KR102831254B1 - Preparation method of in situ crosslinkable copper-containing hydrogel as a controlled nitric oxide-releasing scaffold, and biomedical use thereof - Google Patents

Abstract

The present invention relates to a method for producing a copper-containing in situ hydrogel using a polymer into which a phenol group or a compound containing a phenol group is introduced, a copper compound, an enzyme, and hydrogen peroxide, and to a use thereof. According to the present invention, under the catalytic action of an enzyme, a polymer is oxidized to form a cross-linked network and deposition of Cu nanoparticles, thereby forming a cross-linked copper-containing hydrogel, and the cross-linked copper-containing hydrogel continuously releases copper ions, thereby promoting NO production in the presence of an endogenous NO donor (RSNO) for nearly 3 weeks.
Accordingly, the in vitro bioactivity of copper-containing in situ hydrogels can be confirmed by regulating anti-inflammatory M2 phenotypic polarization and stimulating endothelial cell migration due to the synergistic effect of Cu ions and NO, and the copper-containing hydrogels can promote the wound healing process.

Description

{Preparation method of in situ crosslinkable copper-containing hydrogel as a controlled nitric oxide-releasing scaffold, and biomedical use thereof}

The present invention relates to a method for producing an in situ cross-linked hydrogel using copper for sustained release of nitric oxide and its biomedical use.

Hydrogel is a jelly-like substance formed through physicochemical cross-linking of polymers dissolved in water. It has excellent hydrophilicity, so it can easily absorb water, and its strength and shape can be easily controlled, so it is used as a support for tissue engineering or drug delivery. In addition, hydrogel is being actively studied as an alternative to methods with low drug delivery efficiency in the body due to its excellent biocompatibility and biodegradability, controlled drug release, and minimally invasive properties that can achieve phase transition after injection into the body. The main purpose as a drug delivery vehicle is to release drugs higher than the effective concentration for a long period of time at the site of disease without requiring frequent injections that can cause side effects.

In addition, chronic or non-healing wounds due to extensive inflammatory phase and reduced vascularization pose serious health problems to humans. Therefore, the development of biomaterials that shorten the inflammatory phase and promote angiogenesis is a promising treatment for chronic wounds. Currently, nitric oxide (NO) is an endogenous gas molecule involved in many physiological processes, including immune response, apoptosis, and inflammation. However, the clinical application of NO is limited due to its explosive release from biomaterials.

Accordingly, research is needed on a technology that can continuously form nitric oxide by controlling the release in the early stage using a cross-linked hydrogel.

Republic of Korea Publication Patent No. 10-2014-0037757 (Published on March 27, 2014)

The present invention relates to a method for producing a copper-containing in situ hydrogel using a polymer into which a phenol group or a compound containing a phenol group is introduced, a copper compound, an enzyme, and hydrogen peroxide, and to a use thereof.

In order to achieve the same purpose, the present invention provides a copper-containing in situ hydrogel, which comprises a polymer having at least one of a phenol group and a catechol group introduced therein and a copper compound, wherein copper is coordinated with at least one of the phenols and catechols introduced into a side chain of the polymer to include copper ions in a hydrogel matrix, and wherein at least one of the phenols and catechols introduced into the side chain of the polymer is crosslinked by bonding with each other.

In addition, the present invention provides a method for producing a copper-containing in situ hydrogel, comprising the steps of: preparing a solution in which a polymer substituted with a compound including a phenol group and an enzyme are dissolved; and adding a copper compound and hydrogen peroxide to the solution to crosslink the solution.

In addition, the present invention provides a copper-containing in situ hydrogel manufactured by the method for manufacturing a copper-containing in situ hydrogel described above.

In addition, the present invention provides an anti-inflammatory composition comprising the copper-containing in situ hydrogel described above.

In addition, the present invention provides a composition for tissue regeneration comprising the copper-containing in situ hydrogel described above.

In addition, the present invention provides a composition for angiogenesis comprising the copper-containing in situ hydrogel described above.

