WO2023149692A1

WO2023149692A1 - Method for preparing copper-mediated in situ crosslinkable hydrogel for sustained release of nitric oxide

Info

Publication number: WO2023149692A1
Application number: PCT/KR2023/001166
Authority: WO
Inventors: 박기동; 린 트란디우; 풍 레티
Original assignee: 아주대학교산학협력단
Priority date: 2022-02-07
Filing date: 2023-01-26
Publication date: 2023-08-10
Also published as: KR20230119462A

Abstract

The present invention relates to a method for preparing a copper-containing in situ hydrogel and use thereof, the method using a polymer into which a phenol group or a compound including a phenol group has been introduced, a copper compound, an enzyme, and hydrogen peroxide. According to the present invention, under the catalysis of enzymes, the polymer is oxidized for formation of a crosslinking network and deposition of Cu nanoparticles, so as to form a cross-linked copper-containing hydrogel, and the cross-linked copper-containing hydrogel allows sustained release of copper ions, and thus, for almost three weeks, NO production may be promoted in the presence of an endogenous NO donor (RSNO). Accordingly, due to the synergistic effect between Cu ions and NO, anti-inflammatory M2 phenotype polarization is controlled and the movement of endothelial cells is stimulated, and thus in vitro bioreactivity of the copper-containing in situ hydrogel can be confirmed, and the copper-containing hydrogel can promote a wound healing process.

Description

Manufacturing method of slow-release nitric oxide release in situ cross-linked hydrogel using copper and biomedical use thereof

The present invention relates to a method for preparing a slow-release nitric oxide-releasing in situ crosslinked hydrogel using copper and a biomedical use thereof.

Hydrogel is a jelly-like material through physicochemical crosslinking of polymers dissolved in water. It has excellent hydrophilicity and can easily absorb water, as well as easily control its strength and shape, making it a scaffold or drug for tissue engineering. Used for transmission, etc. In addition, hydrogels are being actively studied as alternatives to methods with low in vivo drug delivery efficiency due to their excellent biocompatibility, biodegradability, control of drug release, and minimally invasive properties capable of achieving phase transition after injection into the body. As a drug delivery system, the main purpose is to release a drug at a drug effective concentration or higher for a long period of time at the disease site so as not to require frequent injection that can cause side effects.

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

Accordingly, there is a need for research into a technology capable of continuously forming nitric oxide by controlling release in an initial stage using a crosslinked hydrogel.

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

In order to achieve the above object, the present invention consists of a polymer and a copper compound in which at least one of a phenol group and a catechol group is introduced, and any one or more of phenol and catechol introduced into the side chain of the polymer and copper are It provides a copper-containing in situ hydrogel, characterized in that it contains copper ions in the hydrogel matrix by coordination, and any one or more of phenol and catechol introduced into the side chain of the polymer is bonded to each other and crosslinked.

In addition, the present invention comprises the steps of preparing a solution in which a compound containing a phenol group is substituted and a polymer and an enzyme are dissolved; and cross-linking by adding a copper compound and hydrogen peroxide to the solution.

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

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

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

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

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

Accordingly, the synergistic effect of Cu ions and NO regulates the polarization of the anti-inflammatory M2 phenotype and stimulates the migration of endothelial cells, confirming the in vitro bioactivity of copper-containing in situ hydrogels. process can be facilitated.

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

Figure 2 is a schematic diagram showing the formation of nitric oxide releasing hydrogel through the catalysis of horseradish peroxidase and tyrosinase, showing that the GH polymer is crosslinked through various interactions between catechol and phenolic groups.

Figure 3 shows the results of testing the properties 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 gelation time by the rheometer It is a graph showing the evaluated mechanical strength, (c) is a graph showing the decomposition rate of lysozyme, (d) is a scanning electron microscope (SEM) image of the cross section.

