KR20220059217A - Hybrid hydrogel and biomedical applications thereof - Google Patents

Abstract

The present invention provides a hybrid hydrogel with excellent mechanical properties and biomedical uses thereof. The hybrid hydrogel according to one embodiment of the present invention has high elastic modulus, compressive strength, tensile strength, and adhesive strength, thereby having excellent physical properties such as mechanical strength and tissue adhesion.

Description

Hybrid hydrogels and their biomedical uses

The present invention relates to hybrid hydrogels and their biomedical uses.

Hydrogels, which are physically or chemically crosslinked polymer networks, can mimic natural extracellular matrices and are under constant development as innovative materials for a wide range of biomedical applications. In particular, in situ-formed injection-type hydrogels have the advantages of easily loading drugs and cells, minimally invasive implantation, and filling irregular wounds. Among various crosslinking strategies for in situ formation such as physical crosslinking and chemical crosslinking, enzyme-mediated crosslinking represents a great advantage for hydrogelation under mild reaction conditions. In particular, Horseradish peroxidase (HRP)-mediated crosslinking reaction has been intensively studied to induce hydrogels in situ. Compared with existing enzyme crosslinking systems such as transglutaminase, tyrosinase, phosphatase, and lysyloxidase, HRP has advantages in fast gelation and high crosslinking efficiency.

The hydrogel developed in this way is widely applied for tissue regeneration, drug delivery, and bioadhesive due to its excellent biocompatibility. Hydrogels formed in situ using an enzyme crosslinking system have been constantly developed, but hydrogels using only natural polymers have limitations in factors required as medical bioadhesives such as elasticity, tensile force, and adhesion. To overcome this, natural polymers (Gelatin, hyaluronic acid, alginate, etc.) with excellent biocompatibility and synthetic polymers (Poly-ethyleneglycol, PEG or PEO-PPO-PEO, Tetronic) that can supplement factors such as elasticity, tensile force, and adhesive force ), a hybrid of bioadhesives is being developed.

Although development has been made through hybridization of various biopolymers and synthetic polymers, research and development under more detailed conditions for controlling the content and molecular weight of synthetic polymers that can be controlled has hardly been done. In the development of bioadhesives through hybridization of natural and synthetic polymers, it is an essential process to research and develop excellent properties of materials by adjusting various conditions of synthetic polymers.

An object of the present invention is to provide a hybrid hydrogel having excellent mechanical properties and a biomedical use thereof.

However, the problems to be solved by the present invention are not limited to the above-mentioned problems, and other problems not mentioned will be clearly understood by those skilled in the art from the following description.

According to an aspect of the present invention, a first polymer containing two or more first reactive groups; and a second polymer comprising a main chain including a repeating unit represented by the following Chemical Formula 1 and a second reactive group bonded to both ends of the main chain, wherein by the reaction of the first reactive group and the second reactive group A hybrid hydrogel is provided in which a first polymer and the second polymer are crosslinked, and the weight average molecular weight of the second polymer is 1,000 or more and 20,000 or less:

[Formula 1]

According to another aspect of the present invention, there is provided a material for tissue adhesion and hemostasis, an implant material for tissue regeneration and filling, or a carrier for a physiologically active material or drug carrier, comprising the hybrid hydrogel.

The hybrid hydrogel according to an embodiment of the present invention has high elastic modulus, compressive strength, tensile force and adhesive force, and thus may have excellent physical properties such as mechanical strength and tissue adhesion.

The hybrid hydrogel according to an embodiment of the present invention may have excellent drug and protein release ability.

The hybrid hydrogel according to an embodiment of the present invention may have excellent cell adhesion and affinity, and thus may have excellent biocompatibility.

A material for tissue adhesion or hemostasis using the hybrid hydrogel according to an embodiment of the present invention; implantable skeleton for tissue engineering; sustained-release drug delivery systems for drugs such as proteins, DNA, growth factors, and cells; tissue filler; wound healing; Alternatively, it can be applied for various biomedical purposes, such as preventing intestinal adhesions.

Effects of the present invention are not limited to the above-described effects, and effects not mentioned will be clearly understood by those skilled in the art from the present specification and accompanying drawings.

1 is a view schematically showing the preparation of a hybrid hydrogel according to an embodiment of the present invention.
Figure 2 is a schematic diagram of the preparation of a hydrogel of the present invention schematically showing a cross-section of a syringe according to an embodiment of the present invention.
3 is a graph showing the physical properties of the hydrogels of Examples 1-1 to 1-3, Comparative Examples 1 and 2-1 to 2-3.
4 is a graph showing the physical properties of the hydrogels of Examples 1-1 to 1-3 and Comparative Examples 3-1 to 3-3.
5 is a graph showing the physical properties of the hydrogels of Example 2-2 and Comparative Examples 4-1 to 4-2.
6 is a graph showing the physical properties of the hydrogels of Examples 2-1 to 2-3, Comparative Examples 1 and 2-3.
7 is a graph showing the physical properties of the hydrogels of Examples 3-1 to 3-3.
8 is a graph showing the physical properties of the hydrogels of Examples 4-1 to 4-3.
9 is a graph showing the physical properties of the hydrogels of Examples 5-1 to 5-3.
10 is a graph showing the physical properties of the hydrogels of Examples 6-1 to 6-3.
11 is a graph showing the physical properties of the hydrogels of Examples 7-1 to 7-3.
12 is an analysis of WST-1 analysis of C2C12 cultured in the hydrogels of Examples 1-1 to 1-3, Examples 2-1 to 2-3, Comparative Example 1 and Comparative Examples 2-1 to 2-3 It is a graph of cell adhesion measured using
13 is a graph showing the cumulative release percentage of the drug according to time of the hydrogels of Examples 1-1 to 1-3, Comparative Examples 1 and 2-1 to 2-3.
14 is a graph showing the cumulative drug release percentage over time of the hydrogels of Examples 2-1 to 2-3, Comparative Examples 1 and 2-3.
15 is a graph showing the cumulative release percentage of the drug over time of dexamethasone or bobbin serum albumin of the hydrogel of Example 2-3.

In the present specification, when a part "includes" a certain component, it means that other components may be further included, rather than excluding other components, unless otherwise stated.

Throughout this specification, "(meth)acrylate" is used in the collective sense of acrylate and methacrylate.

Throughout this specification, "A and/or B" means "A and B, or A or B."

Throughout this specification, the "weight average molecular weight" and "number average molecular weight" of a compound may be calculated using the molecular weight and molecular weight distribution of the compound. Specifically, put tetrahydrofuran (THF) and a compound in a 1 ml glass bottle to prepare a sample sample with a concentration of 1 wt% of the compound, and filter the standard sample (polystyrene, polystyrene) and the sample sample (pore size). After filtration through 0.45 μm), the molecular weight and molecular weight distribution of the compound can be obtained by comparison with the GPC injector (calibration) curve. In this case, Infinity II 1260 (Agilient Co.) can be used as a measuring device, the flow rate can be set to 1.00 mL/min, and the column temperature can be set to 40.0 °C.

Hereinafter, the present invention will be described in more detail.

According to an embodiment of the present invention, a first polymer containing two or more first reactive groups; and a second polymer comprising a main chain including a repeating unit represented by the following Chemical Formula 1 and a second reactive group bonded to both ends of the main chain, wherein by the reaction of the first reactive group and the second reactive group A hybrid hydrogel is provided in which the first polymer and the second polymer are crosslinked, and the weight average molecular weight of the second polymer is 1,000 or more and 20,000 or less.

[Formula 1]

The hybrid hydrogel according to an embodiment of the present invention has a high elastic modulus, compressive strength, tensile force and adhesive force, may have excellent physical properties such as mechanical strength and tissue adhesion, may have excellent drug and protein release ability, and may have excellent cell adhesion. It may have excellent biocompatibility due to excellent properties and affinity.

According to one embodiment of the present invention, the hybrid hydrogel includes a first polymer containing two or more first reactive groups. The first 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 , carrageenan, agarose, and Dodge- may include at least one of the polymer, but is not particularly limited as long as it is used in the art as a polymer capable of forming a hydrogel.

The gelatin may be derived from pig skin or cold water fish skin.

The first polymer may contain two or more first reactive groups, and the first reactive group may be a reaction site that reacts with and binds to a reactive group of another polymer. Accordingly, as the number of first reactive groups included in the first polymer increases, the degree of crosslinking may increase.

