KR20210089899A - Hydrogel of biochemically cross-linked animal cartilage-derived material and method for manufacturing the same and composition for inhibiting angiogenesis using the same - Google Patents

Abstract

The present invention relates to: hydrogel comprising animal cartilage-derived tissues which is obtained by biochemically cross-linking animal cartilage-derived tissues through a polyethylene glycol (PEG)-based cross-linking agent; a manufacturing method of the hydrogel; and a composition for inhibiting angiogenesis using the same.

Description

Hydrogel of biochemically crosslinked animal cartilage-derived tissue, manufacturing method thereof, and composition for inhibiting angiogenesis using the same }

The present invention relates to a biochemically cross-linked hydrogel of animal cartilage-derived tissue, a method for preparing the same, and a composition for inhibiting angiogenesis using the same.

Cartilage, like other connective tissues, is composed of connective tissue cells and acellular matrix, but unlike intrinsic connective tissue, it is a special connective tissue containing a hard and somewhat flexible matrix. Most of the connective tissue cells of cartilage are composed of a single chondrocyte, and these chondrocytes are located in the cartilage cavities in the cartilage matrix.

Acellular matrix is divided into fibrous and amorphous. Most of the fiber component is composed of type II collagen (collagen), but some cartilage is rich in elastic fibers. The intangible substance is mainly composed of protein sugar, which is mainly composed of sulfated glycosaminoglycans. In the cartilage matrix, a large number of protein sugar molecules are linked by linking proteins by hyaluronic acid, one of glycosaminoglycans, to form macromolecules. These macromolecules are also bound to the glue fibers. Protein sugar molecules are attached to the glia by chondronectin, an amorphous glycoprotein.

As other substrates, non-collagen proteins and glycoproteins account for 10-15% of the dry weight of cartilage, and these mainly help to stabilize the structure of matrix macromolecules and form organic tissues.

On the other hand, since the cell antigen is recognized by the host during tissue transplantation, it may cause an inflammatory reaction or immune rejection of the tissue. However, since acellular matrix components are generally resistant to components in allogeneic recipients, various acellular matrix tissues including cardiovascular, blood vessel, skin, nerve, bone muscle, tendon, bladder, and liver can be used for tissue engineering and regenerative medicine applications. A lot of research is being done in this regard.

This natural material derived from animal cartilage tissue is composed of an extracellular matrix (ECM). The extracellular matrix provides an environment for cells to live while supplying chemical factors necessary for cell growth and differentiation, and has high biocompatibility and almost no immune response. However, the short biodegradation period and low physical properties have limitations in practical application. Therefore, in order to solve this limitation, it is necessary to improve the physical properties of cartilage-derived tissues through physical or chemical treatment.

In addition, angiogenesis (angiogenesis) is a biological process that creates a new blood vessel in a tissue or organ. In a normal state, the factors inducing and inhibiting angiogenesis in a living body are in equilibrium, but a situation in which a disease is induced In the disease, the factors that promote vascular growth increase or the inhibitory factors do not function properly, so that the angiogenesis is not regulated autonomously and continues to grow, leading to disease. If such angiogenesis is not controlled normally, pathological disorders such as diabetic retinopathy, rheumatoid arthritis, inflammation, endometriosis, age-related deterioration of vision, psoriasis, and hemangioma are caused.

Therefore, when using a natural material derived from animal cartilage tissue for the purpose of inhibiting angiogenesis, there is a need for research on optimization.

The present invention is a hydrogel that can control the biodegradation period, has improved mechanical properties, and has angiogenesis inhibitory effect. Animal cartilage obtained by biochemically crosslinking animal cartilage-derived tissue through a polyethylene glycol (PEG)-based crosslinking agent. An object of the present invention is to provide a hydrogel and the like containing the derived tissue.

However, the technical problem to be achieved by the present invention is 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.

The present invention provides a hydrogel comprising animal cartilage-derived tissue biochemically crosslinked by using a polyethylene glycol (PEG)-based crosslinking agent.

As a terminal functional group of the PEG-based crosslinking agent, N-hydroxysuccinimide (NHS) is introduced, and the NHS may react with an amine group of the animal cartilage-derived tissue.

