KR20220050278A - Hydrogel composition having alginate coupled methacrylate and manufacturing method of hydrogel - Google Patents

Abstract

The present invention relates to a hydrogel composition including methacrylate-bound alginate and a method for preparing a hydrogel. Particularly, the present invention relates to a hydrogel composition including alginate whose hydroxyl group is bound with methacrylate, and a method for preparing the hydrogel. In the hydrogel composition according to the present invention, the sodium counter ion of alginate is substituted with tetraalkylammonium having oleophilic property, thereby allowing reaction in an organic solvent. Therefore, it is possible to carry out various organic reactions with higher efficiency. In addition, since methacrylate is bound with the hydroxyl group of alginate, it is possible to maintain the function of alginate. Further, it is possible to control the mechanical properties of hydrogel according to a degree of substitution with methacrylate.

Description

Hydrogel composition and hydrogel manufacturing method comprising methacrylate-coupled alginate

The present invention relates to a hydrogel composition comprising alginate to which methacrylate is bound and a method for preparing a hydrogel, and more particularly, to a hydrogel composition comprising alginate in which methacrylate is bound to a hydroxyl group, and the hydrogel It relates to a manufacturing method.

Hydrogels made using natural polysaccharides have long been adopted for biomedical engineering applications such as drug delivery systems and tissue engineering scaffolds, primarily because they can form hydrogels through physical interactions without chemical reactions. For example, agarose hydrogels are readily developed by thermogelling of an agarose solution upon cooling, which induces extensive hydrogen bonding. Polysaccharides with charged functional groups can also develop into hydrogels by ionic crosslinking with polyvalent ions with opposite charges. For example, anionic alginate and carrageenan containing carboxylate and sulfonate groups, respectively, are ionic cross-linked to form a hydrogel with cations such as Ca ²⁺ , whereas chitosan having amine groups is formed with β-glycerophosphate and It can be crosslinked with the same anionic species.

Although these physically cross-linked polysaccharide hydrogels are highly efficient to fabricate and can maintain their natural chemical structure, it is very difficult to control their physicodynamic and biological properties to the desired extent and manner.

Most polysaccharides become highly viscous aqueous solutions, limiting the working concentration range. This inevitably results in hydrogels with a limited range of material properties. In addition, physically crosslinked hydrogels are generally reversible, so structural integrity and physical and mechanical properties can be severely disrupted under certain environmental conditions. For example, thermally-reversible agarose hydrogels dissolve at higher temperatures and ion-crosslinked alginate hydrogels also dissolve in the presence of chelators.

To overcome these challenges, chemical crosslinking strategies are often used to further control material properties and manufacturing and processing. For example, chitosan having an amine group may react with glutaraldehyde through Schiff base formation or with geninpin through nucleophilic substitution to form a hydrogel. Alternatively, polysaccharides are often chemically modified to provide reactive functional groups such as aldehydes, catechols, thiols and (meth)acrylates that are reactive sites for crosslinking reactions. Although this approach has been successfully implemented for hydrogel development, diversifying the conjugation chemistry and introducing multiple chemical reactions to polysaccharides is still a challenge because the solubility of polysaccharides in organic solvents is severely limited.

Republic of Korea Patent Registration No. 10-1360942

Strategies that accommodate different chemical reaction pathways for polysaccharides have been used herein to present different functional moieties. Using the widely used sodium alginate as a model polysaccharide, the sodium alginate counterion was first exchanged with a tetrabutylammonium (TBA) ion to increase hydrophobicity and thereby promote dissolution in polar aprotic solvents such as dimethylsulfoxide. . This made it possible to apply a wider range of chemical reactions to alginate.

Moreover, since alginate has two different functional groups, a hydroxyl group and a carboxyl group, these two different functional groups can be independently conjugated to the alginate using different reaction schemes.

To demonstrate this independent control of multiple conjugation, a methacrylate capable of radical polymerization to form a hydrogel was conjugated to TBA-alginate via ring-opening nucleophilic addition to a hydroxyl group. In contrast, RGD peptides and cell adhesion molecules (CAM) such as gelatin are conjugated to carboxyl groups by amide coupling (Fig. 1a). TBA-alginate was exchanged back to the original sodium alginate.

The resulting CAM-linked methacrylic alginate was used as a crosslinking agent to independently impart cell adhesion properties while creating hydrogels with various mechanical properties controlled by the concentration and degree of methacrylate substitution. (Fig. 1b).

According to an embodiment of the present invention, there is provided a hydrogel composition comprising alginate in which methacrylate is bonded to a hydroxyl group. More preferably, the methacrylate may be bonded to the hydroxyl group of carbon 2 of the alginate.

The alginate may be a compound represented by the following [Formula 1];

[Formula 1]

Here, X is tetraalkylammonium or Na, and m is an integer ≥ 1.

In addition, the alginate may be a compound represented by the following [Formula 2] or [Formula 3];

[Formula 2]

where X is tetraalkylammonium or Na, and n and m are integers ≥ 1,

[Formula 3]

where X is tetraalkylammonium or Na, n and m are integers ≥ 1, and R ₁ is CONH- (cell binding molecule).

The tetraalkylammonium has the formula [NR ₁ R ₂ R ₃ R ₄ ] ⁺ (wherein R ₁ , R ₂ , R ₃ and R ₄ may be the same or different from each other, straight-chain C ₁ -C ₆ alkyl, branched-chain C ₁ -C ₆ alkyl, substituted C ₆ -C ₁₀ aryl, unsubstituted C ₆ -C ₁₀ aryl, and combinations thereof)), and more preferably tetrabutylammonium.

The percentage of m with respect to the sum of n and m in [Formula 3] is preferably 1 to 50%. The percentage of m to the sum of n and m is referred to herein as the degree of methacrylate substitution (DS _MA ).