According to one embodiment of the present invention, the first reactive group may be any one of phenol derivatives, thiol, (meth)acrylamide, cyclodextrin, N-hydroxysuccinimide, furan, and dopa. The phenol derivative may be one selected from the group consisting of phenol, tyramine, hydroxyphenylacetic acid, hydroxyphenylpropionic acid and derivatives thereof.

According to one embodiment of the present invention, the hybrid hydrogel includes a second polymer containing a main chain including a repeating unit represented by the following Chemical Formula 1 and a second reactive group bonded to both ends of the main chain.

[Formula 1]

According to one embodiment of the present invention, the second polymer may have a linear structure. When the second polymer has a linear structure, the elastic modulus, compressive strength, tensile force, and adhesive force of the hybrid hydrogel may be high, and thus mechanical strength may be excellent.

According to one embodiment of the present invention, the second polymer may be polyethylene glycol. When the second polymer includes a repeating unit having 3 or more carbon atoms, solubility may be lowered and thus may not be suitable as a hydrogel.

According to one embodiment of the present invention, the weight average molecular weight of the second polymer may be 1,000 or more and 20,000 or less, 4,000 or more and 20,000 or less, 5,000 or more and 20,000 or less, 8,000 or more and 20,000 or less, or 10,000 or more and 20,000 or less. By adjusting the weight average group molecular weight of the second polymer, it is possible to easily control mechanical properties such as elastic modulus, compressive strength, tensile force and adhesive force, and physical properties such as tissue adhesion of the prepared hybrid hydrogel. In addition, when the weight average molecular weight of the second polymer is adjusted to 1,000 or more and 20,000 or less, 4,000 or more 20,000 or less, 5,000 or more and 20,000 or less, 8,000 or more and 20,000 or less, or 10,000 or more and 20,000 or less, the elastic modulus of the hybrid hydrogel, compressive strength, Due to high tensile and adhesive strength, physical properties such as mechanical strength and tissue adhesion may be excellent, and may be decomposed without accumulation in vivo and may be non-toxic.

According to one embodiment of the present invention, the second polymer may include a second reactive group coupled to both ends. That is, the second polymer may include two second reactive groups, and each second reactive group may be a reaction site that reacts with and binds to a reactive group of another polymer.

According to one embodiment of the present invention, the second reactive group may be any one of a phenol derivative, vinyl sulfone, (meth)acrylate, maleimide, adamantane, amine, and dopa. The phenol derivative may be one selected from the group consisting of phenol, tyramine, hydroxyphenylacetic acid, hydroxyphenylpropionic acid and derivatives thereof.

According to one embodiment of the present invention, the second reactor may be the same as or different from the first reactor.

According to one embodiment of the present invention, the first polymer and the second polymer are cross-linked by the reaction of the first reactive group and the second reactive group. Depending on the type of the first reactor and the second reactor, the type of the reaction may vary, and the conditions of the reaction and the reaction environment such as a catalyst may also vary. An example of a reaction is as follows.

According to one embodiment of the present invention, the first reactive group and the second reactive group are phenol derivatives, and the first polymer and the second polymer are in situ by adding horseradish peroxidase and hydrogen peroxide. (in situ) may be cross-linked. Specifically, when the first reactive group and the second reactive group are phenol derivatives, a bond may be formed between the benzene rings of each phenol derivative. In an aqueous medium, hydrogen peroxide can induce horseradish peroxidase-mediated crosslinking reactions between phenol-rich polymers to form hydrogels.

1 schematically shows the preparation of a hybrid hydrogel according to an embodiment of the present invention.

Referring to FIG. 1 , the first polymer (blue) and the second polymer (orange) contain phenol derivatives as the first and second reactive groups, respectively, and are crosslinked in situ by adding horseradish peroxidase and hydrogen peroxide. and it can be confirmed that a bond between the benzene rings of the first reactive group and the second reactive group is formed.

In forming the hybrid hydrogel, by controlling the concentrations of horseradish peroxidase and hydrogen peroxide to be added, physicochemical properties such as gelation time, decomposition time, mechanical strength, swelling degree or moisture content of the hydrogel can be controlled.

According to one embodiment of the present invention, the first reactive group may be a thiol and the second reactive group may be vinyl sulfone, and the first polymer and the second polymer may be crosslinked by a reaction between the thiol and the vinyl sulfone. . Specifically, when the first reactive group is thiol and the second reactive group is vinyl sulfone, the first polymer and the second polymer may be cross-linked through a Michael addition reaction of thiol and vinyl sulfone by simple mixing.

The Michael addition reaction may be carried out at a pH of 6 to 8, preferably at a neutral condition of pH 7.

According to an embodiment of the present invention, the first reactive group is (meth) acrylamide and the second reactive group is (meth) acrylate, and the first polymer and the second polymer are the (meth) acrylamide and the It may be crosslinked by reaction of (meth)acrylate. Specifically, when the first reactive group is (meth)acrylamide and the second reactive group is (meth)acrylate, the first polymer and the second polymer are (meth)acrylamide and (meth)acrylamide by light irradiation in the presence of a photoinitiator. ) can be photocrosslinked through the reaction of acrylates.

As the photoinitiator, a benzophenone-based initiator, an azo-based initiator, etc. may be used, but the above-mentioned examples are not limited thereto.

The light irradiation may be ultraviolet irradiation, infrared irradiation, E-beam irradiation, etc., and may be irradiating light having a light quantity of less than about 15 mW/cm ² . When irradiating light with an amount of light within the above range, crosslinking can be performed without damaging cells.

According to an embodiment of the present invention, the first reactive group may be a thiol and the second reactive group may be a thiol, and the first polymer and the second polymer may be crosslinked by a reaction between the thiols. Specifically, the first reactive group and the second reactive group may react to form a disulfide bond (S-S), thereby crosslinking.

According to one embodiment of the present invention, the first reactive group may be a thiol and the second reactive group may be maleimide, and the first polymer and the second polymer may be crosslinked by a reaction between the thiol and the maleimide. .

According to one embodiment of the present invention, the first reactive group is cyclodextrin and the second reactive group is adamantane, and the first polymer and the second polymer are cross-linked by reaction of the cyclodextrin with the adamantane. it may have been Specifically, the first polymer and the second polymer may be crosslinked by forming a bond through host-guest interaction.

According to an embodiment of the present invention, the first reactive group is N-hydroxysuccinimide and the second reactive group is an amine, and the first polymer and the second polymer are N-hydroxysuccinimide and the It may be crosslinked by reaction of an amine.

According to one embodiment of the present invention, the first reactive group may be an aldehyde and the second reactive group may be an amine, and the first polymer and the second polymer may be crosslinked by reaction of the aldehyde and the amine.

According to an embodiment of the present invention, the first reactive group may be furan and the second reactive group may be maleimide, and the first polymer and the second polymer may be crosslinked by reaction of the furan with the maleimide. .

According to one embodiment of the present invention, the first reactive group and the second reactive group may be a waveguide, and the first polymer and the second polymer may be crosslinked by a reaction between the waveguides. Specifically, the first polymer and the second polymer may be crosslinked by a reaction between the waveguides in the presence of tyrosinase.

The reaction between the first reactive group and the second reactive group may be a reaction that occurs in the body, and may be a reaction having excellent biocompatibility without toxicity. In addition, the reaction completion time may be several seconds to several minutes, and the reaction rate may be very fast.

According to one embodiment of the present invention, in the first polymer, the first reactive group may be directly connected to the first polymer. Specifically, the first reactive group may be directly connected to the main chain of the first polymer. That is, the first reactive group may not be connected to the first polymer as a separate linker polymer. When the first reactive group is directly connected to the first polymer and crosslinked, the elastic modulus, compressive strength, tensile force, and adhesive force of the hybrid hydrogel are high, and physical properties such as mechanical strength and tissue adhesion force may be excellent.

According to an embodiment of the present invention, the first polymers may be further connected by the reaction between the first reactive groups, and the second polymers may be further connected by the reaction between the second reactive groups. That is, a bond may be formed between the first polymers, and a bond may also be formed between the second polymers to form a hybrid hydrogel by crosslinking.

According to one embodiment of the present invention, the weight ratio of the first polymer to the second polymer may be 3:1 to 1:3, 2:1 to 1:3, or 1:1 to 1:3. By adjusting the weight ratio of the first polymer and the second polymer, it is possible to easily control mechanical properties such as elastic modulus, compressive strength, tensile force, and adhesive force of the prepared hybrid hydrogel, and physical properties such as tissue adhesion. In addition, when the weight ratio of the first polymer and the second polymer is adjusted within the above range, the elastic modulus, compressive strength, tensile force and adhesive force of the hybrid hydrogel are high, and physical properties such as mechanical strength and tissue adhesion force may be excellent. .