According to the present invention, under the catalytic action of an enzyme, a polymer is oxidized to form a cross-linked copper-containing hydrogel for the formation of a cross-linked network and deposition of Cu nanoparticles, and the cross-linked copper-containing hydrogel continuously releases copper ions, thereby promoting NO production in the presence of an endogenous NO donor (RSNO) for nearly 3 weeks.

Accordingly, the in vitro bioactivity of copper-containing in situ hydrogels can be confirmed by regulating anti-inflammatory M2 phenotypic polarization and stimulating endothelial cell migration due to the synergistic effect of Cu ions and NO, and the copper-containing hydrogels can promote the wound healing process.

Figure 1 is a schematic diagram showing that nitric oxide (NO) is continuously released from a copper-containing hydrogel to stimulate angiogenesis and anti-inflammatory processes.
Figure 2 is a schematic diagram showing the formation of nitric oxide releasing hydrogels via the catalytic action of horseradish peroxidase and tyrosinase, and the GH polymers are shown to be cross-linked via various interactions between catechol and phenol groups.
Figure 3 shows the results of an experiment on the characteristics of the hydrogel of the present invention. (a) is a graph showing the gelation time defined by the vial tilting method, (b) is a graph showing the mechanical strength evaluated by a rheometer, (c) is a graph showing the decomposition rate of lysozyme, and (d) is a scanning electron microscope (SEM) image of a cross-section.
Figure 4 shows the cumulative concentrations of copper ions released from the hydrogel and nitric oxide generated. (a) is a graph showing the cumulative concentration of copper ions released from the GH/Cu hydrogel after incubation in PBS, and (b) is a graph showing the cumulative concentration of nitric oxide (NO) released by incubating the GH/Cu hydrogel in the presence of 10 μM GSH as a nitric oxide (NO) donor.
Figure 5 shows the in vitro anti-inflammatory properties. (a) is an immunofluorescence staining image of attached macrophages with DAPI (blue) and CD163 (green), (b) is a graph showing the quantification of the relative mean fluorescence intensity of CD163 compared to the M2 macrophage control, and (c) is a graph showing the concentration of TGF-β released from macrophages by ELISA.
Figure 6 shows in vitro angiogenic activity, where (a) is a scanning electron microscope (SEM) image of live (green)/dead (red) stained cells, (b) is a graph showing the viability of HUVECs after incubation with hydrogel samples for 24 h by WST-1 assay, (c) is a graph showing the quantification of migration rate, and (d) is an optical microscope image showing the migration of HUVECs using a wound scratch migration assay.
Figure 7 shows the angiogenic activity evaluated by in vitro endothelial tube formation assay, where (a) is an optical microscope image showing the formation of a cell network, and (b) is a graph showing quantitative evaluation of various parameters including the number of nodes, (c) the number of branches, and (d) the total branch length.
Figure 8 shows angiogenic activity assessed by the chicken chorionic allantoic membrane (CAM) angiogenesis assay in oocytes, where (a) is an optical image of new blood vessel formation in the CAM, and (b) is a graph quantifying the blood vessels by Image J.
Figure 9 is an image showing the angiogenic activity evaluated by in vivo subcutaneous injection of the hydrogel. (a) is an image showing histological analysis using hematoxylin-eosin (H&E) staining, (b) VEGFA antibody (purple: nuclei, brown: VEGFA), and (c) α-SMA staining for smooth muscle cell staining (purple: nuclei, brown: α-SMA).

Hereinafter, in order to explain the present invention more specifically, preferred embodiments according to the present invention will be described in more detail with reference to the attached drawings. However, the present invention is not limited to the embodiments described herein and may be embodied in other forms.

In the present invention, copper (Cu) ions (NO generating catalysts) were incorporated into phenol-rich gelatin-based hydrogels (GH/Cu) for the in situ generation of NO in the presence of endogenous S-nitrosothiols. Under the catalytic action of tyrosinase and horseradish peroxidase, GH was oxidized for the formation of cross-linked networks and deposition of Cu nanoparticles, and GH/Cu exhibited sustained release of Cu ions, thereby promoting NO production in the presence of endogenous NO donors for almost 3 weeks. The synergistic effect of Cu ions and NO may explain the in vitro bioactivity of GH/Cu by regulating anti-inflammatory M2 phenotype polarization and stimulating the migration of endothelial cells. The GH/Cu hydrogel is suggested as a promising material for promoting wound healing process.