The cell binding molecule is RGD (Arg-Gly-Asp), RGDS (Arg-Gly-Asp-Ser), RGDC (Arg-Gly-Asp-Cys), RGDV (Arg-Gly-Asp-Val), RGES (Arg) -Gly-Glu-Ser), RGDSPASSKP (Arg-Gly-Asp-Ser-Pro-Ala-Ser-Ser-Lys-Pro), GRGDS (Gly-Arg-Gly-Asp-Ser), GRADSP (Gly-Arg- Ala-Asp-Ser-Pro), KGDS (Lys-Gly-Asp-Ser), GRGDSP (Gly-Arg-Gly-Asp-Ser-Pro), GRGDTP (Gly-Arg-Gly-Asp-Thr-Pro), GRGES (Gly-Arg-Gly-Glu-Ser), GRGDSPC (Gly-Arg-Gly-Asp-Ser-Pro-Cys), GRGESP (Gly-Arg-Gly-Glu-Ser-Pro), SDGR (Ser-Asp) -Gly-Arg), YRGDS (Tyr-Arg-Gly-Asp-Ser), GQQHHLGGAKQAGDV (Gly-Gln-Gln-His-His-Leu-Gly-Gly-Ala-Lys-Gln-Ala-Gly-Asp-Val ), GPR (Gly-Pro-Arg), GHK (Gly-His-Lys), YIGSR (Tyr-Ile-Gly-Ser-Arg), PDSGR (Pro-Asp-Ser-Gly-Arg), CDPGYIGSR (Cys- Asp-Pro-Gly-Tyr-Ile-Gly-Ser-Arg), LCFR (Leu-Cys-Phe-Arg), EIL (Glu-Ile-Leu), EILDV (Glu-Ile-Leu-Asp-Val), EILDVPST (Glu-Ile-Leu-Asp-Val-Pro-Ser-Thr), EILEVPST (Glu-Ile-Leu-Glu-Val-Pro-Ser-Thr), LDV (Leu-Asp-Val) and LDVPS (Leu) -Asp-Val-Pro-Ser) may be at least one peptide selected from the group consisting of, but is not limited thereto.

In another example of the cell-binding molecule, the cell-binding molecule is gelatin, collagen, fibronectin, gelatin, laminin, vitronectin, polycaprolactone (PCL), polyethylene oxide (PEO), polyvinyl alcohol (PVA), and polyvinylpyrroly. It may be one or more biocompatible polymers selected from the group consisting of money (PVP), but is not limited thereto.

The hydrogel composition may further include an acrylic monomer and a photoinitiator.

The acrylic monomer is sodium acrylate, sodium methacrylate, acrylamide, C ₁ -C ₁₅ saturated alkyl acrylate or methacrylate, C ₁ - C ₁₅ hydroxyalkyl acrylate or methacrylate; And N,N-di (C ₁ -C ₁₅ saturated or unsaturated alkyl) may be at least one selected from the group consisting of acrylamide, more specifically methacrylate gelatin (methacrylate gelatin), polyethylene glycol ethyl methacrylate ( polyethylene glycol ethyl methacrylate, PEGMA), ethylene glycol ethyl acrylate (PEGA), polyethylene glycol ethyl diacrylate (PEGDA), polyethylene glycol ethyl dimethacrylate, PEGDMA), but is not limited thereto.

The photoinitiator is a water-soluble photoinitiator, 2-hydroxy-4′-(2-hydroxyethoxy)-2-methylpropiophenone), 1-hydroxy-cyclohexyl-phenyl-ketone, 2,2-Dimethoxy-1,2-diphenylethan-1-one, 2-benzyl-2-dimethylamino-1-(4-morpholinophenyl)-1-butanone, 2-methyl-4′-(methylthio)-2-morpholinopropiophenone, TPO-Na (sodium phenyl-2,4,6-trimethylbenzoylphosphinate) , TPO-Li (lithium phenyl-2,4,6-trimethylbenzoylphosphinate), BAPO-ONa (sodium bis(mesitoyl)phosphinate), BAPO-OLi (lithium bis(mesitoyl)phosphinate) may be at least one selected from the group consisting of , but not limited thereto.

Since most photoinitiators are fat-soluble, they cannot be used in hydrogels that must be prepared in aqueous solutions. Therefore, a specially formulated water-soluble photoinitiator must be used. Among Ciba's Irgacure family, Irgacure 2959 (2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone) has the highest solubility and biocompatibility, so it is the most used. In addition, Irgacure 184 (1-hydroxy-cyclohexyl-phenyl-ketone), Irgacure 651 (2,2-Dimethoxy-1,2-diphenylethan-1-one), Irgacure 369 (2-benzyl-2-dimethylamino-1-( 4-morpholinophenyl)-1-butanone), Irgacure 907 (2-methyl-4′-(methylthio)-2-morpholinopropiophenone) may be used.

In addition, compounds including MAPO (mono-acylphosphine oxide) or BAPO (bi-sacylphosphine oxide) are frequently used among phosphines.

Among MAPO, the photoinitiator that has passed water solubility is TPO-Na (sodium phenyl-2,4,6-trimethylbenzoylphosphinate), which is a salt of TPO (diphenyl(2,4,6-trimethylbenzoyl)phosphine oxide), TPO-Li (lithium phenyl -2,4,6-trimethylbenzoylphosphinate (LAP) is mainly used. Among BAPOs, BAPO-ONa (sodium bis(mesitoyl)phosphinate) and BAPO-OLi (lithium bis(mesitoyl)phosphinate) may be used similarly.

According to another embodiment of the present invention, there is provided a hydrogel prepared from the hydrogel composition.

In the hydrogel, as the number of uronic acid residues combined with methacrylate increases, the elastic modulus or cell proliferation rate of the hydrogel may increase. Here, the number of uronic acid residues combined with methacrylate has the same meaning as the degree of methacrylate substitution (DS _MA ).