The average molar mass of the PEG-based crosslinking agent may be 200 g/mol to 10,000 g/mol.

When the biochemically crosslinked animal cartilage-derived tissue is in the form of a film, the contact angle with respect to water may be 40.0 ° to 65.0 °.

The content of the biochemically cross-linked animal cartilage-derived tissue in the hydrogel may be 5 wt% to 30 wt%.

The complex viscosity at 37° C. of the hydrogel may be 1.5 Х 10 ⁰ Pa·s to 7.0 Х 10 ³ Pa·s.

In one embodiment of the present invention, (a) preparing animal cartilage-derived tissue powder by pulverizing animal cartilage-derived tissue; (b) mixing and crosslinking the aqueous solution containing the animal cartilage-derived tissue powder prepared in step (a) with the polyethylene glycol (PEG)-based crosslinking agent-containing solution to prepare a crosslinked animal cartilage-derived tissue; and (c) drying the cross-linked animal cartilage-derived tissue prepared in step (b), re-pulverizing it, and dispersing it in a buffer to prepare a hydrogel.

The content of the crosslinking agent in the crosslinking agent solution in step (b) may be 1 wt% to 10 wt%.

In another embodiment of the present invention, there is provided a pharmaceutical composition for inhibiting angiogenesis comprising the hydrogel.

The pharmaceutical composition may be an injectable formulation.

The hydrogel according to the present invention includes animal cartilage-derived tissue biochemically cross-linked animal cartilage-derived tissue through a polyethylene glycol (PEG)-based crosslinking agent. It may have improved mechanical properties due to the decrease in viscosity. In particular, the hydrogel can be suitably used as an injectable formulation, and has excellent angiogenesis inhibitory efficacy.

Figure 1 (a) schematically shows a method for producing a biochemically crosslinked hydrogel of animal cartilage-derived tissue, Figure 1 (b) shows the result of introducing a carboxyl group (-COOH) into PEG-400. , Figure 1 (c) shows the result of introducing N-hydroxysuccinimide (NHS) into the PEG-400-COOH prepared in Figure 1 (b).
2(a) shows a method of introducing IR783-COOH into the amine group (-NH2) of the pig cartilage-derived tissue prepared in Examples 1 and 2, and FIG. 2(b) shows a carboxyl group (- COOH) was introduced.
3 is a photograph showing that the tissue powder derived from pig cartilage prepared in Examples 1 and 2 can be prepared as a hydrogel and used as an injection formulation.
4 is a photograph showing the contact angle with respect to water when the crosslinking agent of the pig cartilage-derived tissue film prepared in Example 2 is changed and the results thereof.
5 is a photograph before and after swelling and the result when the crosslinking agent of the pig cartilage-derived tissue film prepared in Example 2 is changed.
6 is a result showing the rheological properties according to the degree of crosslinking of the hydrogel of pig cartilage-derived tissue prepared in Example 4.
Figure 7 (a) is a photograph showing the degree of injectability through the measurement of the compressive strength of the hydrogel of the pig cartilage-derived tissue prepared in Example 4, Figure 7 (b) is the received per compression distance in Figure 7 (a) The result of measuring the force is a result showing the compressive strength according to the degree of crosslinking of the prepared pig cartilage-derived tissue hydrogel, FIG. 7(c) is a result showing the maximum force value measured in FIG. 7(b), FIG. 7(d) is a result showing the degree of injection according to the degree of crosslinking of the hydrogel of pig cartilage-derived tissue and the diameter of the syringe needle through the results of FIGS. 7(b) and (c).
8 is a photograph showing the biodegradation behavior over time through an animal experiment using a hydrogel of pig cartilage-derived tissue to which the fluorescent material prepared in Example 3 is introduced.
9 is a photograph of CD31 staining through the tissue extracted in Example 3.

The present inventors can prepare a hydrogel with excellent mechanical properties when a polyethylene glycol (PEG)-based crosslinking agent is used while conducting a study using animal cartilage-derived tissue with excellent biocompatibility, and in particular, inhibiting angiogenesis By confirming that the efficacy is excellent, the present invention was completed.