The “GH/Cu hydrogel” of the present invention refers to a hydrogel prepared by mixing a gelatin-hydroxyphenyl propionic acid (GH) polymer, a copper compound, horseradish peroxidase (HRP), and hydrogen peroxide (H ₂ O ₂ ).

“Angiogenesis” of the present invention refers to the process of new blood vessel formation, that is, the generation and differentiation of new blood vessels into cells, tissues or organs. Angiogenesis of the present invention includes vascular regeneration, vascular restoration and vascular differentiation related to the process of blood vessel formation, including endothelial cell activation, migration, proliferation, matrix remodeling and cell stabilization.

The present invention provides a copper-containing in situ hydrogel, which comprises a polymer having at least one of a phenol group and a catechol group introduced therein and a copper compound, wherein copper is coordinated with at least one of the phenols and catechols introduced into a side chain of the polymer to include copper ions in a hydrogel matrix, and wherein at least one of the phenols and catechols introduced into the side chain of the polymer is crosslinked by bonding with each other.

Figure 1 is a schematic diagram showing that nitric oxide (NO) is continuously released from a copper-containing in situ hydrogel to stimulate angiogenesis and anti-inflammatory processes.

Figure 2 is a schematic diagram showing the formation of nitric oxide releasing hydrogels via the catalytic action of horseradish peroxidase and tyrosinase, and the GH polymers are shown to be cross-linked via various interactions between catechol and phenol groups.

Specifically, in the presence of H ₂ O ₂ , phenol is oxidized to phenol radical by enzyme (horseradish peroxidase, HRP) to enable crosslinking of aromatic rings by C-C or C-O coupling, and in an aerobic environment, phenol is oxidized to catechol by enzyme (tyrosinase) to provide active moieties for crosslinking reaction between phenol-rich gelatin polymers and binding sites for coordination and stabilization of copper ions. Thus, here, two enzyme-catalyzed reactions by horseradish peroxidase (HRP) and tyrosinase occur to form a hydrogel matrix.

The above copper is incorporated into and stabilized in the hydrogel matrix through metal-catecholate coordination bonds. Catechols formed by enzyme (tyrosinase) catalysis can react with each other (hydrogen bonds, π-π interactions, coupling reactions, etc.) to cross-link the polymer network and stabilize copper in the hydrogel matrix through metal coordination with copper ions.

The copper-containing in situ hydrogel according to the present invention can continuously release copper ions by including copper ions in the hydrogel matrix, and can promote nitric oxide production in the presence of an endogenous nitric oxide source for a long period of time (about 3 weeks). Due to the synergistic effect of copper ions and nitric oxide, anti-inflammatory M2 phenotypic polarization can be controlled, and thus anti-inflammatory properties can be exhibited.

In addition, the present invention provides an anti-inflammatory pharmaceutical composition, a pharmaceutical composition for tissue regeneration, and a pharmaceutical composition for angiogenesis comprising the copper-containing in situ hydrogel.

The above pharmaceutical composition may additionally contain a carrier, excipient or diluent commonly used in the manufacture of pharmaceutical compositions.

When the composition of the present invention is a pharmaceutical composition, for administration, it may contain, in addition to the effective ingredients described above, a pharmaceutically acceptable carrier, excipient or diluent. The carrier, excipient and diluent may include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

The pharmaceutical composition of the present invention can be formulated and used in the form of an external preparation, a suppository, or a sterile injection solution according to a conventional method. Specifically, when formulating, it can be prepared using diluents or excipients such as fillers, weighting agents, binders, wetting agents, disintegrating agents, and surfactants that are commonly used. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and tasks. Non-aqueous solvents and suspending agents can be used, such as propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases can be used, such as withepsol, macrosol, Tween 61, cacao butter, laurin butter, and glycerogelatin.