In addition, in the hydrogel, the elastic modulus of the hydrogel may increase as the concentration of the acrylic monomer increases.

The hydrogel may be used for cell culture, differentiation or proliferation.

The cells are adhesion-dependent cells, and may be selected from the group consisting of cardiomyocytes, vascular endothelial cells, adipocytes, epithelial cells, fibroblasts, osteoblasts, chondrocytes, hepatocytes, cervical cells, cancer cells and mesenchymal stem cells. It is not limited thereto.

According to another embodiment of the present invention, 1) through the cation exchange of sodium alginate (sodium alginate) to produce a tetraalkylammonium alginate; And 2) provides a method for producing a hydrogel comprising the step of bonding methacrylate to the hydroxyl group of the alginate.

The tetraalkylammonium has the formula [NR ₁ R ₂ R ₃ R ₄ ] ⁺ (wherein R ₁ , R ₂ , R ₃ and R ₄ may be the same or different from each other, straight-chain C ₁ -C ₆ alkyl, branched C selected from the group consisting of ₁ -C ₆ alkyl, substituted C ₆ -C ₁₀ aryl, unsubstituted C ₆ -C ₁₀ aryl, and combinations thereof), and more preferably tetrabutylammonium.

Step 2) may be performed through a ring-opening nucleophilic addition reaction of tetraalkylammonium alginate and glycidyl methacrylate in an organic solvent, wherein the organic solvent is dimethyl sulfoxide, DMSO), dimethylformamide (DMF), dimethylacetamide, N-methyl-2-pyrrolidone (NMP), and hexamethylphosphoramide (HMPA) ) may be one or more selected from the group consisting of, but is not limited thereto.

The preparation method may further include, after step 2), 3) cation exchange of tetraalkylammonium with Na ⁺ to synthesize methacrylic alginate.

In addition, the preparation method may further include, after step 1), 1-1) coupling a cell-binding molecule to the carboxyl group of alginate through carbodiimide coupling.

In addition, the manufacturing method may further include, after step 3), 4) mixing methacrylic alginate, an acrylic monomer and a photoinitiator, and 5) photocrosslinking by irradiating UV light.

The methacrylic alginate may be added in an amount of 0.1 to 10 wt% based on the total weight of the acrylic monomer, and the concentration of the acrylic monomer is 5 to 50% (w/v), preferably 20 to 40%, most preferably 30 It can be ~ 40%.

The hydrogel composition according to the present invention can be reacted in an organic solvent by replacing the sodium counterion of alginate with tetraalkylammonium having lipophilicity, so that various organic reactions with higher efficacy are possible, as well as methacrylate alginate By bonding with the hydroxyl group of can maintain the unique function of alginate, there is an advantage that can control the mechanical properties of the hydrogel according to the degree of substitution of the methacrylate.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a schematic representation of the synthetic steps of (a) methacryl alginate (MAlg) and (b) the synthesis of cell-adhesive molecule (CAM) conjugated methacryl alginate (MAlg).
2 is a view showing ¹ H-NMR spectrum of Malg according to the change in the degree of methacrylate substitution.
3 is a view showing (a) representative ¹ H-NMR spectra of MAlg, RGD-MAlg and gel-MAlg, and (b) ¹ H-NMR spectrum of RGD-MAlg in various DS _MAs .
4 is a graph showing the mechanical properties of the hydrogel by DS _MA of MAlg; Elastic modulus ( E ) of (b, c) PAAm-MAlg hydrogels and (d, e) PEGMA-MAlg hydrogels in various DS _MAs ; (b, d) 10%, (c, e) 20% PAAm.
Figure 5 is a graph showing the elastic modulus ( E ) of the MGel-MAlg hydrogel in various DS _MA ; (a) 6%, (b) 8%, and (c) 10% MGel.
Figure 6 is a diagram showing the elastic modulus ( E ) of (a) PAAm-MAlg hydrogel and (b) PEGMA-MAlg hydrogel containing different counter ions; TBA ⁺ and Na ⁺ .
7 is a diagram showing the elastic modulus ( E ) of PAAm hydrogels crosslinked with (a, b) RGD-MAlg or (c, d) gel-MAlg; The concentrations of PAAm are (a, c) 10% and (b, d) 20%.
Fig. 8 (a) shows optical and fluorescence microscopy images (scale bar: 100 um) of fibroblasts encapsulated in MGel-MAlg with various DS _MA cells, cells to indicate live (green) and dead (red) cells. fluorescently labeled. (b) A graph showing cell viability at various times during culture up to 7 days. (c) Normalized viable cell counts ( N _t / N ₀ ) were measured over time. (d) It is a graph showing the growth rate ( k _P ) obtained by fitting the graph of (c) with Eq.(1)(*p<0.05).
9 is a view showing cell culture images on the surface of PEGMA hydrogel cross-linked with DS _MA of various RGD-MAlg for up to 5 days for the cell adhesion properties of RGD-MAlg.

The terms used in the present application are only used to describe specific embodiments, and are not intended to limit the present invention. The singular expression includes the plural expression unless the context clearly dictates otherwise. In the present application, terms such as “comprise” or “have” are intended to designate that a feature, number, step, operation, component, part, or a combination thereof described in the specification exists, but one or more other features It is to be understood that it does not preclude the possibility of the presence or addition of numbers, steps, operations, components, parts, or combinations 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 to which this invention belongs. Terms such as those defined in a commonly used dictionary should be interpreted as having a meaning consistent with the meaning in the context of the related art, and should not be interpreted in an ideal or excessively formal meaning unless explicitly defined in the present application. does not

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention in more detail, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .

<Example>

1. Synthesis of tetrabutylammonium (TBA) alginate

TBA alginate was produced from sodium alginate via cation exchange. Sodium alginate was first added to a mixture of ethanol/1M HCl (1:1) and stirred at 4° C. overnight to produce alginic acid. Alginic acid was dispersed in deionized water (DI), and tetrabutylammonium hydroxide (40% in water) was slowly added until the alginate was completely dissolved. After adjusting the pH to 8, the solution was extensively dialyzed against DI water and freeze-dried to obtain TBA-alginate.