Hereinafter, the present invention will be described in detail.

The present invention provides a hydrogel comprising animal cartilage-derived tissue biochemically crosslinked by using a polyethylene glycol (PEG)-based crosslinking agent.

The hydrogel refers to a gel using water as a dispersion medium, and refers to a state in which the hydrosol loses fluidity due to cooling or a hydrophilic polymer having a three-dimensional network structure and microcrystalline structure contains water and expands.

Since the hydrogel has a porous structure, when used as a support including stem cells, it may have the advantage of increasing the survival rate of cells by smoothly supplying sufficient oxygen and nutrients to the cells. In addition, when used as a drug carrier carrying a drug, it is possible to effectively deliver the drug to the target cell while minimizing the dispersion of the drug.

The animal-derived cartilage tissue preferably refers to an animal-derived cartilage acellular matrix (CAM), and includes a fibrous component mostly composed of collagen. The animal from which the cartilage tissue is derived is preferably a mammal, and more preferably any one selected from the group consisting of humans, pigs, cattle, sheep, horses and cats, and more specifically derived from humans or pigs. It is preferred, but not limited thereto.

The PEG-based cross-linking agent is a biochemical cross-linking agent for chemically cross-linking the animal-derived cartilage tissue (natural material), and the PEG-based cross-linking agent is N-hydroxysuccinimide (NHS) is introduced, and the NHS preferably reacts with the amine group of the animal cartilage-derived tissue, but is not limited thereto. The PEG-based cross-linking agent increases water affinity compared to when cross-linking through other cross-linking agents. As such, when the affinity for water is increased, the contact angle with respect to water is decreased, and solubility in water is increased. When the solubility in water is increased, there is an advantage in that it is possible to prepare a hydrogel containing more animal cartilage-derived tissue, which is a raw material, in preparation for crosslinking through other crosslinking agents.

Specifically, the average molar mass of the PEG-based crosslinking agent may be 200 g/mol to 10,000 g/mol, preferably 400 g/mol to 4,000 g/mol, and 400 g/mol to 2000 g/mol. More preferably, it is most preferably 400 g/mol, but is not limited thereto. By maintaining a low average molar mass of the PEG-based crosslinking agent, it is possible to increase the degree of swelling while maintaining a low contact angle with respect to water of the biochemically crosslinked animal cartilage-derived tissue (film form). For example, when the biochemically crosslinked animal cartilage-derived tissue is in the form of a film, the contact angle to water may be 40.0 ° to 65.0 °, preferably 40.0 ° to 50.0 °, and 40.0 ° to 45.0 ° More preferably, but not limited thereto.

The content of the biochemically cross-linked animal cartilage-derived tissue in the hydrogel may be 5 wt% to 30 wt%.

On the other hand, it is characterized in that the complex viscosity of the hydrogel is reduced, the complex viscosity at 37 ℃ of the hydrogel may be 1.5 Х 10 ⁰ Pa·s to 7.0 Х 10 ³ Pa·s, and 1.5 Х 10 ⁰ Pa·s · s to 1.0 Х 10 ² Pa·s, and more preferably 1.5 Х 10 ⁰ Pa·s to 1.0 Х 10 ¹ Pa·s, but is not limited thereto. In order to reduce the complex viscosity of the hydrogel as described above, it is necessary to increase the degree of crosslinking by adjusting the content of the crosslinking agent in the crosslinking agent solution, which will be described later.

In addition, the present invention comprises the steps of (a) pulverizing animal cartilage-derived tissue to prepare an animal cartilage-derived tissue powder; (b) mixing and crosslinking the aqueous solution containing the animal cartilage-derived tissue powder prepared in step (a) with the polyethylene glycol (PEG)-based crosslinking agent-containing solution to prepare a crosslinked animal cartilage-derived tissue; and (c) drying the cross-linked animal cartilage-derived tissue prepared in step (b), re-pulverizing it, and dispersing it in a buffer to prepare a hydrogel.

Since the hydrogel has been described above, a redundant description thereof will be omitted.