The pharmaceutical composition of the present invention can be administered parenterally (for example, intravenously, subcutaneously, intraperitoneally, or topically) depending on the intended method. In addition, the dosage of the pharmaceutical composition of the present invention varies depending on the weight, age, sex, health condition, diet, administration time, administration method, excretion rate, and disease severity of the subject, but is not limited thereto.

The present invention provides a method for producing a copper-containing in situ hydrogel, comprising the steps of: preparing a solution in which a polymer substituted with a compound containing a phenol group and an enzyme are dissolved; and adding a copper compound and hydrogen peroxide to the solution to crosslink the solution.

Specifically, in the cross-linking step, in the presence of hydrogen peroxide (H ₂ O ₂ ), phenol is oxidized to a phenol radical by an enzyme (horseradish peroxidase, HRP) to enable cross-linking of an aromatic ring by C-C or C-O coupling, and in an aerobic environment, phenol is oxidized to catechol by an enzyme (tyrosinase) to provide an activated moiety for a cross-linking reaction between phenol-rich gelatin polymers and a binding site for coordination and stabilization of copper ions. Thus, here, two enzyme-catalyzed reactions by horseradish peroxidase (HRP) and tyrosinase occur to form a hydrogel matrix.

In addition, in the cross-linking step, the copper is incorporated into and stabilized in the hydrogel matrix through metal-catecholate coordination bonds. Catechols formed by enzyme (tyrosinase) catalysis can react with each other to cross-link the polymer network through at least one of hydrogen bonding, π-π interaction, and coupling reaction, and can stabilize copper in the hydrogel matrix through metal coordination with copper ions.

The above polymer comprises at least one selected from the group consisting of gelatin, chitosan, heparin, cellulose, dextran, dextran sulfate, chondroitin sulfate, keratan sulfate, dermatan sulfate, alginate, collagen, albumin, fibronectin, laminin, elastin, vitronectin, hyaluronic acid, fibrinogen and multi-polymer, and at least one of a phenol group and a catechol group may be introduced into a side chain of the polymer.

By including the polymer as described above, copper can be included in the hydrogel matrix by coordination bonding with the phenol group or catechol group introduced to the polymer side chain, and the phenol group or catechol group can be crosslinked by bonding to each other to form a crosslinked hydrogel.

Specifically, the enzyme may include one or more selected from the group consisting of horseradish peroxidase, glutathione peroxidase, haloperoxidase, myeloperoxidase, catalase, hemoprotein, peroxide, peroxiredoxin, animal heme-dependent peroxidases, thyroid peroxidase, vanadiumbromoperoxidase, lactoperoxidase, tyrosinase, and catechol oxidase.

The compound containing the above phenol group may be at least one selected from the group consisting of hydroxyphenyl propionic acid, 4-hydroxyphenyl acetic acid, tyrosine, tyramine, tetronic tyramine, and PEG-tyramine.

A method for producing a copper-containing in situ hydrogel, characterized in that the compound containing the catechol group is at least one selected from the group consisting of dopamine, gallic acid, quercetin, tannic acid, and epigallocatechin gallate (EGCG).

The above copper compound may include at least one selected from copper sulfate (CuSO ₄ ) and copper chloride (CuCl ₂ ).

The copper compound may be present at a concentration of 10 to 75 μM, 25 to 75 μM, 25 to 50 μM or 50 to 75 μM.

The concentration of the enzyme may be 0.001 to 0.010 mg/ml or 0.001 to 0.005 mg/ml.

The concentration of the polymer may be 30 to 100 mg/ml or 30 to 70 mg/ml.

The above cross-linking step may add hydrogen peroxide at a concentration of 0.01 to 0.1 wt% or 0.01 to 0.05 wt%.

In addition, the present invention provides a copper-containing in situ hydrogel manufactured by the above manufacturing method.