2. Synthesis of methacrylic alginate (MAlg)

TBA-alginate (0.15 g) developed from sodium alginate through the cation exchange was mixed with 5% tetrabutylammonium fluoride (TBAF) and 1% 4-dimethylaminopyridine (DMAP, 4-dimethylaminopyridine) It was dissolved in the added 10 mL dimethyl sulfoxide (DMSO, dimethyl sulfoxide). Glycidyl methacrylate was slowly added to the solution and reacted at 40° C. for 48 hours under dry N ₂ gas. The crude product obtained by precipitation in an excess of diethyl ether was dissolved in deionized water (DI) and 1N Na ₂ CO ₃ solution was continuously added until the pH increased to 11 or higher to convert TBA ⁺ to Na ⁺ exchanged. The solution was extensively dialyzed against deionized water and freeze-dried to obtain the final product. The amount of glycidyl methacrylate was varied to control the degree of methacrylate substitution (DS _MA ), defined as the percentage of uronic acid residues bound to the methacrylate; 25 ('DS1'), 51 ('DS2'), 102 ('DS3'), 205 ('DS4'), 409 ('DS5') and 767 ('DS6') mg per 0.15 g TBA-alginate. Chemical structure and DS were analyzed by ¹ H-NMR spectroscopy (400-MR DD2, Agilent).

3. Synthesis of Cell Adhesion Molecule (CAM)-Conjugated MAlg

Cell adhesion molecules, which are gelatin or GRGDS ('RGD') peptides, were conjugated to alginate prior to conjugation of methacrylate to alginate. First, TBA-alginate (0.15 g) was dissolved in 10 mL DMSO/TBAF. 0.04 g diisopropylcarbodiimide, 0.03 g N-hydroxysuccinimide and 0.3 g gelatin (or 0.053 g GRGDS peptide) were added and reacted at room temperature for 24 hours. Methacrylate conjugation to alginate was followed immediately in situ using the procedure described above. Briefly, 0.1 g of DMAP and glycidyl methacrylate are added to the mixture and reacted at 40° C. under dry N ₂ gas for 48 hours. Four different DS _MAs were prepared by varying the amount of glycidylmethactylate; 409 ('DS1'), 614 ('DS2'), 767 ('DS3') and 1278 ('DS4') per 0.15 g TBA-alginate, respectively, 8, 12, 15 and 24 molar equivalents of TBA-uronic acid residues pertains to To prevent larger gelatin molecules from interfering with methacrylate conjugation and subsequent hydrogel formation, the gelatin used here is less than 1000 g mol ⁻¹ in commercially available high molecular weight 300 bloom gelatin using enzymatic hydrolysis with trypsin. was cut short with GRGDS peptides were obtained using standard solid phase peptide synthesis.

4. Fabrication and Characterization of Malg-Linked Hydrogels

A gel precursor solution was prepared containing the monomer, MAlg (or CAM-MAlg) and 0.2% Irgacure ^® 2959 as a photoinitiator. Three different monomers were used; acrylamide (AAm), poly(ethylene glycol) methacrylate (PEGMA) and methacrylic gelatin (MGel). For AAm or PEGMA, MAlg concentrations were varied from 0.1 to 1% and 1-4% for 10% and 20% AAm or PEGMA, respectively. For MGel, MAlg concentrations were varied from 0.1 to 1% for 6%, 8% and 10% MGel. The solution was placed between two glass plates (1 mm thick spacer) and photocrosslinked with UV (2 min, 800 mW cm ^-1 , Model S1500, Omnicure) to prepare a hydrogel. Hydrogel discs (5 mm in diameter) were punctured and soaked in PBS (pH 7.4) for 24 h until further characterization.

Uniaxial compression experiments were performed on the hydrogels for mechanical characterization. Briefly, the hydrogel disc was uniaxially compressed at a rate of 1 mm min ⁻¹ and a strain versus stress curve was obtained (Model 3343, Instron). The modulus of elasticity was determined as the slope (first 10%) of the straight region of the curve. The swelling ratio was measured as the weight ratio of the fully swollen hydrogel to the dried polymer mesh.

5. Characterization

To test the cell adhesion properties of CAM-Malg-linked hydrogels, each hydrogel disc was placed in a well of a 24-well plate and 50 μL cell solution was placed on top of the hydrogel. The cell solution contained NIH3T3 fibroblasts (ATCC) dispersed in cell culture medium, Dulbecco's Modified Eagle medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin at a density of 1 x ¹⁰ cells mL ^-1 . . After 1 hour for cell attachment, the hydrogel was washed with PBS and cultured in a cell culture medium at 37° C. under 5% CO ₂ . Cell adhesion and subsequent proliferation over time were monitored with an inverted microscope (XDS-3FL, Optika).

For 3D cell encapsulation, cells were dispersed at 1 x 10 ⁶ cells mL ^-1 in MGel-MAlg gel precursor solution, placed between two glass plates (0.4 mm thick spacer) and photocrosslinked with UV for 1 min. Hydrogel discs (diameter 5 mm) were punched and cultured in cell culture medium under 5% 37° C. CO ₂ conditions. To determine cell viability, cells were treated with calcein-AM and ethidium homodimer-1 to represent live (green fluorescence) and dead (red fluorescence) cells (LIVE/DEAD Cell Viability Assay, Thermo Fisher), respectively. Viability was reported as a percentage of viable cells at a given time.

Proliferation rate ( k _P ) of encapsulated cells was obtained by fitting a plot of fractional number of viable cells ( N _t / N ₀ ) versus time ( t ) using the following power-law equation was calculated

(1)

(One)

where N _t is the number of living cells at time and N ₀ is the number of first living cells measured immediately after gelation ( t = 0).

