Polymeric compounds of glucuronic acid bearing phenolic groups, gel-forming compositions containing such compounds and methods for producing same - Patents.com

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 generally to polymeric compounds of glucuronic acid and to a method for the hydrogelation of such compounds. 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. [Background technology]

[0002] Polyglucuronic acid (PGU), also called glucuronan, is a saccharide found in the strain Sinorhizobium meliloti M5N1CS [1] It is a homopolymer of glucuronic acid composed of [→4]-β-D-GlcpA-(1→] residues partially acetylated at the C-3 and / or C-2 positions, produced by Mucor rouxii. [2] These polyuronides, first described in the cell walls of Chlorophyceae, have since been [3] Polysaccharides have been isolated from other sources such as the cell walls of Bacillus subtilis. However, the most described polysaccharides have been obtained by rhizobia strains. However, recent advances in the oxidation of primary hydroxyl groups with the 2,2,6,6-tetramethylpiperidine-1-oxyl radical (TEMPO) reagent have made it possible to obtain PGU-mimetic derivatives on a large scale from cellulose, xanthan, curdlan, scleroglucan, chitosan, starch, fungal α-(1,3)-glucans, etc., and concomitantly, 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 acids, the different uses of these compounds, in particular French patent no. 2781673 [6] and International Publication No. 1993 / 18174 [7]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 concerns the immune stimulating properties on human blood monocytes, where low molecular weight PGU enhanced the production of the cytokines IL-1, IL-6, and TNF-α. [8] The application of PGU to cosmetics is described in Lintner (1999) [9] The invention relates to an algae extract from Haematococcus pluvialis, which is claimed by the Publication No. 2010 / 067327

[10] In , an oligo-PGU stimulating the elasticity of the dermis and epidermis has been claimed. The biological activity of these low molecular weight glucuronans modified by sulfonation was also investigated in a model of injured extensor digitorum longus (EDL) in rats, demonstrating that the regenerative activity is not only induced by the presence of sulfate groups, but also by acetyl groups. The regeneration process of cells is regulated by specific signals (or transducing peptides such as growth factors) of the extracellular matrix. These signals are stored, protected and located on a family of large polysaccharides called heparan sulfate (HS). In case of injury, specific enzymes destroy the HS, which no longer protects the specific signals. Other enzymes, called proteases, then destroy the specific signals together with other structural proteins of the extracellular matrix. The biological effect of these modified bacterial polysaccharides could be explained due to their resistance to natural enzymes from the extracellular matrix.

[11] .

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

[0005] HRP-assisted hydrogelation of alginate

[12] , hyaluronic acid

[13] ,gelatin

[14] , dextran

[15] , and poly(vinyl alcohol)

[16] HRP-assisted hydrogelation 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 such as cellulose acetate, cellulose acetate esters, and cellulose acetate esters. Recently, HRP-assisted hydrogelation has been applied to 3D bioprinting.

[17] 、

[18] To create 3D constructs with higher fidelity to the design, rapid curing of the ink ejected from the needle is required. 3D bioprinting is a known technique for the fabrication of 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 has discovered that the addition of Ph clusters to PGU or its derivatives allows the use of a specific (enzymatic) cross-linking route that obviates the need for inorganic cross-linkers, which are slower and tend to blur the structures formed. The hydrogel structures thus obtained form transparent networks and thus retain their printed morphology well. Summary of the Invention

[0007] As a result, the Applicant has developed a method for producing a polymeric compound of glucuronic acid PGU-Ph (hereinafter referred to by the acronym PGU-Ph), which method comprises: - Providing a polymeric compound of glucuronic acid (PGU); - reacting said polymeric compound of glucuronic acid (PGU) with a phenolic compound to form a polymeric compound of glucuronic acid (PGU-Ph) containing at least one phenolic group by radical polymerization or by chemical regioselective grafting of the phenolic group onto the carboxyl group of glucuronic acid.