Specifically, the copper-containing in situ hydrogel can include copper ions within the hydrogel matrix by coordination bonding copper with at least one of phenol and catechol introduced into the side chain of the polymer.

In addition, the copper-containing in situ hydrogel can form a cross-linked hydrogel matrix in which at least one of phenol and catechol introduced into the side chain of the polymer is bonded to each other.

Hereinafter, in order to help understand the present invention, examples will be given and described in detail. However, the following examples are only intended to illustrate the content of the present invention, and the scope of the present invention is not limited to the following examples. The examples of the present invention are provided to more completely explain the present invention to a person having average knowledge in the art.

Example 1

To prepare copper-containing in situ formed hydrogels, GH polymer (50 mg/ml) was completely dissolved in DIW at 37 °C. Then, the polymer solution was mixed with two kinds of enzymes including horseradish peroxidase (HRP) (0.01 - 0.05 mg/ml) and tyrosinase (Tyr) (0.25 kU/ml) in a microtube. In another microtube, the polymer solution was mixed with H ₂ O ₂ (0.02 wt%) and CuSO ₄ (0, 25, 50, 75 μM). Then, the solutions in the two microtubes were mixed (volume ratio = 1:1) to prepare copper-containing in situ hydrogels.

Experimental Example 1 - Measurement of gelation time

The gelation time of the hydrogel was measured as the point at which no flow was observed after the phase transition of the hydrogel in the microtube after uniform mixing of the two solutions, and the results are shown in Fig. 3 (a).

Looking at Fig. 3 (a), which shows the gelation time, it can be confirmed that as the concentration of HRP increases from 0.01 to 0.05 mg/mL, the gelation time decreases from 110 seconds to 20 seconds.

Experimental Example 2 - Rheological Analysis of Hydrogels

To evaluate the mechanical strength of the hydrogel, a total of 300 μL of mechanical strength was measured using a rheometer (Advanced Rheometer GEM-150-050, Bohlin Instruments, Cranbury, NJ, USA).

To evaluate the degradation behavior of the hydrogel, it was cultured at 37°C in PBS containing collagen-decomposing enzyme, the culture medium was replaced at regular intervals, and the residual weight was measured.

In order to analyze the morphological structure of the hydrogel, the formed hydrogel was freeze-dried, and a cross-section was cut to observe the internal structure using FE-SEM (Field Emission Scanning Electron Microscopy). The results are shown in Fig. 3 (b) to (d).

Looking at Figures 3 (b) and (c), the elastic modulus of the hydrogel and the degradation rate of the hydrogel in the presence of lysozyme over time are shown. It can be confirmed that as the content of copper ions increases, the elastic modulus decreases while the degradation rate increases.

Looking at Fig. 3 (d), it can be seen from the image of the cross-section of the hydrogel that as the content of copper ions increases, the degree of cross-linking decreases and the pore size increases.

Experimental Example 3 - Cumulative concentration of nitric oxide

To measure the Cu ions released from the hydrogel, a copper assay kit (Sigma-Aldrich, St. Louis, MO, USA USA) was used. The formed hydrogel was placed in a PBS solution and incubated in a darkroom at 37°C. The supernatant was recovered according to the characteristic time, and new PBS solution was added. The recovered sample was then reacted for 5 minutes at room temperature using a copper assay kit. The absorbance of the standard substance of Cu ions was measured at 359 nm. Since Tyr is an enzyme containing copper, if Cu ions are released from Tyr during the hydrogel degradation process, it may affect the results of this experiment. Therefore, GH hydrogels manufactured without Tyr were used as control samples in this experiment.

To evaluate the NO release behavior of the hydrogel, the hydrogel was placed in a PBS solution containing NO donors (GSNO and GSH) and cultured at 37℃ in a darkroom, and the NO donor solution was withdrawn and replaced at regular intervals. The withdrawn solution was reacted with an equal amount of Griess solution in a darkroom for 15 minutes, and the absorbance was measured at 540 nm with a Cytation™ 3 Cell Imaging Multi-Mode Reader (BioTek™, USA) to evaluate the release behavior of NO released from the hydrogel. Quantitative analysis was performed using a calibration curve obtained with 0 - 1 μM NaNO₂, and the results are shown in Fig. 4.