<Experiment result>

1. Synthesis and Characterization of Methacrylalginate (MAlg) and Cell Adhesion Molecule (CAM)-Conjugated MAlg

Alginate is one of the most widely used natural polymers for drug delivery and biomedical applications such as tissue engineering, food and pharmaceutical additives. However, since it is an extremely hydrophilic anionic polysaccharide that dissolves only in aqueous buffer conditions, the scope of chemical modification is extremely limited. To impart multifunctionality to alginate, alginate was dissolved in an organic solvent by replacing the sodium counter ion (Na-alginate) with a tetramethylammonium counter ion (TBA-alginate) known to be lipophilic. As a result, TBA-alginate was easily dissolved in a polar aprotic solvent such as dimethyl sulfoxide (DMSO).

To utilize as a crosslinking agent for hydrogel preparation, alginate was first conjugated with methacrylate via a ring-opening nucleophilic reaction of glycidyl methacrylate with the hydroxyl group of alginate in DMSO (Fig. 1a). In addition, the crosslinking density was effectively controlled without changing the concentration by changing the degree of methacrylate substitution (DS _MA ).

Previous studies on alginate modification have involved the use of primarily 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) coupling for amide bond formation with the carboxyl group of alginate. However, aqueous-based EDC coupling is generally not efficient enough to control a wide range of degrees of substitution.

In addition, since carboxylates are important properties of alginates and play an important role in ionic interactions, removal of a significant portion of the carboxyl groups through chemical reactions may alter intrinsic alginate functions (e.g. water absorption and ionic crosslinking). . On the other hand, conjugation of methacrylate to the hydroxyl groups of alginate can accommodate a greater degree of substitution because there are more hydroxyl groups than carboxylate groups.

Various amounts of glycidyl methacrylate were reacted with TBA-alginate. The DS _MA result of MAlg(TBA ⁺ ) was confirmed by ¹ H-NMR spectrum. Six different DS _MAs have been developed; 3, 6, 10, 15, 18 and 22%, which are denoted by DS1 to DS6. TBA ⁺ of MAlg was eventually exchanged back to the original Na ⁺ , and removal of TBA ⁺ and residual DMAP were also confirmed by ¹ H-NMR spectrum ( FIG. 2 ).

Two different reaction pathways can be applied to alginates because they have two different types of functional groups: hydroxyl and carboxylate.

To demonstrate the versatility of alginates modified with various functional moieties, cell adhesion molecules (CAM) were conjugated to the carboxylate groups of alginate via carbodiimide coupling, and then generated using the aforementioned methacrylates. The hydrogel (CAM-MAlg) was given a crosslinking function as well as cell adhesion properties. Two different CAMs, GRGDS peptides (RGD-MAlg) and gelatin (gel-MAlg), which are widely used in biomedical applications, are used herein. The presence of CAM and methacrylate in CAM-MAlg was confirmed by ¹ H-NMR spectrum (Fig. 3a). To minimize the change in the properties of sodium alginate, only a portion of the carboxylate was replaced with CAM (3%).

DS _MA control of CAM-MAlg was also confirmed by ¹ H-NMR (Fig. 3b). DS _MA of RGD-MAlg was 6, 11, 16, and 21%, which was lower than that of MAlg under the same reaction conditions. On the other hand, DSMA of gel-MAlg was 24, 28, 37, and 72%, similar to MAlg under the same reaction conditions. This discrepancy is probably because the longer gelatin polypeptide provides an additional reaction site for methacrylate conjugation, effectively increasing conjugation. These results show that methacrylate conjugation to alginate can proceed to varying degrees even in the presence of CAM, demonstrating the convenience of the one-pot dual synthesis method.

2. Mechanical properties of MAlg-linked hydrogels

(1) Effect of monomer type

To evaluate the crosslinking efficacy of MAlg, three types of hydrogels were developed using MAlg with various DS _MAs . polyacrylamide (PAAm), poly(ethylene glycol) methacrylate (PEGMA) and methacrylic gelatin (MGel) (Figure 4a). These hydrogels are made using various types of monomer systems; Monomer (PAAm), macromonomer (PEGMA), multifunctional macromonomer (MGel). The present inventors hypothesized that the mechanical properties of the hydrogel change by varying the degree of interaction between MAlgs due to differences in physical properties. In addition, these mechanical properties of the hydrogel via DS _MA could be controlled even in the presence of CAM.

For the PAAm-MAlg hydrogel, the concentration of acrylamide was maintained at 10% or 20%. At 10%, the MAlg concentration was varied from 0.5 to 2%, and at 20%, the MGel concentration was varied from 1 to 4% while keeping the mass ratio of monomer to cross-linking agent constant. As expected, the modulus of elasticity of the PAAm-MAlg hydrogel increased with both MGel concentration and DS _MA ( FIGS. 4b and 4c ). The modulus at 10% acrylamide was controlled from 5 (± 0.3) to 40.4 (± 2.1) kPa with 2% MAlg using various DS _MAs . Similarly, the modulus at 20% acrylamide varied widely from 20 (± 0.5) to 163 (± 2.3) kPa with 4% MAlg with various DS _MAs . Similar control of the modulus of elasticity using DS _MA was demonstrated at low MAlg concentrations, but to a lesser extent. The trend of the swelling ratio was opposite to that of the modulus of elasticity, a well-known property of elastic networks. This is because the increased crosslink density increases the mechanical stiffness but reduces the molecular weight between the crosslinks, thereby reducing the expansion properties. These results demonstrated that MAlg is very effective in controlling the crosslinking density of PAAm to form hydrogels with various mechanical properties.