In addition, the powdering of animal cartilage-derived tissue in step (a) includes: a) freeze-drying and pulverizing animal cartilage-derived tissue; b) decellularizing the cartilage-derived tissue powder pulverized in step a); and c) treating the decellularized cartilage-derived tissue powder in step b) with an acidic solution and pepsin, and then neutralizing with a basic solution to obtain an aqueous solution of cartilage-derived tissue powder.

Specifically, the powdering of the animal cartilage-derived tissue may mean freeze-drying and freeze-pulverizing the animal cartilage-derived tissue. The freeze-drying and freeze-grinding may be performed in cryogenic conditions using pretreated animal cartilage-derived tissue, and in this case, the cryogenic conditions refer to -100 to -50° C. and 0.01 mTorr to 10 mTorr conditions. In addition, freeze crushing may be performed using a known grinder, and the prepared animal cartilage-derived tissue powder may have a size of 10 μm to 100 μm.

Thereafter, the prepared cartilage-derived tissue powder is dissolved in an aqueous solution containing a proteolytic enzyme, neutralized, and then the proteolytic enzyme is removed by dialysis or the like to prepare a water-soluble cartilage-derived tissue powder. Thereafter, lyophilization at cryogenic temperatures may be added. In this case, as the proteolytic enzyme, an enzyme capable of decomposing collagen constituting the cartilage-derived tissue powder may be used, and pepsin is preferable, but is not limited thereto.

The content of the crosslinking agent in the crosslinking agent solution in step (b) may be 1 wt% to 10 wt%, and as the crosslinking agent content in the crosslinking agent solution increases, the degree of crosslinking can be increased. can reduce In addition, the mixing and crosslinking may be performed at a speed of 10 rpm to 500 rpm, at 20°C to 80°C for 1 hour to 30 hours, for example, the mixing and crosslinking is performed at a speed of 10 rpm to 500 rpm, After being carried out at a first temperature of 20°C to 50°C for 10 hours to 20 hours, at a second temperature exceeding 50°C to 65°C for 5 hours to 10 hours, and then performing a third temperature of more than 65°C to 80°C It can be carried out for 1 hour to 5 hours at the temperature. The animal cartilage-derived tissue has a helix structure in which two chains are twisted as shown in FIG. 1(a), which will be described later. When the temperature is raised, a denaturation phenomenon occurs in which the helix structure is destroyed, and the helix structure is restored at room temperature. will form There is an advantage in that physical properties can be increased by proceeding with this process.

Before dispersion in step (c), it may be further mixed and crosslinked with a solution containing a fluorescent substance having a carboxyl group. Since the fluorescent material has a carboxyl group as a functional group, it has an advantage in that it can chemically react with an amine group of collagen. In this case, as the fluorescent material before the functional group is introduced, IR 783, IR 780, FITC (Fluorescein isothiocyanate), rhodamine, and PI (Propidium iodide) may be used.

In addition, the present invention may provide an injectable formulation comprising the hydrogel.

Since the hydrogel has been described above, a redundant description thereof will be omitted.

The injectable formulation refers to a form that can be injected into a syringe. For example, the injectable formulation is preferably injectable into a syringe having a needle inner diameter of 10G to 36G, and injectable into a syringe having a diameter of 10G to 28G. more preferably, but is not limited thereto. At this time, if the inner diameter of the needle of the syringe is too small, the compression strength per compression distance continuously increases, so there is a problem that it is impossible to inject.

The injectable formulation may be for tissue repair, drug delivery, or cell delivery. Specifically, the hydrogel can be used as a carrier for sustained release of the drug or bioactive factor through injection into the body by carrying the drug or bioactive factor therein. In addition, the hydrogel can be used as a support for bone differentiation when injected into the body by supporting stem cells therein.

In addition, the present invention provides a pharmaceutical composition for inhibiting angiogenesis comprising the hydrogel.

Since the hydrogel has been described above, a redundant description thereof will be omitted. First, the hydrogel uses animal cartilage-derived tissue, and can significantly improve angiogenesis inhibitory efficacy compared to using Small intestinal submucosa (SIS). In addition, the hydrogel is a biochemical crosslinking agent, which is a case of using a PEG-based crosslinking agent, and can improve angiogenesis inhibitory efficacy compared to a case where it is omitted.

