Polymer compounds of glucuronic acid having phenol groups, gel-forming compositions containing such compounds, and methods for producing the same.

Abstract

The present invention relates to a polymeric compound of glucuronic acid bearing a phenolic hydroxyl moiety (hereinafter referred to as PGU-Ph) and more particularly to a method for the hydrogelation of such a compound. The present invention also relates to the use of the hydrogel structures (particles, films or 3D structures) obtained by this method, in particular as three-dimensional cell culture materials for supporting biological molecules used as active ingredients, for cell colonization or for tissue regeneration.

Description

[Technical Field]

[0001] The present invention relates, in general, to polymer compounds of glucuronic acid and methods for hydrogelating such compounds. The present invention also relates to the use of hydrogel structures (particles, films, or 3D structures) obtained by this method, particularly as three-dimensional cell culture materials for supporting biological molecules used as active ingredients, for cell colony formation, or for tissue regeneration. [Background technology]

[0002] Polyglucuronic acid (PGU), also known as glucuronan, is found in the strain Sinorhizobium meliloti M5N1CS. [1] It is a homopolymer of glucuronic acid produced by [→4]-β-D-GlcpA-(1→) residues partially acetylated at the C-3 and / or C-2 positions. Mucor rouxii [2] These polyuronides, first described in the cell walls of green algae, have since been used in green algae. [3] They have been isolated from other sources such as cell walls. However, the most described polysaccharides have been obtained from rhizobia strains. However, recent advances in the oxidation of primary hydroxyl groups with 2,2,6,6-tetramethylpiperidine-1-oxyl radical (TEMPO) reagents have made it possible to obtain PGU-mimicking derivatives on a large scale from cellulose, xanthan gum, curdlan, scleroglucan, chitosan, starch, fungal α-(1,3)-glucan, etc., and, consequently, a new family of polysaccharide lyases capable of degrading these polyglucuronic acids has been identified. [4]、[5] .

[0003] In the field of poly- and oligo-glucuronic acid, the different uses of these compounds, particularly French Patent No. 2781673 [6] and International Publication No. 1993 / 18174 [7]It teaches the biocompatibility of PGU and its use in food, agriculture, pharmaceuticals, cosmetics, or water purification, particularly as a gelling agent, thickening agent, hydrating agent, stabilizing agent, chelating agent, or flocculating agent. Another application relates to the immunostimulatory properties against human blood monocytes, and low molecular weight PGU enhanced the production of cytokines IL-1, IL-6, and TNF-α [8] The cosmetic application of PGU was claimed by Lintner (1999) [9] in relation to algae extracted from Haematococcus pluvialis, or in International Publication No. 2010 / 067327

[10] oligo-PGU that stimulates the elasticity of the dermis and epidermis was claimed. The biological activity of these low molecular weight glucuronans modified by sulfonation was also investigated in a model of the damaged extensor digitorum longus (EDL) muscle of rats, and it was demonstrated that the regenerative activity was induced not only by the presence of sulfate groups but also by acetyl groups. The cell regeneration process is regulated by specific signals in the extracellular matrix (or signaling peptides such as growth factors). These signals are conserved, protected, and arranged on a large family of polysaccharides called heparan sulfate (HS). In case of damage, specific enzymes destroy HS and no longer protect the specific signals. Then, other enzymes called proteases destroy the specific signals together with other structural proteins of the extracellular matrix. Due to their resistance to natural enzymes from the extracellular matrix, the biological effects of these modified bacterial polysaccharides could be explained

[11] .

[0004] In this regard, the applicant recently discovered the hydrogelation properties of PGU or PGU derivatives having phenolic hydroxyl moieties (hereinafter referred to as PGU-Ph) and their potential as components of bioinks for bioprinting. Therefore, the applicant We have further developed a method for hydrogelating these PGU-Ph compounds, which enables the rapid formation of stable hydrogels via horseradish peroxidase (HRP) catalytic crosslinking of the phenolic hydroxyl moiety.

[0005] HRP-assisted hydrogelation is performed using alginates.

[12] hyaluronic acid

[13] ,gelatin

[14] , dextran

[15] , and poly(vinyl alcohol)

[16] It is already known to those skilled in the art as an effective method for obtaining cell-containing hydrogels from various derivatives of natural and synthetic polymers. Recently, HRP-assisted hydrogelation has been applied to 3D bioprinting.