[0008] According to a first embodiment of the invention (hereinafter referred to as the first method for producing PGU-Ph by chemical regioselective grafting of a phenol group onto the carboxyl group of glucuronic acid), the method comprises the following steps: - providing a polymeric compound of glucuronic acid (hereinafter designated by the acronym PGU) of formula (A) or (B) as defined above, - dissolving 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) to form a solution of PGU; - sequentially adding X-(Y-amino-Z-hydroxyalkyl)-n-hydroxyl-phenol or its salt derivative hydrochloride, N-hydroxysuccinimide (NHS) and ethyl-3-(3-dimethylaminopropyl)carbodiimide (WSCD) to the solution of PGU to form the final composition of the reaction medium; - stirring said final composition at room temperature (i.e. between 20°C and 25°C) preferably for at least 4 hours (preferably 20 hours) until a resultant polymer consisting of a polymeric compound of glucuronic acid containing at least one phenolic hydroxyl moiety is obtained.

[0009] After the last step of stirring, the resulting polymer can be precipitated in acetone as shown in Example 1, followed by washing steps with 90% ethanol + 10% water until no absorbance at 275 nm due to the presence of X-(Y-amino-Z-hydroxyalkyl)-n-hydroxyl-phenol can be detected in the washing solution.

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

[0011] The Applicant has also developed another embodiment of the method for producing a polymeric compound of glucuronic acid PGU-Ph according to the invention (hereinafter referred to as the second method for producing PGU-Ph by radical polymerization), which comprises the following steps: - providing a radical of a polymeric compound of glucuronic acid of formula (C),

[0012] [ka] - reacting said radical of formula (C) with a phenolic compound of formula (I) (see above) or (D),

[0013] [ka] By radical coupling to obtain polymeric compounds of glucuronic acid of formula (E) or (F), respectively.

[0014] [ka]

[0015] Preferably, in the method for producing the glucuronic acid polymer 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, preferably tyramine, but any phenolic compound can be used.

[0016] Another object of the invention is an aqueous solution comprising the polymeric compound of glucuronic acid of the invention (typically in an amount of 0.5-8 w / v %), obtainable by any of the methods for producing such a polymeric compound according to the first and second embodiment. Drying of such a solution, once coated on a flat surface, results in a water-soluble, non-gelled film of PGU-Ph (dried but not crosslinked). The crosslinking process is carried out by the addition of divalent ions (preferably Ca 2+ or Mg 2+ M 2+ ) can be used to produce on a dry film. Reticulation of PGU-Ph solutions could be performed using: (1) Divalent cations such as Ca2+ and Mg2+ (M 2+ or (2) Riboflavin, ruthenium II tris(bipyridyl chloride) ([Ru(bpy)3] 2+ ), or their derivatives, using a photoinitiator, such as a photogelation process under visible and / or UV light.

[0017] Yet another object of the present invention is a gel-forming composition comprising: - polymeric compounds of glucuronic acid according to the invention or obtainable by the processes according to the first and second embodiments of the invention, and - A bridged catalyst consisting of an oxidase or a compound (liquid or gas 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, when the compound that contains or can generate H2O2 is glucose (or fructose, or galactose), the cross-linking catalyst is glucose An oxidase (or fructose oxidase, or galactose oxidase) may further be included, so that H2O2 is produced more rapidly.

[0019] According to a first embodiment of the gel-forming composition of the invention, the polymeric compound of glucuronic acid PGU-Ph may be coupled to an oxidase as a cross-linking catalyst, the oxidase being selected from the group consisting of oxidoreductases, peroxidases, catalases, laccases, tyrosinases and / or monosaccharide oxidases, and mixtures thereof.