The angiogenesis inhibition refers to inhibiting a biological process that creates a new blood vessel in a tissue or organ, diabetic retinopathy, retinopathy of prematurity, corneal transplant rejection, neovascularization glaucoma, erythematosus, proliferative retinopathy, psoriasis , hemophilic joints, capillary proliferation within atherosclerotic plaques, keloids, wound granulation, vascular adhesion, rheumatoid arthritis, osteoarthritis, autoimmune diseases, Crohn's disease, restenosis, atherosclerosis, intestinal adhesions, cat scratch disease, Ulcer, liver cirrhosis, glomerulonephritis, diabetic nephropathy, malignant neuropathy, thrombotic microangiopathy, organ transplant rejection, glomerulopathy, diabetes, angiogenesis-dependent cancer, benign tumor, inflammatory disease and neurodegenerative disease It may mean preventing or treating angiogenesis-related diseases selected from.

The pharmaceutical composition may be formulated in an oral or parenteral form according to a conventional method, respectively, and is preferably formulated in an injectable form, but is not limited thereto.

For such formulation, an appropriate carrier, excipient or diluent commonly used in the preparation of the pharmaceutical composition may be included.

The carrier or excipient or diluent may include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, undecided. various compounds or mixtures including vaginal cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

In particular, parenteral formulations (especially, injectable formulations) include sterile aqueous solutions, non-aqueous formulations, suspensions, emulsions, lyophilized formulations, and suppositories. Non-aqueous solvents and suspending agents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. As a base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin, glycerol gelatin and the like can be used.

The preferred dosage of the pharmaceutical composition varies depending on the patient's condition, body weight, degree of disease, drug form, administration route and period, but may be appropriately selected by those skilled in the art. However, for a desirable effect, it may be administered at 0.0001 to 2,000 mg/kg per day, preferably 0.001 to 2,000 mg/kg. Administration may be administered once a day, or may be administered in several divided doses. However, the scope of the present invention is not limited by the dosage.

The pharmaceutical composition may be administered to mammals such as rats, mice, livestock, and humans by various routes. All modes of administration can be administered by, for example, oral, dermal, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or intracerebroventricular injection.

As described above, the hydrogel according to the present invention includes animal cartilage-derived tissues biochemically crosslinked animal cartilage-derived tissues through a polyethylene glycol (PEG)-based crosslinking agent, and the hydrogel has a biodegradation period. While it can be controlled, it can have improved mechanical properties due to a decrease in the complex viscosity. In particular, the hydrogel can be suitably used as an injectable formulation, and has excellent angiogenesis inhibitory efficacy.

Hereinafter, preferred examples are presented to help the understanding of the present invention. However, the following examples are only provided for easier understanding of the present invention, and the contents of the present invention are not limited by the following examples.

Example 1: Isolation of cartilage-derived tissue from pigs and preparation of water-soluble cartilage-derived tissue powder

Articular cartilage was isolated from the forelimbs and hind legs of pigs weighing about 100 kg at 6 months of age under aseptic conditions. At the time of separation, the cartilage part was cut with a blade so that only the cartilage part was separated without including the subchondral bone part, and then washed with a phosphate buffered aqueous solution. The separated articular cartilage tissue was treated with a storage buffer of 10 mM Tris-HCl at pH 8.0 for 12 hours, and then centrifuged using a centrifuge to remove the supernatant. After transferring the precipitated pig cartilage-derived powder to a beaker, SDS buffer was added and stirred at 4°C for 2 hours. After adding sterile tertiary water and centrifuging at 2,000 rpm for 10 minutes using a centrifuge to remove the supernatant, the precipitated pig cartilage joint was transferred to a beaker, DNase solution was added, and the mixture was stirred at 100 rpm for 12 hours. Then, centrifuged at 2,000 rpm for 10 minutes, and after removing the supernatant, freeze-pulverized pig cartilage-derived tissue powder in 3 cycles of pre-cooling 50 sec/grinding (60 Hz) 240 sec/cooling 50 sec in liquid nitrogen. was obtained. The obtained powder was placed in a beaker, a DNase solution was added, and the mixture was stirred at 100 rpm for 12 hours. Thereafter, the solution was centrifuged at 2,000 rpm for 10 minutes using a centrifuge, and after removing the supernatant, a small amount of sterile tertiary water was added to suspend pig cartilage-derived powder in a cryogenic freezer at -70°C and 5 mTorr conditions. Freeze-dried for 48 hours. The freeze-dried pig cartilage-derived powder was stirred by adding 25 ml of an 800 unit/ml pepsin aqueous solution per 1 g to prepare a solution. After neutralization by adding NaOH solution to the prepared solution to pH 7.4, fix only one end of the dialysis membrane, pour the neutralized solution into the dialysis membrane, fix the other end, and put it in 2 L of sterile tertiary water at 4℃ stirred for 24 hours. After dialysis, the aqueous solution of pepsin was placed in a container to be lyophilized and lyophilized in a cryogenic freezer at -70°C and 0 mTorr for 48 hours to prepare a water-soluble pig cartilage-derived powder.