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

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

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

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

Figure 8 shows the angiogenic activity assessed by an intra-oval chicken chorioallantoic angiogenesis (CAM) assay, where (a) is an optical image of new blood vessel formation in the CAM and (b) quantifies blood vessels by Image J it is a graph

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

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

In this study, copper (Cu) ions (NO generating catalyst) were incorporated into phenol-rich gelatin-based hydrogels (GH/Cu) for in situ generation of NO in the presence of endogenous S-nitrosothiol. At this time, under the catalytic action of tyrosinase and horseradish peroxidase, GH is oxidized for the formation of a cross-linked network and deposition of Cu nanoparticles, and GH/Cu exhibits sustained release of Cu ions. NO production was stimulated in the presence of endogenous NO donors for 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 endothelial cell migration. GH/Cu hydrogels are presented as promising materials for accelerating the wound healing process.

The “GH/Cu hydrogel” of the present invention is 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 ). it means.

"Angiogenesis" of the present invention means the process of newly forming blood vessels, that is, the development and differentiation of new blood vessels into cells, tissues or organs. Angiogenesis of the present invention includes revascularization, angiogenesis, and vessel differentiation related to angiogenesis processes including endothelial cell activation, migration, proliferation, matrix remodeling, and cell stabilization.

The present invention consists of a polymer and a copper compound into which at least one of a phenol group and a catechol group is introduced, and copper is coordinated with any one or more of phenol and catechol introduced into the side chain of the polymer to form copper ions in the hydrogel matrix. It provides a copper-containing in situ hydrogel, characterized in that any one or more of phenol and catechol introduced into the side chain of the polymer are bonded to each other and crosslinked.

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

Specifically, in the presence of H ₂ O ₂ , phenol is oxidized to a phenol radical by an enzyme (horseradish peroxidase, HRP), enabling crosslinking of the aromatic ring by CC or CO coupling, and aerobic In the environment, phenol is oxidized to catechol by an enzyme (tyrosinase), providing an active site for cross-linking reactions between phenol-rich gelatin polymers and a binding site for coordinating and stabilizing copper ions. Therefore, here, two enzyme-catalyzed reactions by horseradish peroxidase (HRP) and tyrosinase occur to form a hydrogel matrix.

The copper is included and stabilized in the hydrogel matrix through a metal-catecholate coordination bond. Catechol formed by enzyme (tyrosinase) catalysis can react with each other (hydrogen bond, π-π interaction, coupling reaction, etc.) to cross-link the polymer network and stabilize copper in the hydrogel matrix through metal coordination with copper ions can do.

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 promotes nitric oxide production in the presence of an endogenous nitric oxide source for a long period of time (about 3 weeks). can do. The synergistic effect of copper ions and nitric oxide can modulate the anti-inflammatory M2 phenotype polarization, thereby exhibiting anti-inflammatory properties.

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

The pharmaceutical composition may further include carriers, excipients or diluents commonly used in the preparation of pharmaceutical compositions.

When the composition of the present invention is a pharmaceutical composition, it may contain a pharmaceutically acceptable carrier, excipient or diluent in addition to the above-described active ingredients for administration. The carriers, excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, 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 may be formulated and used in the form of external preparations, suppositories or sterile injection solutions according to conventional methods, respectively. Specifically, when formulated, it may be prepared using diluents or excipients such as commonly used fillers, weighting agents, binders, wetting agents, disintegrants, and surfactants. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized formulations and tablets. Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used as non-aqueous solvents and suspending agents. As a base for suppositories, Witepsol, Macrosol, Tween 61, cacao butter, laurin paper, glycerogelatin, and the like may be used.

The pharmaceutical composition of the present invention may be administered parenterally (eg, intravenous, subcutaneous, intraperitoneal or topical application) according to the desired method. In addition, the dosage of the pharmaceutical composition of the present invention varies depending on the subject's body weight, age, sex, health condition, diet, administration time, administration method, excretion rate, and severity of disease, but is not limited thereto.