[17] 、

[18] Rapid curing of the ink extruded from the needle is required to produce 3D constructs with higher fidelity to the design. 3D bioprinting is a known technique for manufacturing cell-containing constructs based on digital designs. The resulting cell-containing constructs are fabricated for wound dressings and tissue engineering for drug screening and regenerative medicine.

[19] 、

[20] .

[0006] The applicant discovered that the addition of Ph clusters to PGU or its derivatives enables the use of a specific (enzymatic) crosslinking pathway, which is slower and eliminates the need for inorganic crosslinking agents that obscure the formed structure. The hydrogel structure thus obtained forms a transparent network and thus retains its printed form well. [Overview of the project]

[0007] As a result, the applicant has developed a method for producing the glucuronic acid polymer compound PGU-Ph (hereinafter referred to as the acronym PGU-Ph), and this method is - A step of providing a polymer compound of glucuronic acid (PGU), - The process includes the step of reacting the polymer compound of glucuronic acid (PGU) with a phenol compound to form a polymer compound of glucuronic acid (PGU-Ph) containing at least one phenol group by radical polymerization or by chemically selective grafting of a phenol group onto the carboxyl group of glucuronic acid.

[0008] According to the first embodiment of the present invention (hereinafter referred to as the first method for producing PGU-Ph by chemically selective grafting of phenol groups onto the carboxyl groups of glucuronic acid), the method includes the following steps. - A step of providing a polymer compound of glucuronic acid (hereinafter referred to by the acronym PGU) of formula (A) or (B) as defined above, -In order to form a PGU solution, dissolve PGU in an acid buffer solution, preferably in a 1 w / v% 2-(N-morpholino)ethanesulfonic acid (MES) buffer solution (pH 6.0, 100 mM), -In order to form the final composition of the reaction medium, the following steps are taken: X-(Y-amino-Z-hydroxyalkyl)-n-hydroxyl-phenol or its salt derivative hydrochloride, N-hydroxysuccinimide (NHS), and ethyl-3-(3-dimethylaminopropyl)carbodiimide (WSCD) are sequentially added to a solution of PGU. - The final composition is stirred at room temperature (i.e., between 20°C and 25°C) for at least 4 hours (preferably 20 hours) until a polymer is obtained consisting of a polymer compound of glucuronic acid containing at least one phenolic hydroxyl moiety.

[0009] After the final step of stirring, the obtained polymer may be precipitated in acetone, as shown in Example 1, and then washed with 90% ethanol + 10% water until the absorbance at 275 nm due to the presence of X-(Y-amino-Z-hydroxyalkyl)-n-hydroxyl-phenol is no longer detectable in the washing solution.

[0010] Preferably, the glucuronic acid polymer compound PGU-Ph according to the first embodiment of the present invention. In a method for producing, X-(Y-amino-Z-hydroxyalkyl)-n-hydroxyl-phenol may be selected from the group consisting of tyramine, dopamine, and octopamine, and more preferably is tyramine.

[0011] The applicant has also developed another embodiment of a method for producing a polymeric compound of glucuronic acid PGU-Ph according to the present invention (hereinafter referred to as the second method for producing PGU-Ph by radical polymerization), and this method includes the following steps: - A step of providing radicals of a polymeric compound of glucuronic acid of formula (C);

[0012]

Chemical formula

[0013]

Chemical formula

[0014]

Chemical formula

[0015] Preferably, in a method for producing a glucuronic acid polymeric compound PGU-Ph according to the second embodiment of the present invention, the phenolic compound of formula (D) may be selected from the group consisting of tyramine, dopamine, and octopamine, and preferably is tyramine. However, any phenolic compound can be used.

[0016] Another object of the present invention is an aqueous solution containing (atypically in an amount of 0.5-8 w / v%) of the glucuronic acid polymer compound of the present invention, which can be obtained by either of the methods for producing such polymer compounds according to the first and second embodiments. Drying of such a solution once coated onto a flat surface yields a water-soluble, non-gelling film of PGU-Ph (dried but not crosslinked). The crosslinking process involves divalent ions (preferably Ca 2+ or Mg 2+ M 2+ It can be produced on a dry film using a classic crosslinking agent solution composed of ). The networking of the PGU-Ph solution could be carried out using the following: (1) Divalent cations such as Ca2+ and Mg2+ (M 2+ A crosslinking catalyst consisting of ) or (2) Riboflavin, ruthenium-II trisbipyridyl chloride ([Ru(bpy)3] 2+ A photogelation process under visible and / or UV light using a photoinitiator such as a photoinitiator or a derivative thereof.