Example 2: Biochemical crosslinking reaction of pig cartilage-derived tissue using polyethylene glycol 400

0.2 g of the water-soluble pig cartilage-derived powder prepared in Example 1 was added to a 30 mL vial, dispersed in 15 mL of sterile tertiary distilled water, and stirred at room temperature for 24 hours. After stirring, the solution is poured into a Teflon mold (75 mm × 75 mm), and 1.5 mL each of 1%, 3%, 5% and 7% of PEG-400-NHS solution, a biochemical crosslinking agent, is evenly added dropwise with a glass pipette. Then, the reaction was carried out on a shaker at 37°C at 100 rpm for 12 hours, at 60°C at 100 rpm for 6 hours, and at 70°C at 100 rpm for 3 hours on a shaker. The dried solid form of cross-linked pig cartilage-derived tissue was washed alternately with tertiary distilled water and phosphate buffer solution sterilized on a shaker at 100 rpm at 37 ° C for 30 minutes, then lyophilized for 48 hours to biochemically crosslinked. Pig cartilage-derived powder was prepared (Fig. 1 (a) ~ (c)).

Example 3: Preparation of pig cartilage-derived powder containing fluorescent material for near-infrared fluorescence imaging

_{In order to introduce a fluorescent substance into the amine group (-NH 2} ) of the pig cartilage-derived tissue prepared in Examples 1 or 2, first, IR783 (80 mg, 0.11 mmol) and 4-mercaptobenzoic acid (51) in a 50 mL round-bottom flask mg, 0.33 mmol) was dissolved in DMF (3 mL) and stirred at room temperature for 24 h. After the reaction, in order to remove unreacted substances and impurities from the reacted solution, it was precipitated in 30 mL of a mixed solvent of ethanol and diethyl ether (v/v = 1/19) and filtered. The obtained green solid was vacuum dried to obtain IR783-COOH. prepared.

Next, in a 20 mL vial, IR783-COOH (1.4 mg, 1.6 μmol) and DMTMM (0.66 mg, 2.4 μmol) were dissolved in sterile tertiary distilled water (5 mL), and the pig cartilage prepared in Example 1 or 2 The derived powder (0.1 g) was added to the IR783-COOH solution, and the mixed solution was stirred at room temperature for 24 hours. After the reaction, the reaction solution was poured onto a dialysis membrane (molecular weight cut-off: 2.0 kDa, Spectrum Laboratories, CA, USA) and then dialyzed in tertiary distilled water for 3 days to remove unreacted substances, and freeze-dried for 4 days to fluoresce Pig cartilage-derived powder containing the substance was prepared (FIGS. 2(a)-(b)).

Example 4: Preparation of a hydrogel from pig cartilage-derived tissue

A hydrogel was prepared by evenly dispersing the powder derived from pig cartilage prepared in Examples 1 or 2 in a phosphate buffer solution at 10, 15, or 20 wt%, respectively, in a 5 mL vial, and it was confirmed that a hydrogel usable as an injection was formed. (FIG. 3).