The same experiment was performed with PEGMA, a macromonomer, to generate a hydrogel. The modulus of elasticity could be similarly controlled by DS _MA of MAlg, unlike PAAm, but the mechanical properties were generally much lower, and the swelling ratio was much higher at the same concentration for the PEGMA-MAlg hydrogel (Figures 4d and 4e). . In addition, no hydrogels were formed at the low DS _MA of MAlg. For example, in 10% PEGMA, for 0.5%, 1% and 2% MAlg, respectively, the lowest DS _MAs for hydrogel formation were DS2, DS2 and DS4, respectively. This result showed that the crosslinking efficiency of MAlg for PEGMA was significantly lower than that for PAAm, probably due to the steric hindrance of the PEG chain preventing sufficient interaction with MAlg for the crosslinking reaction. Moreover, the modulus of elasticity at 20% PEGMA and 4% MAlg was significantly reduced compared to that at the low MAlg concentration and hydrogels were formed only in DS2 and DS3 and not in the lower (DS1) and higher (DS4-DS6) DS _MAs . . This may be due to the increased phase separation between MAlg and PEGMA at significantly higher concentrations.

While both PAAm and PEGMA increased the DS _MA of MAlg, there was a significant deviation in the overall trend of the count, and a sudden decrease in the count was observed in DS5 (indicated by # in Figures 4c and 4e). Even at low 10% PAAm and PEGMA, the degree of coefficient increase decreased as DS _MA increased. This anomalous behavior may be due to the significant phase separation and insufficient crosslinking reaction between MAlg and monomers due to the significant increase in hydrophobicity causing chain disruption between MAlg molecules with DS _MA . A further increase in DS _MA to DS6 can overcome phase separation in the form of more extended chains via a coil-to-rod transition from DS5 to DS6 with a more densely grafted polymer architecture. As a result, the number of crosslinking sites available for a broader crosslinking reaction is significantly increased.

For MGel hydrogels crosslinked with MAlg with various DS _MAs , the MGel concentration was varied from 6 to 10% and the concentration of MAlg was varied from 0.1 to 1%. Because of the high viscosity of MGel, the MGel concentration range is lower than that of PAAm and PEGMA. At 6% MGel, counts increased all the way to DS4 with DS _MA (Fig. 5a). Between 0.1% and 0.5%, the coefficients were clearly higher at 0.5%, but further increasing the concentration to 1% resulted in lower coefficients than at 0.5%, especially when the DS _MA was high. Because the relative concentrations of MAlg versus MGel (1–6) were higher than those of PAAm and PEGMA (1–10), they were more likely to undergo phase separation, particularly at the higher DS _MA , resulting in insufficient crosslinking. In addition, by having a much higher MW and more dynamic molecular structure than PEG, MGel was more susceptible to insufficient physical interaction with MAlg. Similarly, the counts decreased substantially at the 1% MAlg concentration in the higher DS _MA , although overall counts increased at higher MGel concentrations (Figures 5b and 5c).

MGel hydrogels at 6% and 8% MGel also showed a sudden decrease in counts at high DS _MA (DS4 or DS5) before increasing again at higher DS6 (indicated by # in FIG. 5 ). This is similar to the PAAm and PEGMA hydrogels shown in Figures 4c and 4e, but to a much greater extent. At 10% MGel, the coefficient continued to decrease after DS4 rather than increase at higher DS _MA , especially at higher MAlg concentrations. Also, the change of coefficient using DS _MA was much smaller. This indicates that the increased hydrophobicity with DS _MA interferes with the favorable interaction between MGel and MAlg. Taken together, larger multifunctional macromonomers such as MGel are more likely to be affected by the physical properties of the polymer crosslinking agent (i.e., concentration and DS _MA ) compared to the smaller monomers, which significantly enhances the mechanical properties of the resulting hydrogels. change

(2) the effect of the counterion of MAlg

Na-alginate is a generally accepted form of alginate for a variety of commercial uses, including biomedical products. Thus, the TBA-alginate used in the chemical modification to produce MAlg was ultimately converted back to Na-MAlg. Nevertheless, numerous previous studies have investigated the effects of various counter-ions of alginate on the physicodynamic properties of alginate, including ionic cross-linking. Therefore, the effects of different counterions of MAlg, especially TBA ⁺ and Na ⁺ , on the mechanical properties of the resulting hydrogels were investigated. PAAm-MAlg hydrogels and PEGMA-MAlg hydrogels with various DS _MAs were prepared as previously described. The concentrations of PAAm (or PEGMA) and MAlg were 10% and 2%, respectively ( FIG. 6 ).

For the PAAm-MAlg hydrogel, conversion from TBA ⁺ to Na ⁺ increased the coefficient and decreased the swelling ratio in all DS _MAs (Fig. 6a). This may be due to the more favorable physical interaction between the alginate chain and the acrylamide monomer with smaller Na ⁺ than the large TBA ⁺ which can cause steric hindrance. It is also possible that the local hydrophobicity was increased by TBA ⁺ . On the other hand, in the PEGMA-MAlg hydrogel, the opposite effect was observed, and the coefficient decreased and the swelling ratio increased according to Na ⁺ than TBA ⁺ ( FIG. 6b ). These results further emphasized that the presence of TBA ⁺ could modulate the physical interaction with high molecular weight PEGMA, unlike low molecular weight acrylamide. The tetrabutyl group can interact more favorably with the PEGMA chains through physical entanglement and an increase in van der Waals forces, leading to intrinsic physical crosslinking. Therefore, when TBA ⁺ is replaced with Na ⁺ , the physical crosslinking is effectively removed and mechanical properties are lowered. Overall, these results highlight the importance of the counterion of alginate on the crosslinking efficiency influencing the mechanical properties.

(3) Effect of Cell Adhesion Molecules (CAM) on MAlg

It can be speculated that the presence of CAM in the alginate backbone may interfere with the efficient crosslinking reaction between MAlg and the monomer. To confirm the ability of CAM-MAlg to control the crosslinking density of the hydrogel even in the presence of CAM, a PAAm hydrogel was prepared with CAM-MAlg and mechanical properties were measured (Fig. 7).