Figure 4 shows the cumulative concentrations of copper ions released from the hydrogel and nitric oxide generated. (a) is a graph showing the cumulative concentration of copper ions released from the GH/Cu hydrogel after incubation in PBS, and (b) is a graph showing the cumulative concentration of nitric oxide (NO) released by incubating the GH/Cu hydrogel in the presence of 10 μM GSH as a nitric oxide (NO) donor.

Looking at Figure 4, it shows the cumulative concentration of copper ions released from the hydrogel and nitric oxide generated. In Figure 4 (a), it can be confirmed that as the copper content increases, the copper ions released from the hydrogel increase, and (b) it can be confirmed that as the copper content in the hydrogel increases in the presence of GSNO and GSH, the amount of nitric oxide released also increases.

Experimental Example 4 - Anti-inflammatory Test

Human monocyte-1 (THP-1) cells were cultured in RPMI-1640 medium, collected, and cultured in a medium containing 12-myristate 13-acetate (PMA) for 2 days, and then in a medium without PMA for 1 day to induce differentiation of THP-1 into M0 cells. Hydrogels were treated on the M0 cell layer and cultured depending on the presence or absence of NO donors. After 48 h, the differentiation potential into pro-inflammatory M1 and anti-inflammatory M2 macrophages was analyzed according to the content of released NO. The cells were treated with paraformaldehyde and Triton X-100, and then cultured with primary antibodies of the markers. (M2 marker: CD163, TGF-ß). After reaction with IgG-fluorescent secondary antibodies, DAPI staining was performed, and fluorescence qualitative analysis of the differentiated anti-inflammatory M2 markers was performed using a confocal laser scanning microscope (Zeiss LSM 780, Carl Zeiss, Jena, Germany). The degree of M2 polarization was evaluated by lysing the differentiated M2 cells and quantitative analysis of TGF-ß and CD163 using ELISA, and the results are shown in Figure 5.

(a) Immunofluorescence staining image of attached macrophages with DAPI (blue) and CD163 (green), (b) is a graph showing the quantification of the relative mean fluorescence intensity of CD163 compared to the M2 macrophage control, and (c) is a graph showing the concentration of TGF-β released from macrophages by ELISA.

Figure 5 shows the in vitro anti-inflammatory properties. (a) is an immunofluorescence staining image of attached macrophages with DAPI (blue) and CD163 (green), (b) is a graph showing the quantification of the relative mean fluorescence intensity of CD163 compared to the M2 macrophage control, and (c) is a graph showing the concentration of TGF-β released from macrophages by ELISA.

Looking at Fig. 5 (a) and (b), the immunofluorescence staining image and the quantification of the relative average fluorescence intensity of CD163 are shown. First, in the control group, it can be seen that the green fluorescence is higher in M2 macrophages with high CD163 expression compared to M0 macrophages. Based on this, it can be confirmed that the green expression is significantly greater in the experimental group treated with GH/Cu hydrogel in the presence of NO donors compared to the untreated experimental group. This indicates that polarization toward anti-inflammatory M2 has occurred.

In addition, looking at Fig. 5 (c), it can be seen that the concentration of TGF-β released from macrophages was shown, and it can be confirmed that the experimental group treated with GH/Cu showed a significant increase in the concentration of TGF-β compared to the M0 control group or the experimental group treated with only GH.

Experimental Example 5 - In vitro angiogenic activity

Human umbilical cord blood endothelial cells (HUVECs) were cultured with hydrogels in EGM-2 medium, and the angiogenic ability was evaluated based on the scratch recovery of the cell layer and the NO content released through tube formation of the cells.

The growth of HUVECs according to the NO content released was evaluated using WST-1 and live/dead fluorescence analysis. In addition, HUVECs were cultured to form a cell layer, and then a scratch was formed using a pipette. The hydrogel was placed on the cell layer, and new medium was added and NO donors were supplied every 4 hours. The change in the recovery area of the scratch at a certain time point was measured to evaluate the recovery ability of the vascular endothelial cell scratch according to the NO content.