[0020] Preferably, the oxidase may be an enzyme containing horseradish peroxidase (hereinafter referred to by the acronym HRP) contained in the composition in an amount of at least 0.1 U / mL, preferably 0.1 U / mL to 20 U / mL, 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. The enzyme unit (U) is defined such that 1 pyrogallol unit forms 1.0 mg purpurogallin 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 invention, the polymeric compound PGU-Ph of glucuronic acid can be bound to a compound containing H2O2 in an amount comprised between 0.05 mmol / L and 1 mmol / L, resulting in a well-controlled gelation in the presence of HRP for a gelation time between 2 and 5 seconds. Outside this range, especially between 0.01 and 0.05 mmol / L and above 1 mmol / L, the gelation becomes much slower (gelation time of about 15 seconds), but such amounts of the compound containing H2O2 can be interesting for applications requiring handling times (e.g. hydrogel film preparation and / or injectable hydrogels).

[0022] For 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 can be comprised 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 feasible but very slow. When the glucuronic acid polymer compound is more than 8 w / v%, the gel-forming composition of the present invention is very viscous and difficult to use. Moreover, it is difficult for the crosslinker to penetrate into the composition.

[0023] According to a first variant of the gel-forming composition of the invention, applicable to both embodiments, the gel-forming composition of the invention may further comprise another active biodegradable polymer. Advantageously, the biodegradable polymer may be a polysaccharide, such as a 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 variant of the gel-forming composition, which is applicable to both embodiments, the gel-forming composition of the invention may further comprise suspension cells of animal, bacterial or plant origin. These are as defined in 3.10 5 The cells 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 a first method for producing a hydrogel structure, comprising the following steps: - providing a gel-forming composition according to a first variant, said gel-forming composition, If the gel-forming composition comprises an oxidase, contact with a fluid or gaseous medium containing H2O2, or The gel-forming composition contains H2O2 or generates H2O2 in situ. or, if the compound comprises a compound capable of hydrogelling, contact with an oxidase.

[0026] Yet another 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, comprising the following steps: - providing a gel-forming composition according to the second variant, said gel-forming composition, If the gel-forming composition comprises an oxidase, contact with a fluid or gaseous medium containing H2O2, or Hydrogelling the gel-forming composition by either containing H2O2 or, if it comprises a compound capable of generating H2O2 in situ, by contact with an oxidase.

[0027] Both methods are -One-dimensional hydrogel structures, such as particles (microparticles and nanoparticles), for example, by using various processes (micelle solutions, dropwise deposition in crosslinking solutions), or - two-dimensional hydrogel structures, such as lattices / films, for example by using classical molding processes (spin-coating, tape-casting, etc.), or For example, it may consist of a bioprinting process for producing three-dimensional hydrogel structures, preferably 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 as examples only.

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

[0030] Yet another object of the present invention is a hydrogel construct obtainable by the second method for producing a hydrogel construct, regardless of the bioprinting process used. The hydrogel construct thus obtained can be used for tissue regeneration. [Brief description of the drawings]

[0031] Other innovative features and advantages of the invention will become apparent from a reading of the following description, given by way of example and without limitation, with reference to the accompanying drawings, in which: [Figure 1] 1 shows the UV-Vis absorption spectra of PGU and 0.1 w / w% PGU-Ph (see Example 1). [Diagram 2] 1 shows the shear rate-viscosity profiles of PGU and PGU-HPh at 1 and 2 w / v % (see Example 1). [Diagram 3] a) The effect of 5 U / mL HRP and 0.1 mM H2O2 on gelation of PGU-Ph, and b) the effect of 1 w / v% PGU-HPh and HRP at 0.1 mM H2O2, and c) the effect of 1 w / v% PGU-HPh and H2O2 at 5 U / mL HRP (see Example 2). [Figure 4] FIG. 1 shows the cell morphology and mitochondrial activity of 10T1 / 2 cells cultured for 20 hours in a mixed solution of medium (50% by volume) and PBS (50% by volume) containing 0.5 w / v% PGU or PGU-Ph (see Example 3). [Diagram 5] a) Micrographs of 10T1 / 2 cells seeded on cell culture dishes (dishes), PGU-Ph hydrogels, and PGU-Ph+gelatin-Ph hydrogels (bar: 100 μm) at days 1 and 4, and b) HepG2 cells seeded on days 1 and 3 (see Example 4). [Figure 6]Overlay of micrographs of a) 10T1 / 2 cells and b) HepG2 cells encapsulated in PGU-Ph hydrogels at days 1, 4, and 8 of bioprinting. Calcein-AM (green) and PI (red) were used to stain the cells (bar: 200 μm) (see Example 5). [Figure 7] FIG. 11 is an example of the 3D structure of PGU-Ph hydrogel using blueprint 3D CAD model (see Example 6). [Figure 8] PGU-Ph beads were obtained by adding PGU-Ph+HRP solution dropwise to H2O2 (0.5M) (see Example 7). [Figure 9] Antioxidant PGU-Ph film obtained by drying a solution of PGU-Ph (1 w / v %) (see Example 8).