The present invention comprises the steps of preparing a solution in which a compound containing a phenol group is substituted and a polymer and an enzyme are dissolved; and cross-linking by adding a copper compound and hydrogen peroxide to the solution.

Specifically, in the crosslinking 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 form an aromatic ring by CC or CO coupling. In an aerobic environment, phenol is oxidized to catechol by an enzyme (tyrosinase), providing an active moiety for a crosslinking reaction between phenol-rich gelatin polymers and a binding site for coordinating and stabilizing copper ions. do. Therefore, here, two enzyme-catalyzed reactions by horseradish peroxidase (HRP) and tyrosinase occur to form a hydrogel matrix.

In addition, in the crosslinking step, the copper is included in the hydrogel matrix and stabilized through a metal-catecholate coordination bond. Catechols formed by enzyme (tyrosinase) catalysis can react with each other to cross-link polymer networks through at least one of hydrogen bonds, π-π interactions, and coupling reactions, and form a hydrogel matrix through copper ions and metal coordination. can stabilize copper in

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

By including the polymer as described above, copper may be included in the hydrogel matrix by coordinating a phenol group or catechol group introduced into the side chain of the polymer and copper, and the phenol group or catechol group is bonded to each other and cross-linked to form a cross-linked hydrogel. can form a gel.

Specifically, the enzymes are horseradish peroxidase, glutathione peroxidase, haloperoxidase, myeloperoxidase, catalase, hemoprotein ), peroxide, peroxiredoxin, animal heme-dependent peroxidases, thyroid peroxidase, vanadium bromoperoxidase, It may include one or two or more selected from the group consisting of lactoperoxidase, tyrosinase, and catechol oxidase.

The compound containing the phenol group is hydroxyphenyl propionic acid, 4-hydroxyphenyl acetic acid, tyrosine, tyramine, tetronic tyramine And PEG- may be one or more selected from the group consisting of tyramine (PEG-tyramine).

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) Method for producing a copper-containing in situ hydrogel, characterized in that.

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

The copper compound may be included in 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.

In the crosslinking step, hydrogen peroxide may be added 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 prepared by the above manufacturing method.

Specifically, the copper-containing in situ hydrogel may include copper ions in the hydrogel matrix by coordinating at least one of phenol and catechol introduced into the side chain of the polymer and copper.

In addition, in the copper-containing in situ hydrogel, any one or more of phenol and catechol introduced into the side chain of the polymer may be bonded to each other to form a crosslinked hydrogel matrix.

Hereinafter, examples will be described in detail to aid understanding of the present invention. However, the following examples are merely illustrative of the contents of the present invention, but the scope of the present invention is not limited to the following examples. The embodiments of the present invention are provided to more completely explain the present invention to those skilled in the art.

실시예 1Example 1

To prepare a copper-containing in situ forming hydrogel, 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 a copper-containing in situ hydrogel.

실험예 1-겔화 시간 측정Experimental Example 1 - Measurement of gelation time

The gelation time of the hydrogel was measured at the time when 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 (a) of FIG.

Looking at (a) of Figure 3, it can be seen that the gelation time is shown, and the gelation time decreases from 110 seconds to 20 seconds as the concentration of HRP increases from 0.01 to 0.05 mg / mL.

실험예 2-하이드로젤의 유변학적 분석Experimental Example 2 - Rheological Analysis of Hydrogel

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

In order to evaluate the degradation behavior of the hydrogel, it was cultured at 37 ° C. in PBS containing collagenase, and the remaining weight was measured while replacing the culture medium at regular intervals.

After freeze-drying of the hydrogel formed for the morphological structure analysis of the hydrogel, the cross section was cut and the internal structure was observed with FE-SEM (Field Emission Scanning Electron Microscopy), and the results are shown in FIGS. 3 (b) to (d) ) was shown in

Looking at (b) and (c) of FIG. 3, the elastic modulus of the hydrogel over time and the decomposition rate of the hydrogel in the presence of lysozyme are shown. As the copper ion content increases, the elastic modulus decreases, while the decomposition rate increases. can confirm that

Looking at (d) of FIG. 3, it can be seen from the image of the cross section of the hydrogel that the degree of crosslinking decreases as the copper ion content increases, and the size of the pores increases.