[0017] Another object of the present invention is a gel-forming composition comprising the following: - A polymer compound of glucuronic acid that can be obtained by the method according to the present invention or by the first and second embodiments of the present invention, and - A crosslinking catalyst consisting of an oxidase or compound (in liquid or gaseous form) that contains H2O2 or can generate H2O2 in situ (reducing molecules, e.g., glucose, fructose or galactose (monosaccharides), or lactose and maltose (disaccharides)).

[0018] In this gel-forming composition, if the compound containing or capable of producing H2O2 is glucose (or fructose or galactose), the crosslinking catalyst is glucose. It may further contain oxidase (or fructose oxidase, or galactose oxidase), and H2O2 will be generated more rapidly.

[0019] According to a first embodiment of the gel-forming composition of the present invention, the glucuronic acid polymer compound PGU-Ph may be bound to an oxidase as a crosslinking catalyst, and the oxidase is selected from the group consisting of oxidoreductase, peroxidase, catalase, laccase, tyrosinase and / or monosaccharide oxidase, and mixtures thereof.

[0020] Preferably, the oxidase may be a horseradish peroxidase (hereinafter referred to as the acronym HRP)-containing enzyme present in the composition at an amount of at least 0.1 U / mL, preferably 0.1 U / mL to 20 U / mL, and more preferably about 5 U / mL, for efficient and rapid crosslinking. Below 0.1 U / mL, gelation is very slow, and above 200 U / mL, gelation is too fast and difficult to control. An enzyme unit (U) is defined as one pyrogallol unit forming 1.0 mg of purpurogarin from pyrogallol in 20 seconds at pH 6.0 and 20°C.

[0021] According to a second embodiment of the gel-forming composition of the present invention, the glucuronic acid polymer compound PGU-Ph can be bound to a compound containing H2O2 in an amount of 0.05 mmol / L to 1 mmol / L, resulting in well-controlled gelation during a gelation time of 2 to 5 seconds in the presence of HRP. Outside this range, particularly at 0.01 to 0.05 mmol / L and above 1 mmol / L, gelation becomes much slower (gelation time of about 15 seconds), however, compounds containing such amounts of H2O2 may be interesting for applications requiring handling time (e.g., hydrogel film preparation and / or injectable hydrogels).

[0022] In either the first or second embodiment of the gel-forming composition of the present invention, the amount of the glucuronic acid polymer compound PGU-Ph may be between 0.01 w / v% and 8 w / v%, preferably between 0.1 w / v% and 2 w / v%. When the amount of the glucuronic acid polymer compound PGU-Ph is 0.01 w / v%, gelation is achievable but very slow. When the amount of the glucuronic acid polymer compound exceeds 8 w / v%, the gel-forming composition of the present invention is very viscous and difficult to use. Furthermore, it is difficult for the crosslinking agent to penetrate the composition.

[0023] According to a first modification of the gel-forming composition of the present invention, applicable to both embodiments, the gel-forming composition of the present invention may further comprise another active biodegradable polymer. Advantageously, the biodegradable polymer may be a polysaccharide such as glycosaminoglycan, or a protein selected from the group consisting of collagen, adhesion factors, gelatin, and mixtures thereof. Preferably, the biodegradable polymer may be a gelatin derivative containing a phenolic hydroxyl moiety (gelatin-Ph).

[0024] According to a second modification of the gel-forming composition applicable to both embodiments, the gel-forming composition of the present invention may further comprise suspension cells of animal, bacterial, or plant origin. These are described in 3.10. 5 These may be 10T1 / 2 cells or HepG2 cells present in the composition at a concentration in the range of cells / mL.

[0025] Another object of the present invention is a method for producing a hydrogel structure (hereinafter referred to as the first method for producing a hydrogel structure), which includes the following steps. - A step of providing a gel-forming composition according to a first modification, - The gel-forming composition, ○When the gel-forming composition contains oxidase, contact with a fluid or gaseous medium containing H2O2, or ○ The gel-forming composition contains H2O2 or generates H2O2 in situ. If the compound contains such a compound, the process involves hydrogelation by either contact with oxidase or other means.

[0026] A further object of the present invention is another method for producing a hydrogel structure (hereinafter referred to as the second method for producing a hydrogel structure), the method comprising the following steps. - A step of providing a gel-forming composition according to a second modification, - The gel-forming composition, ○When the gel-forming composition contains oxidase, contact with a fluid or gaseous medium containing H2O2, or ○If the gel-forming composition contains H2O2 or a compound that can generate H2O2 in situ, the process involves hydrogelation by either contact with an oxidase.