In addition, the hybrid hydrogel according to an embodiment of the present invention may be cross-linked in situ by further including a physiologically active material having a phenol, aniline, amine or thiol group, and the physiologically active material is tyrosine. It may be a peptide comprising

According to one embodiment of the present invention, preparing a support on which an enzyme is immobilized; Cross-linking by contacting the first polymer and the second polymer with the support in the presence of hydrogen peroxide; And there is provided a method for producing a hydrogel comprising the step of obtaining a hydrogel by separating the support.

Specific details regarding the first polymer and the second polymer may be as described above.

Enzymes have been used as catalysts for chemical reactions in various fields due to their specific reactivity, high selectivity, and excellent reaction efficiency. However, mass synthesis and purification of enzymes are difficult and expensive, and it has been pointed out that the enzyme activity decreases due to reaction conditions and storage. In addition, since it contains enzymes such as HRP derived from animals and plants, it may cause questions related to biosafety, such as immune response, when injected into the body. In order to improve this, many studies have been conducted on the technology of chemically immobilizing enzymes on the surface of various types and types of supports, such as nano/micro microspheres, and using them for chemical reactions according to the purpose of application. It has been reported that enzyme-immobilized substrates prepared through surface immobilization of enzymes can be easily separated after chemical reaction and can be recycled, and can improve storage stability while maintaining enzyme activity. Therefore, while the inventors of the present invention were studying a method for producing a hydrogel through the surface immobilization of such an enzyme, the reaction target polymer and the enzyme-immobilized support were reacted in the presence of hydrogen peroxide and then the support was separated. The enzyme is not included. It was confirmed that it was possible to prepare an in situ-forming hydrogel that was not, and completed the present invention. Through this, it is possible to overcome safety problems in the body such as an immune response caused by enzymes, thereby expanding the biomedical application range of the hydrogel.

According to one embodiment of the present invention, the enzyme immobilized on the support may be horseradish peroxidase, but is not limited thereto.

According to one embodiment of the present invention, the support may be in the form of fine particles, a porous sponge or a porous sheet, and the fine particle support is in a non-porous or porous form, and glass particles, metal particles, polymer particles, and mixed particles thereof It may be made of, but is not limited thereto.

The mixed particle may be a metal-containing polymer particle, and the metal-containing polymer particle may contain iron.

In addition, the iron-containing polymer particles may be prepared by copolymerizing a monomer having an epoxy group and an iron compound.

In one embodiment of the present invention, the enzyme may be immobilized on a solid phase using techniques known in the art of chromatography and immunoassay, and more specifically, the step of preparing a support on which the enzyme is immobilized includes: A surface modification step of modifying the surface of the support to introduce a functional group; and binding an enzyme to the functional group, wherein the functional group is a carboxyl group, an amine group, a hydroxyl group, an aldehyde group, an epoxy group, a thiol group, a maleimide group, a carbonate ester group, a cyano group, an acryl group, an acetylene group, or It may be a diazo group.

The surface modification is 3,4-dihydroxy hydrocinnamic acid, dopamine, chloroacetylcatechol, aminomethylcatechol, deoxyepinephrine, dihydroxybenzohydrazide, caffeic acid phenethyl ester from the group consisting of and hiruthenone One or more selected catechol derivatives may be used to introduce functional groups, but the present invention is not limited thereto in view of the same chemical bonding when using compounds structurally similar to the above-listed catechol derivatives.

In addition, the surface modification is 3-aminopropyl triethoxysilane, trimethoxy (7-octen-1-yl) silane, vinyl trimethoxysilane, 3-trimethoxysilyl-1-propanethiol, 3-aminopropyl Trimethoxysilane, aminoethyl trimethoxysilyl propylamine, triethoxyvinylsilane, isocyanatopropyl triethoxysilane, cyanoethyl triethoxysilane, mercaptopropyl triethoxysilane and triethoxysilyl One or more silane derivatives selected from the group consisting of propyl diethanolamine may be used to introduce a functional group, but it is not limited thereto in view of the same chemical bonding when using a compound structurally similar to the silane derivative listed above.

However, it will be apparent to those skilled in the art that enzyme binding is possible without the separate surface modification step in the case of using a fine support such as polystyrene beads or SiO ₂ beads, which are polymer beads already containing the functional group on the surface.

The introduced functional group is EDC (N-Ethyl-N′-(3-dimethylaminopropyl) carbodiimide hydrochloride), NHS (N-hydroxysuccinimide), glutaraldehyde, isothiocyanate (diisothiocyanate), isocyanate (isocyanate), It may be activated using any one or more coupling agents selected from the group consisting of dihydoxysuccinimide and avidin/biotin, and the enzyme binds to the activated functional group so that the enzyme is It is immobilized on a micro-particulate support.

In addition, in the embodiment of the present invention, in the case of polymer particles containing iron particles, an epoxy functional group exists on the surface, so that the enzyme is chemically immobilized on the surface by directly adding the enzyme without an additional process to prepare polymer particles.

In another embodiment of the present invention, the concentration of the immobilized enzyme may be an enzyme concentration of 0.001 to 10 mg/g, 0.1 to 10 mg/g, or 0.5 to 7 mg/g, but is not limited thereto.

At this time, in order to increase the activity of the bound enzyme, polyethylene glycol (PEG), polyethylene oxide (PEO), polyethyleneimine (PEI), polyvinyl alcohol (PVA) or polyacrylic acid (PAA) containing a polyfunctional group is used as a spacer (spacer) can be used as

According to one embodiment of the present invention, the process of contacting and separating the polymer and the support can be performed by applying pressure to the polymer present on one side of the support on which the enzyme is immobilized, and passing the polymer through the other side of the support. Specifically, the method may be performed through a syringe kit or a spray kit capable of spraying the tip of the syringe. More specifically, the polymer in the syringe kit or spray kit may be extruded by being cross-linked in situ while passing through a support on which the enzyme is immobilized.

Figure 2 is a schematic diagram of the preparation of the hydrogel of the present invention schematically showing the cross section of the syringe.

According to one embodiment of the present invention, the hydrogen peroxide may be present together with the first polymer and the second polymer, or may be present separately from the polymer using a double injection syringe kit or a spray kit.

According to one embodiment of the present invention, the hydrogel in the form of a sheet or disk may be manufactured using the syringe kit or spray kit and Teflon mold.

According to one embodiment of the present invention, for the method, in a syringe in which a piston is built in a cylinder having an inner space and a discharge force is generated by a sliding operation of the piston to discharge a chemical solution through a needle, the cylinder A syringe for preparing a hydrogel is provided with a support on which an enzyme is immobilized on the inside of the needle side, and a polymer having a phenol or aniline functional group in the side chain is filled in the cylinder.

The hydrogel prepared by the above method may not contain an enzyme, and thus, it is possible to overcome the safety problems in the body, such as an immune response caused by the enzyme, thereby expanding the biomedical application range of the hydrogel.

The hybrid hydrogel according to an embodiment of the present invention has excellent tissue adhesiveness, so that it can be used as a tissue adhesive (bioadhesive) or hemostatic agent (hemostats); implantable skeleton for tissue engineering; sustained-release drug delivery systems for drugs such as proteins, DNA, growth factors, and cells; tissue filler; wound healing; Alternatively, various biomedical applications such as preventing intestinal adhesions are possible.

More specifically, the hybrid hydrogel according to an embodiment of the present invention may be included in a tissue sealant, a material for tissue adhesion and hemostasis. For use as a material for tissue adhesion and hemostasis, i) ease of use, ii) sterilizable, iii) moderate viscosity, iv) low exothermic properties, v) short setting time, vi) strong adhesion, vii) low toxicity, viii ) non-toxicity of the system, ix) an appropriate decomposition time is required, and the hybrid hydrogel according to an embodiment of the present invention can satisfy these requirements as a tissue adhesion material.

Specifically, the hybrid hydrogel according to an embodiment of the present invention has excellent biocompatibility and can be cross-linked through a rapid in situ cross-linking reaction. Since a polymer that can be decomposed by an enzyme or protein is used, it has excellent biodegradability, and it is possible to control the decomposition rate by controlling the crosslinking concentration and the mixing ratio of the first polymer and the second polymer.