실험예 3-산화질소의 누적 농도Experimental Example 3-Cumulative Concentration of Nitric Oxide

A copper analysis kit (Sigma-Aldrich, St. Louis, MO, USA USA) was used to measure Cu ions released from the hydrogel. The formed hydrogel was placed in a PBS solution and incubated at 37° C. in the dark. According to the characteristic time, the supernatant was recovered and fresh PBS solution was added. Then, the recovered sample was reacted at room temperature for 5 minutes using a copper analysis kit. The absorbance of the Cu ion standard was measured at 359 nm. Since Tyr is a copper-containing enzyme, the release of Cu ions from Tyr during the hydrogel degradation process could affect the results of this experiment. Therefore, in this test, the GH hydrogel prepared without Tyr was used as a control sample.

To evaluate the NO release behavior of the hydrogel, the hydrogel was placed in a PBS solution containing NO donors (GSNO and GSH) and incubated at 37 ° C in the dark, and the NO donor solution was recovered and replaced at regular intervals. The recovered solution and the same amount of Griess solution were reacted in the dark for 15 minutes, and the absorbance was measured at 540 nm with a Cytation™ 3 Cell Imaging Multi-Mode Reader (BioTek™, USA) to determine the emission behavior of NO released from the hydrogel. was evaluated. Quantitative analysis was performed through a calibration curve obtained with 0 - 1 μM of NaNO₂, and the results are shown in FIG. 4.

Referring to FIG. 4, it shows the cumulative concentration of copper ions released from the hydrogel and the generated nitric oxide. In FIG. 4 (a), it can be seen that the copper ions released from the hydrogel increase as the copper content increases, , (b) confirms that the amount of nitric oxide released increases as the copper content in the hydrogel increases in the presence of GSNO and GSH.

실험예 4-항염증 테스트Experimental Example 4 - Anti-inflammatory test

After culturing human monocyte cells (THP-1) in RPMI-1640 medium, the cells were collected and cultured in a medium containing 12-myristate 13-acetate (PMA) for 2 days and then cultured in a medium without PMA for 1 day. The differentiation of THP-1 into M0 cells was induced. The hydrogel was treated on the M0 cell layer and cultured according to the presence or absence of NO donor. After 48 hours, the differentiation capacity into pro-inflammatory M1 and anti-inflammatory M2 macrophages according to the released NO content was analyzed. Cells were incubated with primary antibodies of the markers after treatment with paraformaldehyde and Triton X-100. (M2 markers: CD163, TGF-ß). After reaction with an IgG-fluorescent secondary antibody, DAPI staining was performed, and fluorescence qualitative analysis of differentiated anti-inflammatory M2 markers was performed using a confocal laser scanning microscope (Zeiss LSM 780, Carl Zeiss, Jena, Germany). Differentiation-induced M2 cells were lysed and the degree of M2 polarization was evaluated through quantitative analysis of TGF-β and CD163 by ELISA, and the results are shown in FIG. 5 .

(a) is an image of immunofluorescence staining 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 M2 macrophage control, ( c) is a graph showing the concentration of TGF-β released from macrophages by ELISA.

Looking at (a) and (b) of FIG. 5, the immunofluorescence staining image and the relative average fluorescence intensity of CD163 were quantified. First, when viewed in the control group, M2 macrophages with high CD163 expression compared to M0 macrophages, green fluorescence It can be seen that this is high, and based on this, it can be confirmed that the green color expression is significantly greater in the experimental group treated with GH / Cu hydrogel in the presence of NO donor than in the untreated experimental group. This suggests that anti-inflammatory M2 polarization was achieved.

In addition, looking at (c) of FIG. 5, the concentration of TGF-β released from macrophages is shown, and 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. You can check.