[0027] Both methods are - For example, by using various processes (dropping in micelle solution, crosslinking solution), one-dimensional hydrogel structures such as particles (microparticles and nanoparticles), or - For example, two-dimensional hydrogel structures such as lattices / films, by using classical molding processes (spin coating, tape casting, etc.), - For example, this may consist of a bioprinting process for producing a three-dimensional hydrogel structure by using a process selected from the group consisting of inkjet bioprinting, extrusion bioprinting, stereolithography bioprinting, and laser-assisted bioprinting.

[0028] These examples are by no means exhaustive and are given only as examples.

[0029] Another object of the present invention is a hydrogel that can be obtained by a first method for producing a hydrogel structure, regardless of the bioprinting process used. The obtained hydrogel structure can be used as a cell culture material to support cell colony formation (3D application) or as a patch or ointment (2D application).

[0030] A further object of the present invention is a hydrogel structure that can be obtained by a second method for producing a hydrogel structure, regardless of the bioprinting process used. The hydrogel structure thus obtained can be used for tissue regeneration. [Brief explanation of the drawing]

[0031] Other innovative features and advantages of the present invention will become apparent by reading the following description with reference to the examples and corresponding drawings. The following description is illustrative and not limiting. The drawings are shown below. [Figure 1] The UV-Vis absorption spectra of PGU and 0.1 w / w% PGU-Ph are shown (see Example 1). [Figure 2] The shear rate-viscosity profiles of PGU and PGU-HPh at 1 and 2 w / v% are shown (see Example 1). [Figure 3] a) The effect of 5 U / mL HRP and 0.1 mM H2O2 on the gelation of PGU-Ph, b) the effect of 1 w / v% PGU-HPh and 0.1 mM H2O2 on HRP, and c) the effect of 1 w / v% PGU-HPh and 5 U / mL HRP on H2O2 are shown (see Example 2). [Figure 4] This figure shows the morphology and mitochondrial activity of 10T1 / 2 cells cultured for 20 hours in a mixed solution of medium containing 0.5 w / v% PGU or PGU-Ph (50 vol.) and PBS (50 vol.) (see Example 3). [Figure 5] a) Micrographs of 10T1 / 2 cells seeded on cell culture dishes (dishes), PGU-Ph hydrogel, and PGU-Ph+gelatin-Ph hydrogel (bar: 100 μm) on days 1 and 4, and b) Micrographs of HepG2 cells on days 1 and 3 (see Example 4). [Figure 6]a) Micrographs of 10T1 / 2 cells and b) HepG2 cells encapsulated in PGU-Ph hydrogel, superimposed on bioprinting days 1, 4, and 8. Cells were stained with calcein-AM (green) and PI (red) (bar: 200 μm) (see Example 5). [Figure 7] This is an example of a 3D structure of a PGU-Ph hydrogel using a 3D CAD model of the design (see Example 6). [Figure 8] These are PGU-Ph beads obtained by dropping a PGU-Ph+HRP solution into H2O2 (0.5M) (see Example 7). [Figure 9] This is an antioxidant PGU-Ph film obtained by drying a solution of PGU-Ph (1 w / v%) (see Example 8).

[0032] Figures 1-9 are given for illustrative purposes and illustrate the present invention, and are described in more detail in the following embodiments, but do not limit the scope of the present invention. [Examples]

[0033] Materials and methods material -Tyramine hydrochloride purchased from Combi-Blocks (San Diego, California) -Water-soluble carbodiimide (WSCD) purchased from Peptide Institute (Osaka, Japan), -N-hydroxysuccinimide (NHS), HRP (210 units / mg), and H2O2 aqueous solution (31 w / w%) purchased from Fujifilm Wako Pure Chemical Industries (Osaka, Japan). - Mouse fibroblasts (10T1 / 2 cells) and human liver cancer (HepG2 cells) obtained from Riken Cell Bank (Ibaraki, Japan) were grown in a 5% CO2 incubator in Dulbecco's modified Eagle medium (DMEM, Nissui, Tokyo, Japan) supplemented with 10 v / v% fetal bovine serum. -Sinorhizobium meliloti M5N1CS (or S. meliloti M5N1CS strain).