[0032] 1-9 are described in more detail in the following examples and are given by way of illustration to explain the present invention but not to limit the scope of the invention. EXAMPLES

[0033] Materials and Methods material - Tyramine hydrochloride, purchased from Combi-Blocks (San Diego, CA); - 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 fibroblast 10T1 / 2 cells and human hepatoma HepG2 cells obtained from the Riken Cell Bank (Ibaraki, Japan) were grown in Dulbecco's modified Eagle's medium (DMEM, Nissui, Tokyo, Japan) supplemented with 10 v / v% fetal bovine serum in a 5% CO2 incubator. - Sinorhizobium meliloti M5N1CS (or S. meliloti strain M5N1CS).

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

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

[0036] After -72 h incubation, the resulting broth was centrifuged at 33,900 x g for 40 min at 20° C. The supernatant was purified by tangential ultrafiltration on a 100,000 standard molecular weight cut-off (NMWCO) polyethersulfone membrane from Sartorius (Gottingen, Germany) against distilled water. Finally, the retentate solution is lyophilized to obtain PGU of formula B having β-(1,4)-D-polyglucuronic acid chains O-acetylated at carbons 2 and 3 of the β-D-glucuronic acid units. 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, MA) equipped with 25 mm radius parallel plates with a 0.5 mm gap at 20 °C.

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

[0039] cytocompatibility -10T1 / 2 cells were plated in wells of a 96-well cell culture plate at 4 × 10 3 Cells were seeded / well and incubated in culture medium for 20 hours at 37° C. in a humidified 5% CO 2 incubator. The medium was then replaced with medium (0.2 mL) containing 0.5 w / v% PGU or PGU-Ph, and incubated for a further 24 hours. The medium containing the polymer was then replaced with medium (0.2 mL) containing 1 / 20 volume of reagents 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 was measured at 450 nm using a spectrophotometer. -Sodium alginate (Alg) and alginate with 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 wells of a 12-well cell culture dish at 0.5 mL / well. -The plate was then placed into a plastic container. Air containing 8 ppm H2O2, obtained by bubbling air through a 0.5 M H2O2 aqueous solution, was flowed into the plastic container at 10 L / min. After exposure to air containing -H2O2 for 15 min, the hydrogel-coated wells were rinsed sequentially with PBS and medium. -10T1 / 2 cells and HepG2 cells were suspended in a medium containing 0.3 mg / mL catalase, and 6 × 10 4Cells / well were injected into each well.