실험예 5-시험관 내 혈관 신생 활성Experimental Example 5 - Angiogenic activity in vitro

Human umbilical cord blood endothelial cells, HUVEC, were cultured with hydrogel in EGM-2 medium, and the angiogenic ability was evaluated according to the NO content released through cell layer scratch recovery and tube formation.

The growth of HUVECs according to the released NO content was evaluated by WST-1 and live/dead fluorescence analysis. Additionally, HUVECs were cultured to form a cell layer and then scratches were formed using a pipette. The hydrogel is placed on top of the cell layer, fresh medium is added, and NO donor is fed 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 then HUVECs were cultured on it for 1 day. Thereafter, the hydrogel was placed on the cell layer and cultured every 4 hours depending on the presence or absence of a NO donor. After 12 hours, the formation of an angiogenic capillary-like structure of HUVECs was measured with an optical microscope, and the length of the tube was calculated and evaluated using the Image J program. The results are shown in FIGS. 6 and 7 .

Referring to FIG. 6, in the experimental group treated with GH/Cu, it was confirmed that the proliferation of HUVEC cells was significantly increased in the presence of the NO donor, and as the copper ion content increased, NO production increased, resulting in faster migration of HUVECs. can confirm that

Referring to FIG. 7, in the absence of an NO donor, tube-shaped cell sorting was not achieved except for the VEGF-treated experimental group, whereas in the presence of an NO donor, as the copper content increased, tube-shaped cell sorting occurred and At the same time, it can be confirmed that significant increases in the number of nodes, the number of branches, and the total branch length have occurred.

실험예 6-닭 융모요막에서의 혈관 신생 활성Experimental Example 6 - Angiogenic activity in chicken chorioallantoic membrane

The angiogenic properties of the hydrogels were investigated by in ovo Chicken Chorioallantoic Membrane (CAM) assay. First, fertilized eggs (domestic, Korea) were incubated for 7 days (0-7 days) in the presence of 37-38 °C, 40-60% humidity and O2. Then, on the 7th day of hatching, the eggs were taken out and disinfected with 70% alcohol. Then, a window of approximately 1 cm x 1 cm was made in the eggshell, and a preformed 100 μL hydrogel sample was carefully placed into the CAM of the embryo. The window was sealed with sterile parafilm to prevent infection during further incubation. At day 10, images of the CAM-hydrogel composites were taken with a digital camera to assess the initial tissue response. 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 .

Referring to FIG. 8 , it can be seen from the CAM analysis that a large number of new blood vessels are formed in the experimental group treated with VEGF. In comparison, it can be seen that as the copper ion content in the hydrogel increases, the new blood vessels are effectively stimulated.

실험예 7-생체 내 혈관 신생 활성Experimental Example 7 - Angiogenic activity in vivo

A mouse model was used to evaluate the angiogenic ability in vivo using an animal model. Following the ethically approved protocol, hydrogel was subcutaneously injected after mouse anesthesia, and after 3 weeks, the subcutaneous tissue including the hydrogel was recovered to analyze the characteristics of new blood vessels for tissue regeneration in the body. The recovered tissue was immediately fixed in 4% paraformaldehyde for one day and then sectioned in paraffin. 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 .

Referring to FIG. 9, in the experimental group treated with the GH hydrogel, cell invasion did not occur into the hydrogel, whereas in the experimental group treated with the GH / Cu25 hydrogel, a number of cells invaded the hydrogel to form blood vessels. It can be seen that it is in an early stage. In addition, in the experimental group treated with GH/Cu50 and GH/Cu75 hydrogels, it was confirmed that a number of blood vessels were formed at the edge and inside of the cells, similar to the test group treated with VEGF.