[0034] PGU production and extraction - S. meliloti M5N1CS strain was grown at 30°C in a 20L bioreactor (SGI) containing 15L of complete rhizobium medium (RCS medium) supplemented with 1% (w / v) sucrose.

[0035] - For the inoculum, S. meliloti M5N1CS was incubated in 1.5 L of RCS medium on a rotary shaker (120 rpm) at 30°C for 20 hours.

[0036] After incubation for 72 hours, the resulting broth was centrifuged at 33,900 × g for 40 minutes at 20°C. The supernatant was purified by tangential ultrafiltration through a Sartorius (Göttingen, Germany) 100,000 standard molecular weight cutoff (NMWCO) polyethersulfone membrane in distilled water. -Finally, the holding solution is freeze-dried to obtain PGU of formula B, which has a β-(1,4)-D-polyglucuronic acid chain in which carbon 2 and carbon 3 of the β-D-glucuronic acid unit are O-acetylated. Ta.

[0037] test Shear rate-viscosity profile The shear rate-viscosity profile of the solution was measured using a rheometer (HAAKE MARS III, Thermo Fisher Scientific, Waltham, Massachusetts) equipped with parallel plates of 25 mm radius with a 0.5 mm gap at 20°C.

[0038] Hydrogelation time - Gelation time was measured at room temperature using phosphate-buffered saline solution (50-100 mM, pH 7.4) containing PGU-Ph. - This PGU-Ph solution was poured into a 24-well plate at a rate of 0.2 mL / well. Next, 0.1 mL of HRP and 0.1 mL of H2O2 solution were sequentially added to the wells, and the mixture was stirred using a magnetic stirring bar (10 mm in length). -Gelation was confirmed when magnetic stirring was hindered and the surface of the solution swelled.

[0039] cytocompatibility - Place 4 x 10⁶ T1 / 2 cells in the wells of a 96-well cell culture plate. 3 Cells were seeded in wells and incubated in culture medium at 37°C for 20 hours in a humidified 5% CO2 incubator. -The culture medium was then replaced with a medium containing 0.5 w / v% PGU or PGU-Ph (0.2 mL), and incubated for a further 24 hours. Next, the culture medium containing the polymer was replaced with a medium (0.2 mL) containing 1 / 20th the volume of the reagent from a colorimetric mitochondrial activity assay kit (purchased from Dojindo, Kumamoto, Japan, under the trade name Cell Counting Kit-8). - After 2 hours of incubation, the absorbance at 450 nm was measured using a spectrophotometer. -Sodium alginate (Alg) and alginate containing a pH moiety (Alg-Ph) were used as controls.

[0040] Cell behavior on hydrogels A solution containing -1 w / v% PGU-Ph, or 1 w / v% PGU-Ph + 1 w / v% gelatin-Ph, and 5 U / mL HRP was poured into the wells of a 12-well cell culture dish at a rate of 0.5 mL / well. Next, the plates were placed in a plastic container. Air containing 8 ppm of H2O2, obtained by aerating air in a -0.5 M H2O2 aqueous solution, was introduced into a plastic container at a rate of 10 L / min. After exposure to air containing H2O2 for 15 minutes, the wells coated with hydrogel were sequentially rinsed with PBS and culture medium. -10T1 / 2 cells and HepG2 cells were suspended in a medium containing 0.3 mg / mL of catalase, and 6 × 10⁶ cells were prepared. 4Cells were injected into each well at a rate of one cell per well.

[0041] Printing process and system - An extrusion 3D printing system developed by modifying a commercially available 3D printing system (purchased from Anycubic Corporation (Guangdong, China) under the product name Anycubic i3 Mega) will be used for 3D bioprinting. -This extrusion 3D printing system includes a syringe pump for injecting ink, a 27-gauge stainless steel needle for extruding the ink, a bubbling system for supplying air containing 8 ppm H2O2, and a spool for layering the extruded ink. It consisted of a stage and a needle. The ink flow rate in the needle and the movement speed of the stage were fixed at 22 mm / s. - The printing of cell-containing 3D hydrogel constructs was performed inside a biological safety cabinet. An ink containing -1 w / v% PGU-Ph, or 1 w / v% PGU-Ph and 1 w / v% gelatin-Ph, and 5 U / mL of HRP was used. The effect of ink-induced extrusion on cells was determined by measuring the viability of 10T1 / 2 cells and HepG2 cells suspended in ink at a concentration of -3 × 10⁵ cells / mL. - The ink containing cells was collected at the tip of a needle, and the cells were stained with trypan blue dye for measurement using a hemocytometer. - The viability of cells encapsulated in hydrogels obtained through the printing process was determined by staining the cells with fluorescent dyes, calcein-AM, and propidium iodide (PI).