The material for tissue adhesion and hemostasis may include cranial nerve surgery including a vascular surgical area; orthopedic surgery, including bone adhesion; hemostasis in lacerations; suture of the femoral artery; cataract incision sutures, cartilage and articular cartilage healing; skin bonding; organ/secretory incision hemostasis; gastrointestinal division; And it can be applied to any one selected from the group consisting of tendon and ligament healing.

The hybrid hydrogel according to an embodiment of the present invention can be used as an artificial extracellular matrix as an effective scaffold for tissue engineering. At this time, since the degradation rate of the hydrogel plays a very important role in the differentiation and growth of cells inside the gel, proper control of the degradation rate is essential.

For example, gelatin is specifically degraded by matrix metalloproteinases (MMPs) secreted by cells, particularly MMP-2 and MMP-9. The hydrogel matrix containing gelatin is decomposed by these enzymes and re-formed into the extracellular matrix (ECM) secreted by the cells, which is effective for effectively growing and differentiating the cells inside the hydrogel.

In addition, the matrix stiffness of the hydrogel used as an effective scaffold for tissue engineering also has a great influence on the growth and differentiation of cells inside the gel. In addition, appropriate matrix stiffness is required for each cell. For example, osteocytes have been shown to grow well in a rigid matrix, and soft tissue cells, such as fibroblasts and myoblasts, are known to grow well in a soft matrix. In a system using an enzymatic reaction, crosslinking of the hydrogel can be easily controlled by controlling the amount of hydrogen peroxide, and thus the stiffness of the hydrogel can be easily controlled.

In addition, according to one embodiment of the present invention, there is provided an implant material for tissue regeneration and filling comprising the hybrid hydrogel.

The implant material includes cartilage regeneration, bone regeneration, periodontal bone regeneration, skin regeneration, cardiac tissue regeneration, artificial intraocular lens, spinal nerve regeneration Cord regeneration, cranial regeneration, vocal regeneration and augmentation, adhesion barrier, urinary incontinence treatment, wrinkle removal filler, wound dressing), tissue augmentation, and intervertebral disc treatment may be applied to any one selected from the group consisting of.

In addition, according to one embodiment of the present invention, there is provided a carrier for a physiologically active material or drug carrier comprising the hybrid hydrogel.

The physiologically active substance or drug is a peptide or protein drug; antibacterial agents; anticancer drugs; and anti-inflammatory agents; may include one or more selected from the group consisting of.

The peptide or protein pharmaceuticals include fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), transforming growth factor (TGF), bone growth factor (bone). morphogenetic protein; BMP), human growth hormone (hGH), porcine growth hormone (pGH), leukocyte growth factor (G-CSF), erythrocyte growth factor (EPO), macrophage growth factor (M-CSF), tumor necrosis factor ( TNF), epithelial growth factor (EGF), platelet-derived growth factor (PDGF), interferon-α, β, γ, interleukin-2 (IL-2), calcitonin, nerve growth factor (NGF), growth hormone releasing factor, Angiotensin, Luteinizing Hormone Releasing Hormone (LHRH), Luteinizing Hormone Releasing Hormone Agonist (LHRH Agonist), Insulin, Thyroid Stimulating Hormone Releasing Hormone (TRH), Angiostatin, Endostatin, Somatostatin, Glucagon, Endorphin, Basi It may be selected from the group consisting of tracin, megain, colistin, single antibody, vaccines, and mixtures thereof.

The antibacterial agent includes minocycline, tetracycline, ofloxacin, fosfomycin, mergain, profloxacin, ampicillin, penicillin, doxycycline, thienamycin, cephalosporin, norkadisin, gentamicin, neomycin, ghana Mycin, paromomycin, micronomycin, amikacin, tobramycin, dibecacin, cefotaxin, cefaclor, erythromycin, cyprofloxacin, levofloxacin, enoxacin, vancomycin, imipenem, fusidic acid and their It may be selected from the group consisting of mixtures.

The anticancer agents include paclitaxel, texotere, adriamycin, endostatin, angiostatin, mitomycin, bleomycin, cisplatin, carbopletin, doxorubicin, daunorubicin, idarubicin, 5-fluorouracil, methotrexate, actinomycin-D and mixtures thereof.

The anti-inflammatory agents include acetaminophen, aspirin, ibuprofen, dicrofenac, indomethacin, piroxicam, fenoprofen, flubiprofen, ketoprofen, naproxen, suprofen, loxoprofen, synoxy It may be selected from the group consisting of cam, tenoxicam, and mixtures thereof.

In addition, the hybrid hydrogel according to an embodiment of the present invention can be used as an artificial extracellular matrix as an effective scaffold for drug delivery. For example, by introducing tyramine into heparin, which physically binds various growth factors, it is possible to effectively support growth factors and enable sustained release behavior (growth factor binding site).

Cell adhesion within the hydrogel matrix can be improved by using a cell adhesion peptide or protein to which phenol is bound, for example, RGDY or YIGSR. Tissue-adhesive artificial ECM can be made in situ using these components necessary for effective cell growth and differentiation using an enzyme mechanism.

Hereinafter, examples will be given to describe the present invention in detail. However, the embodiments according to the present invention may be modified in various other forms, and the scope of the present invention is not to be construed as being limited to the embodiments described below. The embodiments of the present specification are provided to more completely explain the present invention to those of ordinary skill in the art.

Preparation Example 1: Preparation of the first polymer

1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC, Sigma-Aldrich Co., Ltd.) 3.83 g (20 mmol) dissolved in a co-solvent of 15 ml of water: dimethylformamide (DMF) , Junsei (Tokyo, Japan) (3:2) was added to a solution containing 3.32 g (20 mmol) of 3-(4-hydroxy-phenyl)propionic acid (HPA, Sigma-Aldrich) in 125 ml of cosolvent. . After activation for 1 hour, 3.45 g (30 mmol) of N-hydroxysuccinimide (NHS, Sigma-Aldrich) dissolved in 15 ml of water in the solution: DMF (3:2) cosolvent was stirred for 1 hour. was put in. The activated HPA solution was added to a gelatin solution (40° C.) containing 5 g of gelatin (pig skin type A; >300 Bloom, Sigma-Aldrich) in 150 ml of deionized water. The reaction between the activated HPA solution and the gelatin solution was performed for 24 hours, and the temperature was maintained at 40° C. so that the gelatin was homogeneously dissolved in the HPA solution. The reacted mixture was ultrafiltered from a dialysis membrane (Spectrum laboratories, Inc. (CA., L.S., USA)) having a molecular weight cutoff (MWCO) of 3,500 kDa in distilled water at 40° C., and the medium was filtered for 3 days. It was replaced 3 times a day. As the first polymer, gelatin-hydroxyphenyl propionic acid was obtained in a yield of 90.8% after freeze-drying.

Preparation Example 2-1: Preparation of the second polymer

N dissolved in methylene chloride in 1.21 g (6 mmol) of p-nitrophenyl chloroformate (PNC, Sigma-Aldrich) dissolved in 20 ml of methylene chloride (MC, J.T. Baker (PA, USA)) under nitrogen gas conditions A solution of 0.73 g (6 mmol) of ,N-dimethylpyridin-4-amine (DMAP, Alfa Aesar (Ward Hill, MA, USA)) was added. 10 g (1 mmol) of 10 kDa linear polyethylene glycol (PEG; Sigma-Aldrich Co., Ltd.) was dissolved in 100 ml of MC and dropped into a mixed solution of PNC and DMAP. The mixed solution was reacted at room temperature for 24 hours under nitrogen gas conditions. The reaction solution was added dropwise to 4 L ethyl ether, condensed and precipitated. The white precipitate was then filtered off and dried in a vacuum oven for 3 days. PEG-PNC2 is obtained as a white powder. White solid PEG-PNC2 modified with 90% or more of PNC was completely dissolved in 150 ml of N-dimethylsulfoxide (DMSO, Junsei (Tokyo, Japan)) under a nitrogen environment. Tyramine (0.69 g, 5 mmol) was also dissolved in 30 ml of DMSO, and 0.70 g (5 mmol) of triethylamine (TEA, KANTO Chemical CO., INC., (Japan)) was added under nitrogen condition. The dissolved tyramine solution was added dropwise to the PEG-PNC2 solution and reacted at room temperature for 24 hours under nitrogen condition. The reacted mixture was ultrafiltered from a dialysis membrane having a MWCO of 3,500 kDa on metalol, and the medium was changed 3 times a day for 3 days. After 3 days, the dialyzed solution was evaporated and precipitated in 4L ethyl ether. The obtained polyethylene glycol-tyramine was filtered and dried in a vacuum oven for 3 days. Completely dried polyethyleneglycol-tyramine showed the formation of a white solid.