[0041] Printing process and system An extrusion 3D printing system developed by modifying a commercially available 3D printing system (purchased from Anycubic Co., Ltd., Guangdong, China, under the trade name Anycubic i3 Mega) is used for 3D bioprinting. -The extrusion 3D printing system includes a syringe pump to flow the ink, a 27-gauge stainless steel needle to extrude the ink, a bubbling system to deliver air containing 8 ppm H2O2, and a stirrer to layer the extruded ink. The ink flow rate in the needle and the moving speed of the stage were fixed at 22 mm / s. -Printing of cell-containing 3D hydrogel constructs was performed in a biological safety cabinet. Ink containing -1 w / v % PGU-Ph, or 1 w / v % PGU-Ph and 1 w / v % gelatin-Ph, and 5 U / mL HRP was used. The effect of ink extrusion on cells was determined by measuring the viability of 10T1 / 2 and HepG2 cells suspended in ink at -3x105 cells / mL. - The ink containing the cells was collected at the tip of the needle and the cells were stained with trypan blue dye for measurement using a hemocytometer. The viability of cells encapsulated in the 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 the first method for producing PGU-PH This synthesis is achieved according to the first method for producing PGU-Ph as follows. This was dissolved in 2-(N-morpholino)ethanesulfonic acid (MES) buffer solution (pH 6.0) at 1 w / v %. -Tyramine hydrochloride, NHS, and WSCD were added successively at 45 mM, 10 mM, and 20 mM, respectively, and the mixture was stirred at room temperature for 20 hours. - The resulting polymer was precipitated in acetone and then washed with 90% ethanol and 10% water until no absorbance at 275 nm, due to the presence of tyramine, was detectable in the washings. The resulting phenolized PGU is PGU-Ph according to the first embodiment of the 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 in particular shows that the PGU-Ph solution has a peak at 275 nm due to the Ph moiety, whereas the PGU solution does not have a peak at 275 nm. The content of the Ph moiety calculated based on a calibration curve obtained from tyramine solutions of known proportions is 3.7 x 10 -4 It is in mol-Ph / g.

[0044] Figure 2 shows the shear rate-viscosity profiles of the 1 and 2 w / v% PGU-Ph solutions and compares them to the shear rate-viscosity profiles of the 1 and 2 w / v% PGU solutions. Figure 2 notably shows 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 Solutions The hydrogel was produced according to the first method for producing a hydrogel structure according to the present invention, by HRP catalysis 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 hydrogelation time with 5 U / mL HRP and 0.1 mM H2O2. The gelation time of a 0.5 w / v% PGU-Ph solution is 6.0 seconds.

[0047] 3b and 3c show the gelation times measured for the 1.0 w / v% PGU-Ph solution. Figure 3 shows the effect of HRP and H2O2 concentrations on gelation. The gelation time decreases from 71 s to 2 s as the HRP concentration increases from 0.1 U / mL to 20 U / mL (Figure 3b). The gelation time decreases as the H2O2 concentration increases from 0.05 mM to 1 mM, but increases as the H2O2 concentration increases further. It is noted that higher concentrations of H2O2 lead to depolymerization of PGU-Ph, which then significantly reduces gelation.

[0048] Example 3: Cytocompatibility 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 cell morphology and mitochondrial activity after 20 hours of culture in a mixed solution of medium (50% by volume) and PBS (50% by volume) containing 0.5 w / v% of either the example PGU-Ph or its corresponding PGU.

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

[0051] The mitochondrial activity of cells incubated in solutions containing PGU and PGU-Ph was approximately 20% higher than that of those incubated in solutions containing Alg and Alg-Ph (p<0.03).

[0052] Example 4: Cell behavior on PGU-Ph hydrogels 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 hydrogels containing PGU-Ph. The day after seeding, most of the 10T1 / 2 cells and HepG2 cells were floating on the PGU-Ph hydrogel, and the HepG2 cells formed aggregates (Figures 5a, 5b).

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

[0054] No significant morphological differences were found between 10T1 / 2 cells on PUG-Ph+gelatin-Ph hydrogels and 10T1 / 2 cells on cell culture dishes. HepG2 cells seeded on PGU-Ph+gelatin-Ph hydrogels also attached, extended, and proliferated (Fig. 5b). However, their morphology was obviously different from that on cell culture dishes. HepG2 cells on cell culture dishes attached to the substrate and formed small aggregates the day after seeding. The cells then grew as a monolayer, and the size of the aggregates increased. HepG2 cells on PGU-Ph+gelatin-Ph hydrogels did not form obvious aggregates the day after seeding.

[0055] During the subsequent incubation period, HepG2 cells grew on the PGU-Ph+gelatin-Ph hydrogels and did not form obvious aggregates, which were quite different from those formed on cell culture dishes.