[0042] Example 1: PGU-PH synthesis achieved according to a first method for producing PGU-PH This synthesis is carried out according to the first method for producing PGU-Ph, as follows: -This was dissolved at a 1 w / v% concentration in 2-(N-morpholino)ethanesulfonic acid (MES) buffer solution (pH 6.0). -Tyramine hydrochloride, NHS, and WSCD were added sequentially at concentrations of 45 mM, 10 mM, and 20 mM, respectively, and the mixture was stirred at room temperature for 20 hours. - The obtained polymer was precipitated in acetone, and then washed with 90% ethanol and 10% water until the absorbance at 275 nm due to the presence of tyramine was no longer detectable in the washing solution. - The obtained phenolized PGU is PGU-Ph according to the first embodiment of the present invention.

[0043] Figure 1 shows the UV-vis spectrum of the 0.1 w / w% PGU-Ph solution (PGU-Ph solution) described above, which is compared to that of 0.1 w / w% PGU (PGU solution). Figure 2 shows, in particular, that the PGU solution does not have a peak around 275 nm, whereas the PGU-Ph solution has a peak around 275 nm due to the Ph portion. The content of the Ph portion calculated based on the calibration curve obtained from a known proportion of tyramine solution is 3.7 × 10⁻⁶. -4 It is expressed as mol-Ph / g.

[0044] Figure 2 shows the shear rate-viscosity profiles of 1 and 2 w / v% PGU-Ph solutions, and compares them with the shear rate-viscosity profiles of 1 and 2 w / v% PGU solutions. Figure 2 shows, in particular, that the viscosity of the PGU-Ph solution is higher than that of the PGU solution, and that the viscosity of the 2 w / v% PGU-Ph solution is higher than that of the 1 w / v% PGU-Ph solution.

[0045] Example 2: Hydrogelation of PGU-Ph solution The hydrogel was produced according to the first method for producing a hydrogel structure according to the present invention, by an HRP-catalyzed reaction in the presence of H2O2 using the PGU-Ph solution obtained in Example 1.

[0046] Figure 3a shows the effect of PGU-Ph concentration on the hydrogelation time with 5 U / mL HRP and 0.1 mM H2O2. The gelation time for a 0.5 w / v% PGU-Ph solution is 6.0 seconds.

[0047] Figures 3b and 3c show the gelation time measured for a 1.0 w / v% PGU-Ph solution. The effects of HRP and H2O2 concentrations are shown. Gelation time decreases from 71 seconds to 2 seconds as the HRP concentration increases from 0.1 U / mL to 20 U / mL (Figure 3b). Gelation time decreases as the H2O2 concentration increases from 0.05 mM to 1 mM, but increases further as the H2O2 concentration increases. Note that higher concentrations of H2O2 lead to depolymerization of PGU-Ph, which then significantly reduces gelation.

[0048] Example 3: Cell compatibility of PGU-Ph To evaluate the cytocompatibility of PGU-Ph obtained in Example 1, 10T1 / 2 cells were incubated in a solution containing PGU-Ph. Solutions containing PGU, Alg, or Alg-Ph were used as controls.

[0049] Figures 4a, b, and c show the morphology and mitochondrial activity of cells cultured for 20 hours in a mixed solution of medium (50 vol%) containing either 0.5 w / v% of the example PGU-Ph or the corresponding PGU, and PBS (50 vol%).

[0050] There were no significant differences in cell morphology specific to exposure to PGU-Ph. Furthermore, there was no significant decrease in mitochondrial activity in cells incubated in the mixed solution containing 0.5 w / v% Alg and Alg-Ph (p=0.28, Figure 4c), similar to cells incubated in the mixed solution (p=0.45), caused by the Ph moiety introduced into PGU.

[0051] Mitochondrial activity in cells incubated in a solution containing PGU and PGU-Ph was approximately 20% higher than that in cells incubated in a solution containing Alg and Alg-Ph (p<0.03).

[0052] Example 4: Cell behavior on PGU-Ph hydrogel Hydrogels containing only PGU-Ph (obtained in Example 1) and hydrogels containing both PGU-Ph (obtained in Example 1) and gelatin-Ph were used to evaluate the cytocompatibility and cell adhesion of the PGU-Ph-containing hydrogel. The day after seeding, the majority of 10T1 / 2 cells and HepG2 cells were suspended on the PGU-Ph hydrogel, and HepG2 cells formed aggregates (Figures 5a, 5b).