Preparation 2-2

Polyethylene glycol-tyramine was prepared in the same manner as in Preparation Example 2-1, except that 15 mmol of PNC, 2.5 mmol of 4 kDa linear polyethylene glycol, and 15 mmol of DMAP were used.

Preparation 2-3

Polyethylene glycol-tyramine was prepared in the same manner as in Preparation Example 2-1, except that 4 mmol of PNC, 0.5 mmol of 20 kDa linear polyethylene glycol, and 4 mmol of DMAP were used.

Preparation Example 3: Heparin-Phenol

1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC, 8 mmol) and N-hydroxysuccinimide (NHS, 5 mmol) were reacted with heparin (1 g) in 100 mL of distilled water Heparin with an activated carboxyl group can be prepared, and heparin-phenol can be prepared by reacting tyramine (3 mmol) with an amine group with the activated carboxyl group. After the reaction at room temperature for 24 hours, heparin-phenol was obtained in the same manner as in Preparation Example 1.

Preparation Example 4: hyaluronic acid-phenol

It was prepared in the same manner as in Preparation Example 3, except that hyaluronic acid (1 g), EDC (15 mmol), NHS (15 mmol) and tyramine (20 mmol) were used.

Preparation Example 5: Alginate-phenol

It was prepared in the same manner as in Preparation Example 3, except that alginate (1 g), EDC (12 mmol), NHS (15 mmol) and tyramine (15 mmol) were used.

Preparation 6-1: Gelatin-thiol

After completely dissolving 5 g of gelatin (pig skin type A; >300 bloom) in 150 ml of deionized water and 40° C., cystamine hydrochloride (20 mmol, Sigma-Aldrich) was added. After adjusting the pH of the reaction solution to 4.5 using 1 mol of hydrochloric acid solution (HCl), EDC (20 mmol) and NHS (20 mmol) were immediately added and dissolved, followed by reaction. After the reaction for 24 hours, the pH of the reaction solution was adjusted to 8 using 4 moles of sodium hydroxide (NaOH), and an excess of dithiothreitol (DTT, 40 mmol, Sigma-Aldrich) was added to prevent the disulfide bond of cystamine with thiol. was reduced to After additional 24 hours of reaction, the pH was adjusted to 4 using HCl, and then obtained in the same manner as in Preparation Example 1.

Preparation Examples 6-2 to 6-4: PEG-VS

4 g (1 mmol) of 4 kDa linear polyethylene glycol (PEG; Sigma-Aldrich) was dissolved in 100 ml of 0.1 M NaOH, and divinyl sulfone (10 mmol) was added thereto, followed by reaction at room temperature for 6 hours. After the reaction, the pH was adjusted to 4 using HCl and then dialyzed through a dialysis membrane having a MWCO of 3.5 kDa in distilled water. The polyethylene glycol-vinyl sulfone of Preparation Example 6-2 was obtained through freeze-drying after dialysis while replacing distilled water for 3 days.

Polyethylene glycol-vinylsulfone of Preparation Examples 6-3 and 6-4 was prepared in the same manner as above except that 10 g (1 mmol) of 10 kDa linear PEG or 20 g (1 mmol) of 20 kDa linear PEG were used. .

Preparation 7-1: Gelatin-MA

After completely dissolving 5 g of gelatin (pig skin type A; >300 bloom) in 100 ml of deionized water and 60° C., methacrylate (4 μmol) was added and reacted for 1 hour. After the reaction, 400 mL of DPBS at 40°C was added, and after the reaction was terminated, it was dialyzed through a dialysis membrane having a MWCO of 12-14 kDa. Distilled water was replaced for 3 days, and after dialysis, gelatin-methacrylate was obtained through freeze-drying.

Preparation Examples 7-2 to 7-4: PEG-A

2 g (0.5 mmol) of 4 kDa PEG and triethylamine (5 mmol) were completely dissolved in methylene chloride (MC, 60 mL, and acryloyl chloride (Sigma-Aldrich, 1.2 mmol) dissolved in 5 mL of MC was slowly dissolved After the reaction, the reaction was carried out for 12 hours in a nitrogen atmosphere, blocking light at 5 ° C. After the reaction, the colorless and sticky liquid reactant was washed with ammonium chloride and dried to prepare polyethylene glycol-acrylate of Preparation Example 7-2 prepared.

Polyethylene glycol-acryl of Preparation Examples 7-3 and 7-4 in the same manner as above, except that 10 kDa PEG-DA 5 g (0.5 mmol) or 20 kDa PEG-DA 10 g (0.5 mmol) of PEG was used. rate was prepared.

Preparation Examples 8-1 to 8-3: Linker

질소 분위기에서 24시간 동안 반응을 진행하였다. 반응 후 증류수 상에서 투석막(dialysis membrane)을 통해 투석하였다. 3일간 증류수를 교체해주며 투석 후에 동결건조를 통해 수득하였다. 용액을 동결 건조하여 분자량 별 PEG가 링커로 존재하는 백색의 분말 생성물인 ㅈ제젤라틴-폴리에틸렌글리콜-티라민(Gelatin-PEG-Tyramine, GPT)을 수득하였다. To 100 mL of DMSO in which PEG-PNC2 prepared in Preparation Examples 2-1 to 2-3 was dissolved, 50 mL of DMSO in which tyramine was dissolved was added, and the reaction was carried out in a nitrogen atmosphere for 6 hours. At this time, the molecular weight of PEG is 4 kDa (Preparation Example 8-1), 10 kDa (Preparation Example 8-2), and 20 kDa (Preparation Example 8-3), respectively, and the molar ratio of PEG-PNC2: tyramine is 1: 1 is After 6 hours, it was mixed with Gelatin solution (1 g/200 ml in DMSO) and

The reaction was carried out in a nitrogen atmosphere for 24 hours. After the reaction, it was dialyzed through a dialysis membrane on distilled water. It was obtained through lyophilization after dialysis while replacing distilled water for 3 days. The solution was freeze-dried to obtain gelatin-polyethylene glycol-tyramine (GPT), which is a white powder product in which PEG according to molecular weight exists as a linker.

Preparation 9-1: Tetronic-Tyramine

After dissolving 30 g (1.67 mmol) of Tetronic in 300 mL of dioxane, 8.33 mmol of N,N-dimethylpyridin-4-amine (DMAP, Alfa Aesar (Ward Hill, MA, USA)) and trie in this solution A solution of 8.33 mmol of thiamine (TEA) in 40 mL of dioxane and a solution of 8.33 mmol of p-nitrophenyl chloromate (PNC) in 50 mL of dioxane were sequentially mixed. The molar ratio of Tetronic:PNC:DMAP:TEA is 1:5:5:5. At this time, the reaction temperature was 30 °C, and the reaction was carried out in a nitrogen atmosphere for 24 hours. After completion of the reaction, the remaining reagents are removed from the solution using a filter, and the reaction solution is concentrated using a rotary evaporator and slowly dropped into 2 L of cold ether to form a precipitate, and the precipitate is filtered using a filter to obtain a product did After drying in a vacuum oven for 24 hours to remove the residual organic solvent, Tetronic-PNC was prepared. Thereafter, a solution of 5.55 mmol of tyramine in 150 mL of THF was added to a solution of 20 g (1.11 mmol) of Tetronic-PNC in 200 mL of tetrahydrofuran (THF), followed by a reaction. Tetronic-PNC: The molar ratio of tyramine is 1:5. At this time, the reaction temperature was 30 °C and the reaction was carried out in a nitrogen atmosphere for 24 hours. After completion of the reaction, the solution was dialyzed on methanol through a dialysis membrane having a MWCO of 3.5 kDa. Methanol was replaced for 3 days and after dialysis, the solution was concentrated using a rotary evaporator, and then slowly dropped into 2 L of cold ether to form a precipitate, and the precipitate was filtered using a filter to obtain a product. After drying for 24 hours in a vacuum oven to remove the residual organic solvent, tetronic-tyramine was prepared.

Preparation 9-2: 4-ARM PEG-Tyramine

It was prepared in the same manner as in Preparation Example 9-1, except that 33.4 g (1.67 mmol) of 4-ARM PEG of 20 kDa was used instead of Tetronic.