To evaluate the formation of angiogenic capillary structures of HUVECs, a thin gel layer was formed on TCP with Corning Matrigel Matrix solution, and HUVECs were cultured on it for 1 day. Afterwards, the hydrogel was placed on the cell layer and cultured every 4 hours depending on the presence or absence of NO donors. After 12 hours, the formation of angiogenic capillary-like structures of HUVECs was measured under an optical microscope, and the length of the tubes was calculated and evaluated using the Image J program, and the results are shown in Figs. 6 and 7.

Figure 6 shows in vitro angiogenic activity, where (a) is a scanning electron microscope (SEM) image of live (green)/dead (red) stained cells, (b) is a graph showing the viability of HUVECs after incubation with hydrogel samples for 24 h by WST-1 assay, (c) is a graph showing the quantification of the migration rate, and (d) is an optical microscope image showing the migration of HUVECs using a wound scratch migration assay.

Figure 7 shows the angiogenic activity evaluated by in vitro endothelial tube formation assay, where (a) is an optical microscope image showing the formation of a cell network, and (b) is a graph showing quantitative evaluation of various parameters including the number of nodes, (c) the number of branches, and (d) the total branch length.

Looking at Figure 6, it can be confirmed that the proliferation of HUVEC cells in the presence of NO donors was clearly increased in the experimental group treated with GH/Cu, and as the content of copper ions increased, the production of NO increased, which resulted in faster migration of HUVECs.

Looking at Figure 7, it can be seen that in the absence of NO donors, tube-shaped cell alignment did not occur except in the experimental group treated with VEGF, whereas in the presence of NO donors, as the copper content increased, tube-shaped cell alignment clearly occurred and at the same time, a significant increase in the number of nodes, branches, and total branch length occurred.

Experimental Example 6 - Angiogenic activity in chicken chorioallantoic membrane

The angiogenic properties of the hydrogel were investigated using an in ovo chicken chorioallantoic membrane (CAM) assay. First, fertilized eggs (domestic, Korea) were hatched for 7 days (days 0–7) at 37–38°C, 40–60% humidity, and O2 presence. After 7 days of hatching, the eggs were removed and disinfected with 70% alcohol. Then, a window measuring approximately 1 cm × 1 cm was created in the eggshell, and 100 μL of preformed hydrogel sample was carefully placed on the CAM of the embryo. The window was sealed with sterilized parafilm to prevent infection during further culture. On day 10, images of the CAM-hydrogel composites were captured with a digital camera to evaluate the initial tissue response. The quantitative analysis of blood vessels around the hydrogel was calculated using the Image J program. In this experiment, GH hydrogel containing 10 ng/mL of VEGF was used as a positive control, and the results are shown in Fig. 8.

Figure 8 shows angiogenic activity assessed by the chicken chorionic allantoic membrane (CAM) angiogenesis assay in oocytes, where (a) is an optical image of new blood vessel formation in the CAM, and (b) is a graph quantifying the blood vessels by Image J.

Looking at Figure 8, it can be confirmed through CAM analysis that a large number of blood vessels were formed in the experimental group treated with VEGF, and compared to this, it can be confirmed that as the content of copper ions in the hydrogel increases, it effectively stimulates blood vessel formation.

Experimental Example 7 - In Vivo Angiogenic Activation

A mouse model was used to evaluate the in vivo angiogenic ability using an animal model. Following an ethically approved protocol, mice were anesthetized, injected subcutaneously with hydrogel, and 3 weeks later, subcutaneous tissues containing hydrogels were harvested to analyze the angiogenic characteristics for in vivo tissue regeneration. The harvested tissues were immediately fixed in 4% paraformaldehyde for one day, embedded in paraffin, and sectioned. The tissue sections were subjected to histological analysis using hematoxylin and eosin (H&E) and immunohistochemical analysis using anti-αSMA and VEGFA antibodies, which are angiogenesis-related factors, and the results are shown in Fig. 9.