[0053] During the subsequent incubation period, the cells remained suspended on the PGU-Ph hydrogel. A small number of cells adhered to the hydrogel but did not elongate. In contrast, 10T1 / 2 cells seeded on the PUG-Ph + gelatin-Ph hydrogel adhered, elongated, and proliferated similarly to those on the cell culture dish (Figure 5a).

[0054] No significant morphological differences were observed between 10T1 / 2 cells on PGU-Ph+gelatin-Ph hydrogel and 10T1 / 2 cells on a cell culture dish. HepG2 cells seeded on PGU-Ph+gelatin-Ph hydrogel also adhered, elongated, and proliferated (Figure 5b). However, their morphology was clearly different from that of those on a cell culture dish. HepG2 cells on a cell culture dish adhered to the substrate and formed small aggregates the day after seeding. Subsequently, the cells proliferated as a monolayer, and the size of the aggregates increased. HepG2 cells on PGU-Ph+gelatin-Ph hydrogel did not form any significant aggregates the day after seeding.

[0055] During the subsequent incubation period, HepG2 cells proliferated on the PGU-Ph+ gelatin-Ph hydrogel and did not form any distinct aggregates that were significantly different from those formed on the cell culture dish.

[0056] Example 5: 3D Hydrogel as a Cell Culture Material to Support Cell Colony Formation Printing. The effects of the 3D printing process and PGU-Ph hydrogel on cells were evaluated by printing hydrogel constructs encapsulating 10T1 / 2 cells and HepG2 cells. The viability of 10T1 / 2 cells and HepG2 cells the day after bioprinting, stained with calcein-AM and PI, was 92.3% and 91.6%, respectively. This result demonstrates that the printing process using PGU-Ph solution as ink was not harmful to these cells.

[0057] Regarding the morphology of the encapsulated cells, 10T1 / 2 cells maintained a round shape without the formation of cell aggregates during the 8-day study (Figure 6a). In contrast, HepG2 cells formed aggregates in the hydrogel construct, and the size of the aggregates increased with increasing culture duration (Figure 6b). There was no apparent increase in dead cells in either cell type.

[0058] Example 6: 3D printing of hydrogels. To evaluate the feasibility of a PGU-Ph solution as a bioprinting ink, a 1 w / v% PGU-Ph solution containing 5 U / mL of HRP was extruded onto a substrate. As shown in Figure 7, when the solution was extruded in air containing 8 ppm H2O2, a transparent 3D hydrogel construct with good fidelity to the design drawing (3D CAD model) was obtained. These results demonstrate the feasibility of a gellable PGU-Ph solution as a 3D printing ink through HRP-mediated hydrogelation.

[0059] Example 7: Particle synthesis. To evaluate the feasibility of synthesizing PGU-Ph particles such as microbeads, a 1 w / v% PGU-Ph solution containing 5 U / mL of HRP was added dropwise to H2O2 (0.5 M) and stirred for 1 minute. The microbeads were then washed with water and stored in ethanol / aqueous solution (70 / 30). As shown in Figure 8, transparent PGU-Ph beads with a good spherical shape were obtained.

[0060] Example 8: Synthesis of antioxidant PGU-Ph dry film. To evaluate the feasibility of synthesizing antioxidant PGU-Ph films, a 1 w / v% PGU-Ph solution was poured into a plastic Petri dish and dried at 50°C for 24 hours. A PGU film without the pH moiety was used as a control. To evaluate the antioxidant effect of the PGU-Ph film, free radical scavenging activity was measured using 1,1-diphenyl-2-picrylhydrazyl (DPPH). Briefly, 100 mg of PGU-Ph film was added to 5.0 mL of DPPH solution (0.1 mM DPPH 96° in ethanol). The solution was left in the dark at room temperature with stirring for 24 hours. After that, the absorbance was measured at 517 nm using a Shimadzu UV-1700 spectrophotometer.

[0061] The DPPH radical scavenging activity was calculated as an inhibition rate based on the following formula (1). DPPH radical inhibition rate (%) = ((A control -A sample ) / A control ) × 100 (1) Here, A sample and A control These values ​​represent the absorbance at 517 nm for the PGU-Ph film and the control without the PGU-Ph film (i.e., DPPH solution), respectively.