Example 1-1: Preparation of hybrid hydrogel

111.1 mg/ml of gelatin-hydroxyphenyl propionic acid prepared in Preparation Example 1 and 111.1 mg/ml of polyethylene glycol-tyramine prepared in Preparation Example 2-1 Dulbecco's phosphate buffered saline (DPBS, Wisent (Saint-Bruno, Quebec, Canada)). A final polymer solution was prepared by mixing the gelatin-hydroxyphenyl propionic acid and polyethylene glycol-tyramine solution dissolved in the above in a volume ratio of 1:1.

Horseradish oxidase (HRP, type VI, 250-330 U/mg solid, Sigma-Aldrich) 0.04 mg/ml and hydrogen peroxide (30% by weight in water, Sigma-Aldrich) 0.6 wt% were mixed with Dulbecco's phosphate buffered saline. (DPBS, Wisent (Saint-Bruno, Quebec, Canada)).

The hydrogel was prepared by mixing each HRP and H ₂ O ₂ solution (polymer: HRP and H ₂ O ₂ = 9: 1 volume ratio) mixed with the polymer at the same volume ratio (final concentration: GH 5 wt%, PT 5 wt%).

A hydrogel with a capacity of 300 ul is formed in a 1 ㎖ vial, and the gelation possibility is measured by measuring the time until the point where flow is not observed according to gelation while tilting the vial by the vial-tilting method. The gelation time was determined.

Example 1-2

A hybrid hydrogel was prepared in the same manner as in Example 1-1, except that 44.4 mg/ml (4 kDa) of polyethylene glycol-tyramine prepared in Preparation Example 2-2 was used.

Examples 1-3

A hybrid hydrogel was prepared in the same manner as in Example 1-1, except that 222.2 mg/ml (20 kDa) of polyethylene glycol-tyramine prepared in Preparation Example 2-3 was used.

The molar concentration of polyethylene glycol-tyramine in Examples 1-1, 1-2, and 1-3 is a fixed value at 5 mM.

Example 2-1

Gelatin-hydroxyphenyl propionic acid prepared in Preparation Example 1 and polyethylene glycol-tyramine prepared in Preparation Example 2-3 were each added to 166.6 mg/ml Dulbecco's phosphate buffered saline (DPBS, Wisent (Saint-Bruno, Quebec, Canada)) ), a hybrid hydrogel was prepared in the same manner as in Example 1-1, except that it was mixed so that the weight ratio of the solution was 3:1.

Example 2-2

Gelatin-hydroxyphenyl propionic acid prepared in Preparation Example 1 and polyethylene glycol-tyramine prepared in Preparation Example 2-3 were each added to 166.6 mg/ml Dulbecco's phosphate buffered saline (DPBS, Wisent (Saint-Bruno, Quebec, Canada)) ), a hybrid hydrogel was prepared in the same manner as in Example 1-1, except that it was mixed so that the weight ratio of the solution was 1:1.

Example 2-3

Gelatin-hydroxyphenyl propionic acid prepared in Preparation Example 1 and polyethylene glycol-tyramine prepared in Preparation Example 2-3 were each added to 166.6 mg/ml Dulbecco's phosphate buffered saline (DPBS, Wisent (Saint-Bruno, Quebec, Canada)) ), a hybrid hydrogel was prepared in the same manner as in Example 1-1, except that it was mixed so that the weight ratio of the solution was 1:3.

Examples 3-1 to 7-3

As shown in Table 1 below, a hybrid hydrogel was prepared in the same manner as in Example 2-3, except that the first polymer and the second polymer were used.

first polymer second polymer Example 3-1 Preparation 3 Preparation 2-1 Example 3-2 Preparation 3 Preparation 2-2 Example 3-3 Preparation 3 Preparation 2-3 Example 4-1 Preparation 4 Preparation 2-1 Example 4-2 Preparation 4 Preparation 2-2 Example 4-3 Preparation 4 Preparation 2-3 Example 5-1 Preparation 5 Preparation 2-1 Example 5-2 Preparation 5 Preparation 2-2 Example 5-3 Preparation 5 Preparation 2-3 Example 6-1 Preparation 6-1 Preparation 6-2 Example 6-2 Preparation 6-1 Preparation 6-3 Example 6-3 Preparation 6-1 Preparation 6-4 Example 7-1 Preparation 7-1 Preparation 7-2 Example 7-2 Preparation 7-1 Preparation 7-3 Example 7-3 Preparation 7-1 Preparation 7-4

Comparative Example 1

111.1 mg/ml of gelatin-hydroxyphenyl propionic acid prepared in Preparation Example 1 was dissolved in Dulbecco's phosphate buffered saline (DPBS, Wisent (Saint-Bruno, Quebec, Canada)). The final polymer solution was prepared by mixing the gelatin-hydroxyphenyl propionic acid dissolved in the above and Dulbecco's phosphate buffered saline (DPBS, Wisent (Saint-Bruno, Quebec, Canada)) in a volume ratio of 1:1. A hybrid hydrogel was prepared in the same manner as in Example 1-1.

Comparative Examples 2-1 to 2-3

Polyethylene glycol-tyramine prepared in Preparation Examples 2-1 to 2-3, respectively, 44.4 mg / ml (4 kDa, Comparative Example 2-1), 111 mg / ml (10 kDa, Comparative Example 2-2), 222.2 mg /ml (20 kDa, Comparative Example 2-3) was dissolved in Dulbecco's phosphate buffered saline (DPBS, Wisent (Saint-Bruno, Quebec, Canada)). Example 1 except that the final polymer solution was prepared by mixing polyethylene glycol-tyramine dissolved in the above and Dulbecco's phosphate buffered saline (DPBS, Wisent (Saint-Bruno, Quebec, Canada)) in a volume ratio of 1:1. A hybrid hydrogel was prepared in the same manner as in -1.

Comparative Examples 3-1 to 3-3

The gelatin-polyethylene glycol-tyramine prepared in Preparation Examples 8-1 to 8-3 was added to Dulbecco's phosphate buffered saline (DPBS, Wisent (Saint-Bruno, A hybrid hydrogel was prepared in the same manner as in Example 1-1, except that one dissolved in Quebec, Canada)) was used as the final polymer solution.

Comparative Examples 4-1 and 4-2

Instead of polyethylene glycol-tyramine prepared in Preparation Example 2-3 in Example 1-3, prepared in Preparation Example 9-1 (Comparative Example 4-1) or Preparation Example 9-2 (Comparative Example 4-2) A hybrid hydrogel was prepared in the same manner as in Example 1-1, except that 222.2 mg/ml of the topographical polyethylene glycol derivative was used.

Reference Example 1

Fibrin glue (Greenplast®) was purchased from Green Cross Co. and prepared.

Experimental Example 1: Evaluation of mechanical properties

According to the following Measurement Examples 1 to 4, Examples 1-1 to 1-3, 2-1 to 2-3, 3-1 to 3-3, 4-1 to 4-3, 5-1 to 5-3 , 6-1 to 6-3, 7-1 to 7-3 and Comparative Examples 1, 2-1 to 2-3, 3-1 to 3-3, 4-1 to 4-2 of the hydrogel prepared in The physical properties were measured. In addition, the adhesive force of the fibrin glue of Reference Example 1 was measured according to Measurement Example 4 and shown in the adhesive force graph of FIGS. 3 to 11 .

Measurement Example 1: Measurement of elastic modulus

The modulus of elasticity of the hydrogel was measured with a rheometer (GEM-150-050, Bohlin Instruments, USA) in vibration mode. 300 μl hydrogel was prepared according to the molecular weight and content of PEG on the plate (diameter = 25 mm, gap = 0.5 mm). Rheological analysis of the hydrogel was performed in a dynamic time sweep, and the G' values were recorded 10 minutes after the measurement.

Measurement Example 2: Measurement of Compressive Strength

Compressive strength was measured by a universal testing machine (UTM) (H50K-T UTM, GEN-M-128, Tinius Olsen, Norway). First, a hydrogel (600 μl, n = 3) was formed into a square cube (10 Х 10 Х 10 mm) and then incubated in 5 ml of 0.01M PBS for 24 hours. Then, the prepared hydrogel cube was placed on a UTM plate and pressure was applied at a constant rate of 10 mm/min (1000 N). The force (N) at which the hydrogel cube broke was recorded as the compressive strength.

Measurement example 3: Measurement of tensile strength

Tensile strength was measured by a universal testing machine (UTM) (H50K-T UTM, GEN-M-128, Tinius Olsen, Norway). First, a hydrogel (800 μl, n = 3) was formed into a rectangular rod (10 Х 30 Х 1 mm) and then incubated in 5 ml of 0.01M PBS for 24 hours. Then, the prepared hydrogel bar was placed on a UTM plate and tensile force was applied at a constant rate of 50 mm/min (20 N). The force (N) at which the hydrogel bar broke was recorded as the tensile strength.