Having described specific parts of the present invention in detail above, it is clear to those skilled in the art that these specific descriptions are only preferred embodiments, and the scope of the present invention is not limited thereby. do. That is, the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims

It consists of a polymer and a copper compound into which at least one of a phenol group and a catechol group is introduced, and copper is coordinated with any one or more of the phenol and catechol introduced into the side chain of the polymer to contain copper ions in the hydrogel matrix, , Copper-containing in situ hydrogel, characterized in that any one or more of phenol and catechol introduced into the side chain of the polymer are bonded to each other and crosslinked.
According to claim 1,

Copper-containing in situ hydrogel, characterized in that the phenol introduced into the side chain of the polymer is oxidized to a phenol radical by an enzyme to link the aromatic ring by any one or more of C-C and C-O bonds to crosslink the polymer network.
According to claim 1,

The catechol introduced into the side chain of the polymer is oxidized phenol introduced into the polymer by an enzyme in an aerobic environment,

The catechol is a copper-containing in situ hydrogel, characterized in that for stabilizing copper in the hydrogel matrix through a coordination bond with copper ions.
An anti-inflammatory pharmaceutical composition comprising the copper-containing in situ hydrogel according to claim 1.
A pharmaceutical composition for tissue regeneration comprising the copper-containing in situ hydrogel according to claim 1.
A pharmaceutical composition for angiogenesis comprising the copper-containing in situ hydrogel according to claim 1.
preparing a solution in which a compound containing a phenolic group is substituted and a polymer and an enzyme are dissolved; and

Method for producing a copper-containing in situ hydrogel comprising the step of crosslinking by adding a copper compound and hydrogen peroxide to the solution.
According to claim 7,

The crosslinking step is a method for producing a copper-containing in situ hydrogel, characterized in that copper is included in the hydrogel matrix and stabilized through a metal-catecholate coordination bond.
According to claim 7,

The crosslinking step is a method for producing a copper-containing in situ hydrogel, characterized in that the catechol formed by the catalytic action of the enzyme reacts with each other to crosslink the polymer network.
According to claim 7,

In the crosslinking step, phenol is oxidized to a phenol radical by the catalytic action of an enzyme to crosslink an aromatic ring by at least one of C-C and C-O bonds to crosslink the polymer network. Method for producing a copper-containing in situ hydrogel .
According to claim 7,

The polymer is gelatin, chitosan, heparin, cellulose, dextran, dextran sulfate, chondroitin sulfate, keratan sulfate, dermatan sulfate, alginate, collagen, albumin, fibronectin, laminin, elastin, vitronectin, hyaluronic acid, fibrinogen and -A method for producing a copper-containing in situ hydrogel containing at least one selected from the group consisting of polymers.
According to claim 7,

The enzymes are horseradish peroxidase, glutathione peroxidase, haloperoxidase, myeloperoxidase, catalase, hemoprotein, peroc Peroxide, peroxiredoxin, animal heme-dependent peroxidases, thyroid peroxidase, vanadium bromoperoxidase, lactoperoxy A method for preparing a copper-containing in situ hydrogel containing one or two or more selected from the group consisting of lactoperoxidase, tyrosinase, and catechol oxidase.
According to claim 7,

The copper compound is a method for producing a copper-containing in situ hydrogel containing at least one selected from copper sulfate (CuSO 4 ) and copper chloride (CuCl 2 ).
According to claim 7,

The copper compound is a method for producing a copper-containing in situ hydrogel, characterized in that it comprises a concentration of 10 to 75 μM.
According to claim 7,

Method for producing a copper-containing in situ hydrogel, characterized in that the concentration of the polymer is 30 to 100 mg / ml.
According to claim 7,

The compound containing the phenol group is hydroxyphenyl propionic acid, 4-hydroxyphenyl acetic acid, tyrosine, tyramine, tetronic tyramine And a method for producing a copper-containing in situ hydrogel, characterized in that at least one selected from the group consisting of PEG-tyramine.
According to claim 7,

The crosslinking step is a method for producing a copper-containing in situ hydrogel, characterized in that by adding hydrogen peroxide at a concentration of 0.01 to 0.05 wt%.