[0062] As shown in Figure 9a, a transparent PGU-Ph film was obtained. As observed in Figure 9b, these results demonstrate the feasibility of PGU-Ph solutions as antioxidant films for food packaging and biomaterials.

[0063] List of References 1-Heyraud, A., Courtois, J., Dantas, L., Colin-Morel, P., and Courtois, B. (1993). "Structural characterization and rheological properties of extracellular glucuronans produced by Rhizobium meliloti M5N1 mutant." Carbohydrate Research, 240(24), 71-78.

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Claims

1. A method for producing a polymer compound of glucuronic acid, - Includes the step of providing a radical of the glucuronic acid polymer compound of formula (C), 【Chemistry 1】 - Reaction of the radical of formula (C) with the phenol compound of formula (D), 【Chemistry 2】 A polymer compound of glucuronic acid of formula (E) is obtained by radical coupling. 【Transformation 3】 (In the formula, X = monovalent ion M+ of a metal belonging to the alkali metal group,) R represents H and / or a sulfate group and / or an acyl group containing COCH3, n is an integer selected such that the molar mass of the polymer compound is between 10 and 1000 kilodaltons. method.

2. The method according to claim 1, wherein the phenol compound of formula (D) is selected from the group consisting of tyramine, dopamine, and octopamine.

3. A polymer compound of glucuronic acid containing at least one phenol group, obtained by the method described in claim 1 or 2.

4. A solution comprising water and the polymer compound of glucuronic acid described in claim 3.

5. A gel-forming composition, A polymer compound of glucuronic acid according to claim 3, Oxidase, or H ２ O ２ Contains or H ２ O ２ A gel-forming composition comprising a crosslinking catalyst consisting of a compound that can generate gel in situ.

6. The gel-forming composition according to claim 5, wherein the polymer compound of glucuronic acid is bound to an oxidase as a crosslinking catalyst, selected from the group consisting of oxidoreductase, peroxidase, catalase, laccase, tyrosinase, and monosaccharide oxidase, and mixtures thereof.

7. The gel-forming composition according to claim 6, wherein the oxidase is horseradish peroxidase (HRP) present in the composition at an amount of at least 0.01 U / mL.

8. The aforementioned glucuronic acid polymer compound is present in an amount of 0.05 mmol / L to 1 mmol / L of H ２ O ２ The gel-forming composition according to claim 5, which is bonded to a compound containing the compound.

9. The gel-forming composition according to claim 5, wherein the polymer compound of glucuronic acid is present in an amount of 0.01 w / v% to 8 w / v%.

10. The gel-forming composition according to claim 5, further comprising another biodegradable polymer.

11. The gel-forming composition according to claim 10, wherein the biodegradable polymer is a polysaccharide or protein selected from the group consisting of collagen, adhesion factors, gelatin, and mixtures thereof.

12. The gel-forming composition according to claim 11, wherein the biodegradable polymer is a gelatin derivative containing a phenolic hydroxyl moiety (gelatin-Ph).

13. The gel-forming composition according to claim 5, further comprising suspension cells of animal, bacterial, or plant origin.

14. A method for producing a hydrogel structure, To provide the gel-forming composition described in claim 5, The gel-forming composition is If the gel-forming composition contains oxidase, H ２ O ２ Contact with a fluid or gaseous medium containing, or The gel-forming composition contains H ２ O ２ or contains a compound capable of generating H ２ O ２ in situ, and comprises hydrogelation by any of contact with an oxidase. A method comprising this.

15. A method for producing a hydrogel structure, - To provide the gel-forming composition described in claim 13, - The gel-forming composition, - If the gel-forming composition contains oxidase, H ２ O ２ Contact with a fluid or gaseous medium containing, or - The gel-forming composition is H ２ O ２ Contains or H ２ O ２ A method comprising hydrogelation by either contact with an oxidase or by any other means, in which the compound can generate hydrogel in situ.

16. The method according to claim 14, comprising a bioprinting process for producing a one-dimensional hydrogel structure, a two-dimensional hydrogel structure, or a three-dimensional hydrogel structure.

17. A hydrogel structure obtained by the method of claim 14.

18. A cell-containing hydrogel structure obtained by the method of claim 15.

19. A three-dimensional cell culture material for supporting cell colony formation, comprising the hydrogel structure described in Claim 17.

20. A patch or ointment comprising the hydrogel structure described in Claim 17.

21. A cell-containing hydrogel structure according to claim 18, for tissue regeneration and / or tissue engineering.

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