Measurement Example 4: Measurement of adhesive strength

The adhesive strength was measured by a universal testing machine (UTM) (H50K-T UTM, GEN-M-128, Tinius Olsen, Norway). The decellularized pig skin obtained from Hans Bio-Mad was cut into a 10 x 25 mm rectangle and immersed in 0.01M DPBS for 30 minutes to create an environment similar to that of real pig skin. Ethyl cyano acrylate was used to attach to the ends of a polyvinyl chloride (PVC) substrate (60 x 25 mm). A polymer solution containing HRP (75ul) is applied to the pig skin area, and a polymer solution containing H ₂ O ₂ (75ul) is applied to the other pig skin, and then the two surfaces are overlapped and pressed with a weight of 100g for 10 minutes. gave. Both ends of the PVC of the prepared substrate were fixed to UTM and pulled with a constant force at a constant speed of 5 mm/min (20 N). The force (N) at which the pigskin broke was recorded as the adhesive strength.

Results analysis

3 shows the physical properties of the hydrogels of Examples 1-1 to 1-3, Comparative Examples 1 and 2-1 to 2-3. Specifically, the elastic modulus (upper left), compressive strength (upper right), and tensile strength (lower left) of the hydrogels of Examples 1-1 to 1-3, Comparative Examples 1 and 2-1 to 2-3 and adhesive strength (bottom right).

Referring to FIG. 3, the physical properties of the hybrid hydrogel prepared in Examples 1-1 to 1-3 are comparatively superior to those of the hydrogel prepared with a single polymer of Comparative Examples 1 and 2-1 to 2-3. It can be confirmed, and in particular, it can be confirmed that the physical properties of the hybrid hydrogels of Examples 1-2 and 1-3 using polyethylene glycol having a large molecular weight are excellent.

4 shows the physical properties of the hydrogels of Examples 1-1 to 1-3 and Comparative Examples 3-1 to 3-3. Specifically, the elastic modulus (top left), compressive strength (top right), tensile strength (bottom left) and adhesive strength (top left) of the hydrogels of Examples 1-1 to 1-3 and Comparative Examples 3-1 to 3-3 (bottom left) bottom right) is shown.

4, even when polyethylene glycol of the same molecular weight is used, compared to Comparative Examples 3-1 to 3-3 in which polyethylene glycol acts as a linker, the first reactive group is directly bonded to the first polymer and cross-linked Example 1- It can be seen that the physical properties of the hybrid hydrogels of 1 to 1-3 are better.

5 shows the physical properties of the hydrogels of Example 2-2 and Comparative Examples 4-1 to 4-2. Specifically, the elastic modulus (upper left), compressive strength (upper right), tensile strength (lower left) and adhesive strength (lower right) of the hydrogels of Examples 2-2 and Comparative Examples 4-1 to 4-2 indicated.

Referring to FIG. 5, it can be confirmed that the physical properties of the hydrogel of Example 2-2 prepared using linear polyethylene glycol are superior to those of the hydrogel of Comparative Examples 4-1 to 4-2 prepared using multi-regional polyethylene glycol. can

6 shows the physical properties of the hydrogels of Examples 2-1 to 2-3, Comparative Examples 1 and 2-3. Specifically, the elastic modulus (upper left), compressive strength (upper right), tensile strength (lower left) and adhesive strength (upper left) of the hydrogels of Examples 2-1 to 2-3, Comparative Examples 1 and 2-3 bottom right) is shown.

6, it can be seen that the physical properties of the hybrid hydrogels prepared in Examples 2-1 to 2-3 are superior to those of the hydrogels prepared with a single polymer of Comparative Examples 1 and 2-3, and in particular, It can be seen that the physical properties of Examples 2-2 and 2-3 in which the weight ratio of the first polymer to the second polymer is 1:1 or 1:3 are excellent.

7 shows the physical properties of the hydrogels of Examples 3-1 to 3-3. Specifically, the elastic modulus (upper left), compressive strength (upper right), tensile strength (lower left) and adhesive strength (lower right) of the hydrogels of Examples 3-1 to 3-3 were shown.

8 shows the physical properties of the hydrogels of Examples 4-1 to 4-3. Specifically, the elastic modulus (upper left), compressive strength (upper right), tensile strength (lower left) and adhesive strength (lower right) of the hydrogels of Examples 4-1 to 4-3 were shown.

9 shows the physical properties of the hydrogels of Examples 5-1 to 5-3. Specifically, the elastic modulus (upper left), compressive strength (upper right), tensile strength (lower left) and adhesive strength (lower right) of the hydrogels of Examples 5-1 to 5-3 were shown.

10 shows the physical properties of the hydrogels of Examples 6-1 to 6-3. Specifically, the elastic modulus (upper left), compressive strength (upper right), tensile strength (lower left) and adhesive strength (lower right) of the hydrogels of Examples 7-1 to 7-3 were shown.

11 shows the physical properties of the hydrogels of Examples 7-1 to 7-3. Specifically, the elastic modulus (upper left), compressive strength (upper right), tensile strength (lower left) and adhesive strength (lower right) of the hydrogels of Examples 7-1 to 7-3 were shown.

7 to 9 , it can be seen that excellent physical properties can be achieved even if the first polymer is changed to heparin, hyaluronic acid, or alginate, respectively, and referring to FIGS. 10 to 11 , the first reactor and the second Even if the reactor is changed, it can be confirmed that the excellent physical properties of the hydrogel are also secured.

Summarizing the above, it can be seen that the hybrid hydrogel according to an embodiment of the present invention has excellent mechanical properties due to high elastic modulus, compressive strength, tensile strength and adhesive strength. In particular, as the molecular weight of the second polymer increases, the mechanical It can be seen that the physical properties are excellent.

Biocompatibility Assessment

Experimental Example 2: Evaluation of cell viability

C2C12 was trypsinized, centrifuged, counted, and 10% fetal bovine serum (Wisent, Saint-Bruno, Quebec, Canada) and 1% penicillin-streptomycin (P/S, Gibco BRL (Grand Island, NY)). USA))) was carefully suspended in DMEM growth medium to obtain a homogeneous cell solution at a density of 5 x 10 ³ cells/ml. The hydrogels in triplicate were prepared in 24-well plates according to the method of the hydrogel formation section. However, Dulbecco's phosphate buffered saline was used to prepare polymer, HRP and H ₂ O ₂ solutions, which were filtered using a syringe filter (pore size 0.2 m) for sterilization. After gelation and stabilization within 30 min, the hydrogel was incubated with 1 mL cell solution prepared under standard culture conditions (37 °C and 5% CO ₂ ). The medium was changed every 2 days. Cell proliferation was quantified by the WST-1 assay method. Each hydrogel well was replaced with 1 mL of fresh medium and 100 μL WST-1 reagent was added to each sample well. The plate was incubated for 1 hour at 37 °C under 5% CO ₂ , and then 100 μL of the reaction solution was transferred to a 96-well plate. Optical density (OD) was measured at 450 nm using a microplate spectrophotometer (VersaMax Tunable Microplate reader, Molecular Devices, USA). The measurement was performed three times.

The hydrogels were tested in the same manner as above using the hydrogels of Examples 1-1 to 1-3, Examples 2-1 to 2-3, Comparative Examples 1 and 2-1 to 2-3, respectively, Cell adhesion was measured.

In FIG. 12, the WST-1 analysis of C2C12 cultured in the hydrogel of Examples 1-1 to 1-3, Examples 2-1 to 2-3, Comparative Example 1 and Comparative Examples 2-1 to 2-3 was used. A graph of the measured cell adhesion is shown.

12, it can be seen that the hybrid hydrogels prepared in Examples 1-1 to 1-3 show similar levels of cell adhesion and affinity to Comparative Example 1, which is a gelatin hydrogel having excellent cell affinity. It can be confirmed that the hybrid hydrogels prepared in Examples 2-1 to 2-3 also show adhesion and cell affinity similar to those of Comparative Example 1, which is a gelatin hydrogel having excellent cell affinity.