The coefficient ranges controlled by 1%, 2% and 4% RGD-MAlg at 10% PAAm ranged from 1.5 to 4.2, 3.7 to 13.5 and 5.3 to 36.3 kPa, respectively (Fig. 7a). At a higher PAAm concentration of 20%, the counting ranges were 2.0 to 7.7, 5.5 to 18.4, and 13.7 to 50.4 kPa at the same RGD-MAlg concentration (Fig. 7b). Similarly, the ranges of coefficients controlled by 1%, 2% and 4% gel-MAlg were 0.6 to 3.1, 0.8 to 12.7, and 2.6 to 43.8 kPa at 10% PAAm, respectively (Fig. 7c). At 20% PAAm, the range of coefficients increased from 0.5 to 10.9, 1.8 to 35.3, and 5.7 to 90.7 kPa at the same gel-MAlg concentration (Fig. 7d). It should be noted that the range of mechanical properties of hydrogels controlled by CAM-MAlg is not as broad as that by MAlg at a given DS _MA concentration due to increased steric hindrance of the CAM. Nevertheless, CAM-MAlg was able to successfully control the concentration and crosslink density of hydrogels generated with DS _MA . In addition, the coefficient range controlled by RGD-MAlg up to DS3 was larger than that controlled by gel-MAlg, probably due to the steric hindrance of the bulkier gelatin. However, the highest coefficients obtained by DS4 were greater in gel-MAlg.

3. Biocompatibility of MAlg-binding hydrogels

The biocompatibility of MAlg-binding hydrogels was first assessed by encapsulating cells within the hydrogel and measuring viability and proliferation. The effect of the mechanical properties controlled by DS _MA on cells was investigated. First, as shown in Fig. 5, fibroblasts were encapsulated in MGel-MAlg hydrogels using various DS _MAs while maintaining MGel and MAlg concentrations at 6% and 1%, respectively (Fig. 8). Since gelatin provides cell adhesion and matrix degradation sites necessary for cell activity, photocrosslinked gelatin hydrogels have been widely used as cell culture platforms for biomedical engineering. The initial survival rates taken on day 1 were all above 90%, suggesting that the hydrogel was highly biocompatible ( FIGS. 8a and 8b ). Subsequent cell proliferation also demonstrated that the MGel-MAlg hydrogel provided a suitable microenvironment for the encapsulated cells (Figures 8a and 8c). Interestingly, the cell proliferation rate showed a significant increase from DS _MA , especially DS1 to DS4, which is thought to be due to the effect of greater mechanical properties of the hydrogel that promote cell proliferation through increased mechanotransduction. . Overall, these results clearly highlighted the biocompatibility of MAlg-binding hydrogels.

The ability to induce cell adhesion by CAM-MAlg while controlling the mechanical properties was further evaluated by culturing cells in PEGMA hydrogels cross-linked with CAM-MAlg (Fig. 9). The CAM-free control, PEGMA-MAlg hydrogel, did not show significant cell adhesion and subsequent proliferation as expected. On the other hand, PEGMA hydrogels crosslinked with RGD-MAlg or gel-MAlg showed stable initial cell adhesion, followed by diffusion and proliferation over time. In general, cell proliferation increased with DS _MA by day 3 for both gel-MAlg or RGD-MAlg bound hydrogels. However, the extent of proliferation was greater in RGD-MAlg, possibly due to the higher density of cell adhesion peptides per mass of RGD-MAlg compared to gel-MAlg. In addition, at higher stiffness (higher DS _MA ), the cells became longer, characterized by stress fiber formation and polarization of fibroblasts through mechanical transitions. Taken together, these results effectively demonstrate the dual functional properties of CAM-MAlg as a crosslinking agent and cell adhesion site, enabling convenient fabrication of cell culture platforms.

As described above in detail a specific part of the present invention, for those of ordinary skill in the art, this specific description is only a preferred embodiment, and it is clear that the scope of the present invention is not limited thereto. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (23)

A hydrogel composition comprising alginate in which methacrylate is bonded to a hydroxyl group.
According to claim 1,
The alginate is a hydrogel composition comprising a compound represented by the following [Formula 1];
[Formula 1]

Here, X is tetraalkylammonium or Na, and m is an integer ≥ 1.
According to claim 1,
The alginate is a hydrogel composition comprising a compound represented by the following [Formula 2] or [Formula 3];
[Formula 2]

where X is tetraalkylammonium or Na, and n and m are integers ≥ 1,
[Formula 3]