Experimental Example 1: Evaluation and analysis of contact angle and swelling degree control of a tissue film derived from pig cartilage according to the type of biochemical crosslinking agent

(1) Evaluation of the contact angle of the film according to the type of biochemical crosslinking agent

For the evaluation of the contact angle of the porcine cartilage-derived tissue film prepared in Example 2, 0.01 mL of tertiary distilled water was dropped on the film and the measurement results were shown in FIG. 4 using a contact angle goniometer (Phoenix 150, Suwon, Korea).

As shown in Figure 4, as a biochemical crosslinking agent, when a PEG-based crosslinking agent is used (CAM-PEG-400, CAM-PEG-2000, and CAM-PEG-4000), when glutaraldehyde is used (CAM-GA) In comparison, it is confirmed that the contact angle with respect to water is greatly reduced. In addition, it is confirmed that, among the cases of using a PEG-based crosslinking agent, the contact angle with respect to water also tends to decrease as the length decreases.

(2) Evaluation of film swelling according to the type of biochemical crosslinking agent

To evaluate the swelling degree of the porcine cartilage-derived tissue film prepared in Example 2, the porcine cartilage-derived tissue cut to a size of 15 mm X 15 mm was freeze-dried in a cryogenic freezer at -70° C. and 0 mTorr for 48 hours to retain moisture. After the film was completely removed, the weight of the film was measured, and the weight of the film containing moisture was measured after 3 h, 6 h, and 12 h by immersion in tertiary distilled water, and the results are shown in FIG. 5 .

As shown in FIG. 5 , as a biochemical crosslinking agent, when a PEG-based crosslinking agent is used (CAM-PEG-400, CAM-PEG-2000, and CAM-PEG-4000), when glutaraldehyde is used (CAM-GA) In comparison, it is confirmed that the degree of swelling is greatly increased. In addition, in the case of using a PEG-based crosslinking agent, it is confirmed that the swelling degree increases too much as the length thereof increases, thereby having a problem according to the decomposition behavior. Specifically, CAM-PEG-400 maintains a constant weight containing moisture after 6 hours, but CAM-PEG-4000 has low crosslinking efficiency within the same time period, so the weight of CAM-PEG-4000 is reduced due to structural destruction after 6 hours. is found to decrease. In addition, the decomposition behavior according to the content of the cross-linking agent is shown in FIG. 8, which will be described later. Through this, the decomposition behavior according to the length of the cross-linking agent can be inferred. That is, when the concentration of the crosslinking agent increases, the water content of the hydrogel increases. Similarly, when the length of the crosslinking agent increases, the water content of the hydrogel increases, and it is determined that the decomposition period will be shortened as shown in FIG. 8 . do.

Experimental Example 2: Evaluation and Analysis of Control of Mechanical Properties of Pig Cartilage-Derived Tissue Hydrogels According to the Degree of Biochemical Crosslinking

(1) Evaluation of rheological properties of hydrogels according to the degree of biochemical crosslinking

Elastic modulus and complex viscosity at 37 ° C using a rheometer (MCR 102, Anton Paar, Ostfildern, Germany) to measure the rheological properties according to the weight ratio and the degree of crosslinking of the hydrogel derived from pig cartilage prepared in Example 4 The values were measured, and the measurement results are shown in FIG. 6 .

As shown in FIG. 6 , it was confirmed that the complex viscosity of the hydrogel decreased as the degree of crosslinking of the pig cartilage-derived tissue increased, and the possibility of controlling the mechanical properties of the hydrogel by controlling the degree of crosslinking of the pig cartilage-derived tissue was confirmed.

(2) Evaluation of compressive strength and injectability of hydrogels according to the degree of biochemical crosslinking

In order to quantitatively determine the injectability by measuring the compressive strength according to the degree of crosslinking of the hydrogel of the pig cartilage-derived tissue prepared in Example 4, the method shown in FIG. 7(a) was used. The hydrogel was loaded in a 2 mL syringe and compressed at a rate of 10 mm/min using UTM (H5KT), and the inner diameter of the syringe needle was measured using 30 and 26G, and the measurement results are shown in FIG. 7(b). , the maximum force value measured during compression is shown in FIG. 7( c ).

As shown in Fig. 7(b), the scanability was easily observed for the curve in which the force received per compression distance was kept constant, and it was confirmed that the curve in which the force received per compression distance was continuously increased was not scannable. and the injectability of the results is shown in FIG. 7(d).