[0056] Example 5: 3D hydrogels as cell culture materials to support cell colony formation Printing. The effect of the 3D printing process and PGU-Ph hydrogel on cells was evaluated by printing hydrogel constructs encapsulating 10T1 / 2 and HepG2 cells. The viability of 10T1 / 2 and HepG2 cells the day after bioprinting was 92.3% and 91.6%, respectively, as stained with calcein-AM and PI. This result demonstrates that the printing process using PGU-Ph solution as the 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 constructs, and the size of the aggregates increased with increasing culture period (Figure 6b). There was no obvious increase in dead cells in both cells.

[0058] Example 6: 3D printing of hydrogels. To evaluate the feasibility of 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, transparent 3D-hydrogel constructs with good fidelity to the design (3D CAD model) were obtained when the solution was extruded in air containing 8 ppm HO. These results demonstrate the feasibility of a gellable PGU-Ph solution through HRP-mediated hydrogelation as a 3D printing ink.

[0059] Example 7: Synthesis of particles. To evaluate the feasibility of synthesizing PGU-Ph particles 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 min. The microbeads were then washed with water and stored in an ethanol / water solution (70 / 30). As shown in Figure 8, transparent PGU-Ph beads with good spherical shape were obtained.

[0060] Example 8: Synthesis of antioxidant PGU-Ph dry films. To evaluate the viability of antioxidant PGU-Ph film synthesis, 1 w / v% PGU-Ph solution was poured into a plastic Petri dish and dried at 50 °C for 24 h. A PGU film without the Ph moiety was used as a control. To evaluate the antioxidant effect of the PGU-Ph film, the free radical scavenging activity was measured using 1,1-diphenyl-2-picrylhydrazyl (DPPH). Briefly, the PGU-Ph film (100 mg) 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 h. The absorbance was then 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 are the absorbance at 517 nm for the PGU-Ph film and the control without the PGU-Ph film (i.e., DPPH solution), respectively.

[0062] A transparent PGU-Ph film was obtained, as shown in Figure 9a, and 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 the extracellular glucuronan produced by the Rhizobium meliloti M5N1 mutant." Carbohydrate Research, 240(24), 71-78.

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Claims

1. 1. A method for producing a polymeric compound of glucuronic acid containing at least one phenolic group, comprising: Providing a polymeric compound of glucuronic acid (PGU); reacting the polymeric compound of glucuronic acid with a phenolic compound to form a polymeric compound of glucuronic acid containing at least one phenolic group by radical polymerization or by chemical regioselective grafting of the phenolic group to a carboxyl group of glucuronic acid.

2. 2. A method for producing the polymeric compound of glucuronic acid according to claim 1, comprising: providing a radical of a polymeric compound of glucuronic acid of formula (C), 【Chemistry 1】 - reaction of said radical of formula (C) with a phenolic compound of formula (I) or (D), 【Chemistry 2】 by radical coupling to obtain a polymeric compound of glucuronic acid of formula (E) or (F) 【Chemistry 3】 method.

3. 2. A method for producing the polymeric compound of glucuronic acid according to claim 1, comprising: Polymeric compounds of glucuronic acid (PGU) of formula (B): or providing a monovalent ion carboxylate of formula (A) thereof; 【Chemistry 4】 Combined into one of the following: -(1)β-(1,4), and / or -(2) α-(1,4), and / or -(3) β-(1,3), and / or -(4) α-(1,3), and / or -(5) α-(1,2), and / or －（６）β－（１，４） (X = a monovalent ion M of a metal belonging to the alkali metal group) ＋ indicates, R = H and / or sulfate group and / or COCH ３ represents an acyl group containing n is an integer selected such that the molar mass of the polymeric compound is between 10 and 1000 kilodaltons; - dissolving said polymeric compound of glucuronic acid (B) or its monovalent ionic carboxylate of formula (A) in an acid buffer solution to form a solution of PGU; - adding sequentially to said solution of PGU, a phenol group consisting of X-(Y-amino-Z-hydroxyalkyl)-n-hydroxyl-phenol or a salt derivative thereof, NHS and WSCD to form a final composition of reaction medium; - stirring said final composition at room temperature for at least 4 hours until a resultant polymer consisting of a polymeric compound of glucuronic acid containing a phenolic hydroxyl moiety is obtained.