Figure 9 is an image showing the angiogenic activity evaluated by in vivo subcutaneous injection of the hydrogel. (a) is an image showing histological analysis using hematoxylin-eosin (H&E) staining, (b) VEGFA antibody (purple: nuclei, brown: VEGFA), and (c) α-SMA staining for smooth muscle cell staining (purple: nuclei, brown: α-SMA).

Looking at Figure 9, it can be seen that in the experimental group treated with GH hydrogel, no cell invasion occurred inside the hydrogel, whereas in the experimental group treated with GH/Cu25 hydrogel, a large number of cells invaded inside the hydrogel and were in the initial stage of forming blood vessels. In addition, in the experimental groups treated with GH/Cu50 and GH/Cu75 hydrogels, it can be seen that, similar to the experimental group treated with VEGF, a large number of blood vessels were formed at the edges and inside of the cells.

While the specific parts of the present invention have been described in detail above, it is obvious to those skilled in the art that such specific description is merely a preferred embodiment and that the scope of the present invention is not limited thereby. In other words, the actual scope of the present invention is defined by the appended claims and their equivalents.

Claims (17)

A hydrogel matrix comprising a gelatin-hydroxyphenyl propionic acid (GH) polymer having at least one phenol group and a catechol group introduced therein and a copper compound,
Copper is coordinated with at least one of phenol and catechol introduced into the side chain of the polymer to include copper ions in the hydrogel matrix,
The above hydrogel matrix is crosslinked by binding at least one of phenol and catechol introduced into the side chain of the polymer to each other,
The phenol introduced into the above polymer side chain is oxidized into a phenol radical by an enzyme, and an aromatic ring is connected by at least one of CC and CO bonds to form a polymer network.
The catechol introduced into the above polymer side chain is phenol introduced into the polymer by enzymes in an aerobic environment, and is oxidized.
A copper-containing in situ hydrogel characterized in that the catechol stabilizes copper in a hydrogel matrix through coordination bonding with copper ions.
delete delete An anti-inflammatory pharmaceutical composition comprising a copper-containing in situ hydrogel according to claim 1. delete delete A step for preparing a solution in which gelatin-hydroxyphenyl propionic acid (GH) polymer and enzyme are dissolved; and
A step of cross-linking by adding a copper compound and hydrogen peroxide to the above solution;
The copper compound is contained at a concentration of 50 to 75 μM,
A method for producing a copper-containing in situ hydrogel, characterized in that the cross-linking step comprises forming a polymer network by cross-linking aromatic rings through at least one of CC and CO bonds by oxidizing phenol into phenol radicals through the catalytic action of an enzyme, and copper is incorporated into and stabilized in the hydrogel matrix through metal-catecholate coordination bonds.
delete In paragraph 7,
A method for producing a copper-containing in situ hydrogel, characterized in that the cross-linking step comprises cross-linking a polymer network by reacting catechols formed by the catalytic action of an enzyme. delete delete In paragraph 7,
A method for producing a copper-containing in situ hydrogel, wherein the enzyme comprises one or more selected from the group consisting of horseradish peroxidase, glutathione peroxidase, haloperoxidase, myeloperoxidase, catalase, hemoprotein, peroxide, peroxiredoxin, animal heme-dependent peroxidases, thyroid peroxidase, vanadiumbromoperoxidase, lactoperoxidase, tyrosinase, and catechol oxidase. In paragraph 7,
A method for producing a copper-containing in situ hydrogel, wherein the copper compound comprises at least one selected from copper sulfate (CuSO ₄ ) and copper chloride (CuCl ₂ ). delete In paragraph 7,
A method for producing a copper-containing in situ hydrogel, characterized in that the concentration of the polymer is 30 to 100 mg/ml. delete In paragraph 7,
A method for producing a copper-containing in situ hydrogel, characterized in that the cross-linking step comprises adding hydrogen peroxide at a concentration of 0.01 to 0.05 wt%.