7(d), as the degree of crosslinking of the pig cartilage-derived tissue increases, the degree of injectability tends to increase, and as the length of the inner diameter of the syringe needle decreases, the pressure applied to the inside of the syringe needle increases. A tendency to decrease the degree of injectability was confirmed.

Experimental Example 3: Observation and evaluation of biodegradation period of porcine cartilage-derived tissue hydrogel through near-infrared fluorescence imaging

After sterilizing the pig cartilage-derived tissue powder into which the IR783 fluorescent material prepared in Example 3 was introduced using UV and 70% ethanol, 20% by weight of hydrogel was prepared in a phosphate buffer solution having a pH of 7.4, and nude mouse (male) , 8 weeks of age), 0.15 mL was injected with a 1 mL syringe (26 G) subcutaneously, and the results of observation of fluorescence imaging according to each time are shown in FIG. 8 .

As shown in FIG. 8 , the degree of fluorescence expression decreases over time as a whole, and as the degree of crosslinking of porcine cartilage-derived tissue increases, it was confirmed that the fluorescence retention period decreases, and the degradation rate in vivo according to the degree of crosslinking. The possibility of control was confirmed. That is, as the degree of crosslinking increases, it contains a lot of moisture, and accordingly, more substances used for decomposition of animal cartilage-derived tissues are contained in the hydrogel, which can be seen to increase the decomposition rate in vivo.

Experimental Example 4: Evaluation of In vivo Immunological Angiogenesis Inhibition

The tissue extracted in Experimental Example 2 was fixed in 10% formalin, and the fixed tissue was made into a paraffin block, cut to a thickness of 4 μm, fixed on a slide, and immunologically anti-CD31 antibody ab28364: CD31 (anti-CD31 antibody ab28364: abcam, UK) was stained and shown in FIG. 9 .

As shown in FIG. 9 , it was confirmed that angiogenesis could be inhibited since almost no blood vessels appeared in the hydrogel of pig cartilage-derived tissue crosslinked according to the present invention.

Claims (10)

A hydrogel comprising animal cartilage-derived tissue biochemically crosslinked by using a polyethylene glycol (PEG)-based crosslinking agent.
According to claim 1,
The PEG-based crosslinking agent is a terminal functional group, N-hydroxysuccinimide (NHS) is introduced, and the NHS is characterized in that it reacts with the amine group of the animal cartilage-derived tissue, hydrogel.
According to claim 1,
The hydrogel, characterized in that the average molar mass of the PEG-based crosslinking agent is 200 g / mol to 10,000 g / mol.
According to claim 1,
When the biochemically crosslinked animal cartilage-derived tissue is in the form of a film, the hydrogel, characterized in that the contact angle to water is 40.0 ° to 65.0 °.
According to claim 1,
The content of the biochemically crosslinked animal cartilage-derived tissue in the hydrogel is 5% to 30% by weight, the hydrogel.
According to claim 1,
The complex viscosity at 37° C. of the hydrogel is 1.5 Х 10 ⁰ Pa·s to 7.0 Х 10 ³ Pa·s, the hydrogel.
(a) pulverizing animal cartilage-derived tissue to prepare animal cartilage-derived tissue powder;
(b) preparing a crosslinked animal cartilage-derived tissue by mixing and crosslinking the aqueous solution containing the animal cartilage-derived tissue powder prepared in step (a) with the polyethylene glycol (PEG)-based crosslinking agent-containing solution; and
(c) drying the cross-linked animal cartilage-derived tissue prepared in step (b), re-pulverizing it, and dispersing it in a buffer to prepare a hydrogel
Method for producing hydrogel.
8. The method of claim 7,
The method for producing a hydrogel, characterized in that the content of the crosslinking agent in the crosslinking agent solution in step (b) is 1% to 10% by weight.
A pharmaceutical composition for inhibiting angiogenesis comprising the hydrogel according to any one of claims 1 to 6.
10. The method of claim 9,
The pharmaceutical composition is an injectable formulation, characterized in that the pharmaceutical composition for inhibiting angiogenesis.

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