where X is tetraalkylammonium or Na, n and m are integers ≥ 1, and R ₁ is CONH- (cell binding molecule).
4. The method of claim 3,
The percentage of m relative to the sum of n and m is 1 to 50% of the hydrogel composition.
4. The method of claim 3,
The cell binding molecule is RGD (Arg-Gly-Asp), RGDS (Arg-Gly-Asp-Ser), RGDC (Arg-Gly-Asp-Cys), RGDV (Arg-Gly-Asp-Val), RGES (Arg) -Gly-Glu-Ser), RGDSPASSKP (Arg-Gly-Asp-Ser-Pro-Ala-Ser-Ser-Lys-Pro), GRGDS (Gly-Arg-Gly-Asp-Ser), GRADSP (Gly-Arg- Ala-Asp-Ser-Pro), KGDS (Lys-Gly-Asp-Ser), GRGDSP (Gly-Arg-Gly-Asp-Ser-Pro), GRGDTP (Gly-Arg-Gly-Asp-Thr-Pro), GRGES (Gly-Arg-Gly-Glu-Ser), GRGDSPC (Gly-Arg-Gly-Asp-Ser-Pro-Cys), GRGESP (Gly-Arg-Gly-Glu-Ser-Pro), SDGR (Ser-Asp) -Gly-Arg), YRGDS (Tyr-Arg-Gly-Asp-Ser), GQQHHLGGAKQAGDV (Gly-Gln-Gln-His-His-Leu-Gly-Gly-Ala-Lys-Gln-Ala-Gly-Asp-Val ), GPR (Gly-Pro-Arg), GHK (Gly-His-Lys), YIGSR (Tyr-Ile-Gly-Ser-Arg), PDSGR (Pro-Asp-Ser-Gly-Arg), CDPGYIGSR (Cys- Asp-Pro-Gly-Tyr-Ile-Gly-Ser-Arg), LCFR (Leu-Cys-Phe-Arg), EIL (Glu-Ile-Leu), EILDV (Glu-Ile-Leu-Asp-Val), EILDVPST (Glu-Ile-Leu-Asp-Val-Pro-Ser-Thr), EILEVPST (Glu-Ile-Leu-Glu-Val-Pro-Ser-Thr), LDV (Leu-Asp-Val) and LDVPS (Leu) -Asp-Val-Pro-Ser) at least one hydrogel composition selected from the group consisting of.
4. The method of claim 3,
The cell-binding molecule is gelatin, collagen, fibronectin, gelatin, laminin, vitronectin, polycaprolactone (PCL), polyethylene oxide (PEO), polyvinyl alcohol (PVA) and polyvinylpyrrolidone (PVP) in the group consisting of One or more biocompatible polymers selected from the hydrogel composition.
According to claim 1,
The composition is a hydrogel composition further comprising an acrylic monomer and a photoinitiator.
8. The method of claim 7,
The acrylic monomer is sodium acrylate, sodium methacrylate, acrylamide, C ₁ -C ₁₅ saturated alkyl acrylate or methacrylate, C ₁ - C ₁₅ hydroxyalkyl acrylate or methacrylate; And N,N-di (C ₁ -C ₁₅ saturated or unsaturated alkyl) at least one hydrogel composition selected from the group consisting of acrylamide.
8. The method of claim 7,
The acrylic monomer is methacrylate gelatin (methacrylate gelatin), polyethylene glycol ethyl methacrylate (polyethylene glycol ethyl methacrylate, PEGMA), ethylene glycol ethyl acrylate (polyethylene glycol ethyl acrylate, PEGA), polyethylene glycol ethyl diacrylate (polyethylene A hydrogel composition which is at least one selected from the group consisting of glycol ethyl diacrylate, PEGDA), and polyethylene glycol ethyl dimethacrylate (polyethylene glycol ethyl dimethacrylate, PEGDMA).
8. The method of claim 7,
The photoinitiator is a water-soluble photoinitiator, 2-hydroxy-4′-(2-hydroxyethoxy)-2-methylpropiophenone), 1-hydroxy-cyclohexyl-phenyl-ketone, 2,2-Dimethoxy-1,2-diphenylethan-1-one, 2-benzyl-2-dimethylamino-1-(4-morpholinophenyl)-1-butanone, 2-methyl-4′-(methylthio)-2-morpholinopropiophenone, TPO-Na (sodium phenyl-2,4,6-trimethylbenzoylphosphinate) , TPO-Li (lithium phenyl-2,4,6-trimethylbenzoylphosphinate), BAPO-ONa (sodium bis (mesitoyl) phosphinate), BAPO-OLi (lithium bis (mesitoyl) phosphinate) at least one hydrogel selected from the group consisting of composition.
A hydrogel prepared from the composition according to any one of claims 1 to 10.
12. The method of claim 11,
A hydrogel in which the elastic modulus or cell proliferation rate of the hydrogel increases as the number of uronic acid residues combined with methacrylate increases.
12. The method of claim 11,
A hydrogel in which the elastic modulus of the hydrogel increases as the concentration of the acrylic monomer increases.
12. The method of claim 11,
The hydrogel is a hydrogel used for cell culture, differentiation or proliferation.
1) producing tetraalkylammonium alginate through cation exchange of sodium alginate; and
2) A method for producing a hydrogel comprising the step of bonding methacrylate to a hydroxyl group of the alginate.
16. The method of claim 15,
The tetraalkylammonium has the formula [NR ₁ R ₂ R ₃ R ₄ ] ⁺ (wherein R ₁ , R ₂ , R ₃ and R ₄ may be the same or different from each other, straight-chain C ₁ -C ₆ alkyl, branched C ₁ -C ₆ alkyl, substituted C ₆ -C ₁₀ aryl, unsubstituted C ₆ -C ₁₀ aryl, and a method for producing a hydrogel represented by a combination thereof).
16. The method of claim 15,
Step 2) is a method for producing a hydrogel that is carried out through a ring-opening nucleophilic addition reaction of tetraalkylammonium alginate and glycidyl methacrylate in an organic solvent.
18. The method of claim 17,
The organic solvent is dimethyl sulfoxide (DMSO), dimethylformamide (DMF), dimethylacetamide, N-methyl-2-pyrrolidone (N-methyl-2-pyrrolidone, NMP) , And hexamethylphosphoramide (hexamethylphosphoramide, HMPA) at least one hydrogel manufacturing method selected from the group consisting of.
16. The method of claim 15,
After step 2) above
3) cation exchange of tetraalkylammonium with Na ⁺ to synthesize methacrylic alginate.
16. The method of claim 15,
After step 1) above
1-1) Hydrogel production method further comprising the step of binding a cell-binding molecule to the carboxyl group of alginate through carbodiimide coupling.
20. The method of claim 19,
After step 3) above
4) mixing methacrylic alginate, acrylic monomer and photoinitiator; and
5) The hydrogel manufacturing method further comprising the step of photocrosslinking by irradiating UV.
22. The method of claim 21,
The methacrylic alginate is a hydrogel preparation method in which 0.1 to 10 wt% is added based on the total weight of the acrylic monomer.
22. The method of claim 21,
The concentration of the acrylic monomer is 5 to 50% (w / v) hydrogel production method.

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