Experimental Example 3: Evaluation of drug release ability

The precursor solutions of the hydrogels of Examples 1-1 to 1-3, Examples 2-1 to 2-3, Comparative Example 1 and Comparative Examples 2-1 to 2-3 were dissolved in PBS, and a drug (Dexamethasone, Dex): 10 mg/ml) and divided into two aliquots. HRP (0.04 mg/ml) was added to one aliquot and H ₂ O ₂ (0.6 wt%) was added to the other aliquot (polymer : Dex : HRP / H ₂ O ₂ = 8 : 1 : 1 by volume) ). The drug loaded hydrogel was formed by mixing two aliquots. The final experimental conditions of HRP, H ₂ O ₂ and drug were 0.004 mg/ml, 0.01-0.03 wt%, and 1 mg/ml (Dex), respectively. Then, 1 ml PBS buffer was added to the hydrogel and incubated at 37 °C. At predetermined time intervals, the supernatant was collected, stored at -20 °C and fresh PBS buffer was added. The concentration of Dex in the collected buffer was analyzed by measuring the absorbance at 241 nm. The cumulative release percentage of Dex was calculated by the following equation (C: Dex release concentration in the imported buffer, Co: initial Dex concentration).

[Equation]

Dex emission (%) = C / Co Х 100

The hydrogel was evaluated for drug release ability by using the hydrogels of Examples 1-1 to 1-3, Comparative Examples 1 and 2-1 to 2-3, respectively, and Examples 1-1 to 1 in FIG. 13 -3, the hydrogels of Comparative Examples 1 and 2-1 to 2-3, and the hydrogels of Examples 2-1 to 2-3, Comparative Examples 1 and 2-3 in FIG. 14 according to time A graph of the cumulative release percentage is shown.

Referring to FIG. 13 , it was confirmed that the drug release level increased as the molecular weight of PEG increased.

Referring to FIG. 14 , it was confirmed that the drug release level increased as the content of PEG increased.

Experimental Example 4: Evaluation of release ability according to molecular weight of drug

Using the hydrogel of Example 2-3, and using dexamethasone having a molecular weight of 392.464 g/mol or bobbin serum albumin having a molecular weight of 66.5 kg/mol, the cumulative release percentage was measured in the same manner as in Experimental Example 3 derived.

15 shows the cumulative drug release percentage with time of dexamethasone or bobbin serum albumin of the hydrogel of Example 2-3.

Referring to FIG. 15 , a drug with a small molecular weight, such as dexamethasone, was relatively more easily released from the hydrogel, indicating a faster release rate than a macromolecular drug such as bobbin serum albumin.

Claims (22)

a first polymer containing two or more first reactive groups; and
A second polymer comprising a main chain including a repeating unit represented by the following formula (1) and a second reactive group bonded to both ends of the main chain;
The first polymer and the second polymer are cross-linked by the reaction of the first reactive group and the second reactive group,
A hybrid hydrogel having a weight average molecular weight of 1,000 or more and 20,000 or less of the second polymer:
[Formula 1]

According to claim 1,
The weight ratio of the first polymer to the second polymer is 3:1 to 1:3 hybrid hydrogel.
According to claim 1,
The second polymer is a hybrid hydrogel having a linear structure.
According to claim 1,
The first 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 , A hybrid hydrogel comprising at least one of carrageenan and agarose.
According to claim 1,
The first reactive group is a phenol derivative, thiol, (meth) acrylamide, cyclodextrin, N-hydroxysuccinimide, furan, hybrid hydrogel of any one of dopa.
According to claim 1,
The second reactive group is a phenol derivative, vinyl sulfone, (meth) acrylate, maleimide, adamantane, amine, hybrid hydrogel of any one of dopa.
According to claim 1,
The first reactive group and the second reactive group are phenol derivatives,
The hybrid hydrogel wherein the first polymer and the second polymer are cross-linked in situ by horseradish peroxidase and hydrogen peroxide addition.
According to claim 1,
wherein the first reactive group is a thiol and the second reactive group is vinyl sulfone;
The first polymer and the second polymer is a hybrid hydrogel that is cross-linked by the reaction of the thiol and the vinyl sulfone.
According to claim 1,
The first reactive group is (meth) acrylamide and the second reactive group is (meth) acrylate,
The first polymer and the second polymer is a hybrid hydrogel that is cross-linked by the reaction of the (meth) acrylamide and the (meth) acrylate.
According to claim 1,
The hybrid hydrogel that the first polymer is further connected by the reaction between the first reactive groups, and the second polymer is further connected by the reaction between the second reactive groups.
According to claim 1,
The hydrogel is a hybrid hydrogel that is crosslinked in situ including a physiologically active material having an additional phenol, aniline, amine or thiol group.
According to claim 1,
The hydrogel is a hybrid hydrogel that is cross-linked in situ including a physiologically active material that is a peptide containing tyrosine additionally.
A material for tissue adhesion and hemostasis comprising the hybrid hydrogel according to any one of claims 1 to 12.
14. The method of claim 13,
The material is a cranial nerve surgery including a vascular surgical area; orthopedic surgery, including bone adhesion; hemostasis in lacerations; suture of the femoral artery; cataract incision sutures, cartilage and articular cartilage healing; skin bonding; organ/secretory incision hemostasis; gastrointestinal division; And material for tissue adhesion and hemostasis applied to any one selected from the group consisting of tendon and ligament healing.
An implant material for tissue regeneration and filling comprising the hybrid hydrogel according to any one of claims 1 to 12.
16. The method of claim 15,
The implant material includes cartilage regeneration, bone regeneration, periodontal bone regeneration, skin regeneration, cardiac tissue regeneration, artificial intraocular lens, spinal nerve regeneration Cord regeneration), cranial regeneration, vocal regeneration and augmentation, adhesion barrier, urinary incontinence treatment, wrinkle removal filler, burn treatment agent ( wound dressing), tissue augmentation, and an implant material for tissue regeneration and filling that is applied to any one selected from the group consisting of intervertebral disc treatment.
A carrier for a physiologically active substance or drug carrier comprising the hybrid hydrogel according to any one of claims 1 to 12.
18. The method of claim 17,
The physiologically active substance or drug is a peptide or protein drug; antibacterial agents; anticancer drugs; and an anti-inflammatory agent; a carrier for a physiologically active substance or drug carrier comprising at least one selected from the group consisting of.
19. The method of claim 18,
The peptide or protein drug is fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), transforming growth factor (TGF), bone morphogenetic growth factor (bone morphogenetic) protein; BMP), human growth hormone (hGH), porcine growth hormone (pGH), leukocyte growth factor (G-CSF), red blood cell growth factor (EPO), macrophage growth factor (M-CSF), tumor necrosis factor (TNF) ), epithelial growth factor (EGF), platelet-derived growth factor (PDGF), interferon-α,β,γ, interleukin-2 (IL-2), calcitonin, nerve growth factor (NGF), growth hormone releasing factor, angio Tensin, luteinizing hormone releasing hormone (LHRH), luteinizing hormone releasing hormone agonist (LHRH agonist), insulin, thyroid stimulating hormone releasing hormone (TRH), angiostatin, endostatin, somatostatin, glucagon, endorphin, bacitra A carrier for a physiologically active substance or drug carrier, which is any one selected from the group consisting of syn, mergain, colistin, single antibody, vaccines, and mixtures thereof.
19. The method of claim 18,
The antibacterial agent is minocycline, tetracycline, ofloxacin, fosfomycin, megain, profloxacin, ampicillin, penicillin, doxycycline, thienamycin, cephalosporin, norkadisin, gentamicin, neomycin, kanamycin , paromomycin, micronomycin, amikacin, tobramycin, dibecacin, cefotaxin, cefaclor, erythromycin, cyprofloxacin, levofloxacin, enoxacin, vancomycin, imipenem, fusidic acid and mixtures thereof A carrier for a physiologically active substance or drug delivery that is any one selected from the group consisting of.
19. The method of claim 18,
The anticancer agent is paclitaxel, texotere, adriamycin, endostatin, angiostatin, mitomycin, bleomycin, cisplatin, carbopletin, doxorubicin, daunorubicin, idarubicin, 5-fluorouracil, methotrexate, ect A carrier for a physiologically active substance or drug carrier, which is any one selected from the group consisting of tinomycin-D and mixtures thereof.
19. The method of claim 18,
The anti-inflammatory agent is acetoaminephen, aspirin, ibuprofen, dicrofenac, indomethacin, piroxicam, fenoprofen, flubiprofen, ketoprofen, naproxen, suprofen, loxoprofen, synox Sicam, tenoxicam, and any one selected from the group consisting of mixtures thereof, a carrier for a physiologically active substance or drug delivery.

Images