4. 4. The method according to any one of claims 1 to 3, wherein the X-(Y-amino-Z-hydroxyalkyl)-n-hydroxyl-phenol or its salt derivative or the phenolic compound of formula (D) is selected from the group consisting of tyramine, dopamine and octopamine, preferably tyramine.

5. A polymeric compound of glucuronic acid containing at least one phenolic group obtainable by the process according to claim 1.

6. A solution comprising water and the polymer compound of glucuronic acid according to claim 5.

7. 1. A gel-forming composition comprising: A polymer compound of glucuronic acid according to claim 5; oxidase, or H ２ O ２ or H ２ O ２ and a crosslinking catalyst comprising a compound capable of generating in situ a gel-forming composition.

8. 8. The gel-forming composition of claim 7, wherein the polymeric compound of glucuronic acid is bound to an oxidase as a cross-linking catalyst selected from the group consisting of oxidoreductases, peroxidases, catalases, laccases, tyrosinases, and monosaccharide oxidases, and mixtures thereof, preferably horseradish peroxidase (HRP).

9. 9. The gel-forming composition of claim 8, wherein the oxidase is horseradish peroxidase (HRP) present in the composition in an amount of at least 0.01 U / mL, preferably between 0.1 U / mL and 20 U / mL, and more preferably around 5 U / mL.

10. The glucuronic acid polymer compound is added in an amount of 0.05 mmol / L to 1 mmol / L. ２ O ２ 8. The gel-forming composition of claim 7, wherein the compound is linked to a compound containing:

11. 11. A gel-forming composition according to any one of claims 7 to 10, wherein the polymeric compound of glucuronic acid is present in an amount of from 0.01% to 8% w / v, preferably from 0.1% to 2% w / v.

12. 8. The gel-forming composition of claim 7, further comprising another biodegradable polymer.

13. The biodegradable polymer is selected from collagen, adhesion factors, gelatin, and mixtures thereof.

13. The gel-forming composition of claim 12, wherein the gel-forming composition is a polysaccharide or a protein selected from the group consisting of:

14. 14. The gel-forming composition of claim 13, wherein the biodegradable polymer is a gelatin derivative containing a phenolic hydroxyl moiety (gelatin-Ph).

15. 8. The gel-forming composition of claim 7, further comprising suspension cells of animal, bacterial, or plant origin.

16. A method for producing a hydrogel structure, comprising the steps of: Providing a gel-forming composition according to claim 7; The gel-forming composition is When the gel-forming composition comprises an oxidase, ２ O ２ Contact with a fluid or gaseous medium containing The gel-forming composition is ２ O ２ or H ２ O ２ or, in the case of a compound capable of generating in situ an oxidase, by contacting the hydrogel with the hydrogel.

17. A method for producing a hydrogel structure, comprising the steps of: - providing a gel-forming composition according to claim 15, - said gel-forming composition - if the gel-forming composition comprises an oxidase, H ２ O ２ Contact with a fluid or gaseous medium containing - the gel-forming composition is ２ O ２ or H ２ O ２ or, in the case of a compound capable of generating in situ an oxidase, by contacting the hydrogel with the hydrogel.

18. 18. The method of any one of claims 16 or 17, comprising a bioprinting process for producing a one-dimensional hydrogel structure, or a two-dimensional hydrogel structure, or a three-dimensional hydrogel structure.

19. A hydrogel structure obtained by combining the methods of claims 16 and 18.

20. A cell-containing hydrogel structure obtained by combining the methods according to claims 17 and 18.

21. 22. Use of the hydrogel structure of claim 21 as a three-dimensional cell culture material for supporting cell colony formation.

22. 22. Use of the hydrogel structure of claim 21 as a patch or salve.

23. 24. Use of the cell-containing hydrogel construct of claim 23 for tissue regeneration and / or tissue engineering.

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