hydroxyethyl acrylate are graft polymerized; Poly (ethylene glycol) diacrylate; And a HA-p (2-HEA) -PEG hydrogel comprising a radical polymerization initiator,
The radical polymerization initiator generates a hyaluronic acid-O radical from the hyaluronic acid to generate the hyaluronic acid-O radical and hydroxyethyl acrylate. Graft copolymer)
The HA-p (2-HEA) -PEG hydrogel is characterized in that produced by the reaction of Figure 1 below, biocompatible hydrogel.

delete delete The method of claim 1,
The hydrogel has a porous structure, biocompatible hydrogel.
The method of claim 1,
The hydrogel has a pH-dependent drug release, biocompatible hydrogel.
(a) preparing a mixture of hyaluronic acid and hydroxyethyl acrylate;
(b) adding a radical polymerization initiator to the mixture to prepare a graft copolymerized copolymer; And
(c) adding poly (ethylene glycol) diacrylate to the graft copolymer; In the manufacturing method of the HA-p (2-HEA) -PEG hydrogel comprising,
In the step (b), the hyaluronic acid-O radical is generated from the hyaluronic acid to produce the hyaluronic acid-O radical and the hydroxyethyl acrylate ( hydroxyethyl acrylate) to form a graft copolymer,
The HA-p (2-HEA) -PEG hydrogel manufacturing method according to the reaction step of Figure 1 below, the method of producing a biocompatible hydrogel.

delete delete The method of claim 6,
The radical polymerization initiator is potassium persulfate (KPS, Potassium persulfate), a method for producing a biocompatible hydrogel.
delete

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