KR101552387B1 - Method of Manufacturing Crosslinked Hydrogel for Drug Delivery system - Google Patents

Abstract

TECHNICAL FIELD The present invention relates to a medical material, and more particularly, to a medical material comprising a catechol-based functional group having an excellent adhesive force and coating power as an effective ingredient and being capable of being provided as an efficient drug delivery material, Moisture permeability and can prevent invasion of bacteria, bacteria and foreign substances from the outside, and provides a proper wetting environment to the wound to promote wound healing, minimize scars remaining after wound healing, To a medical material capable of preventing adhesion.

Description

Technical Field [0001] The present invention relates to a cross-linked hydrogel for drug delivery,

The present invention relates to matrix materials for drug delivery. More particularly, the present invention relates to a crosslinked hydrogel which can be used for a medical material containing a bio-material conjugated with a catechol having excellent adhesive strength.

Drug Delivery System (DDS) is a Dosage Form designed to minimize the side effects of existing medicines, maximize efficacy and effectiveness, and efficiently deliver the required amount of medication. Biomolecules for biocompatible and biodegradable hydrogels that can be injected for efficient drug delivery of macromolecules such as proteins and genes have been developed.

These polymers are chemically and physically cross-linked for stability and stability in vivo, but some crosslinking agents may be used for chemical bonding. Such crosslinked materials may cause side effects such as inflammation due to increased toxicity and residual materials. In the case of hydrogels, variables such as the rate of drug encapsulation and persistence are important.

In the case of hydrogels, natural wound healing mechanism proceeds when the tissue damage occurs due to drug delivery, surgery, or inflammation. If fibrous tissue is excessively generated in this process, the surrounding tissue and abnormal tissue It can also be used as an anti-adhesion material to interfere with bonding. Adhesion after abdominal surgery may lead to aftereffects such as intestinal dysfunction, intestinal obstruction, and chronic pain. In particular, adhesions after obstetric surgery will cause infertility.

In the case of obstetrics and gynecology, adherence to the endometrium occurs in 20 ~ 50% after curettage or aspiration for incomplete abortion, stillbirth and recurrent miscarriage. It is known that infertility, amenorrhea and habitual abortion are caused by such adhesion, Recently, a method of using physical barriers has been developed and used.

Physical barriers that are effective in preventing adhesion are required to have the flexibility of being able to act as a tissue or physical barrier while the wound heals and to wrap the wound area and isolate it from surrounding tissues. In addition, after the regeneration of damaged tissue, it must be resolved and absorbed and released in the body, and physical barriers and products decomposed therefrom should not cause toxic or inflammatory reactions in the body. A biodegradable polymer is used as a material for such an adhesion inhibitor.

Biodegradable polymers are collectively referred to as polymers that are chemically degraded in the body and whose shape and weight gradually disappear. Natural biodegradable polymers include collagen, fibronectin, gelatin, chitosan Poly (lactic acid) (PLA), poly (glycolic acid) (PGA), poly (D, L), and alginic acid -latide-co-glycolide (PLGA) and similar copolymers and poly (ε-caprolactone) (PCL), polyanhydrides, and polyesters.

Hyaluronic acid is a non-sulfated glycosaminoglycan that is widely distributed throughout connective tissue, epithelial tissue and nervous tissue. Hyaluronic acid is composed of D-glucuronic acid and D-N-acetylglucosamine, and is a disaccharide polymer in which β-1,4 and β-1,3 glucosidic bonds are alternately bonded. Pluronic F127 adhesion inhibitors also have the advantage of ease of use, which is a sol form at room temperature and changes from a body temperature to a gel form, but it has drawbacks that it must be used only on dry, bleeding-free surfaces.

In addition, PLA or PGA, which is a synthetic biodegradable polymer, has a long biodegradation period of 4 weeks or more and high mechanical strength. However, since it is a hydrophobic material, it has low water absorption and is not attached to the surface of living tissue. The hydrolysis results in the release of the acidic degradation product, which may cause an inflammatory reaction which causes adhesion.

(Patent Document 0001) Korean Patent Laid-Open No. 10-2008-0104228

(Non-Patent Document 0001)

Soft Matter, Yuhan Lee, Hyun Jung Chung, Sangho Yeo, Cheol-Hee Ahn, Heashin Lee, Phillip B. Messersmith and Tae Gwan Park, 977-983

(Non-Patent Document 0002)

Chem. Commun., Yeon Jeong Oh, a Il Hwan Cho, b Haeshin Lee, c Ki-Jung Park, Hyukjin Lee and Sung Young Park, 2012, 48, 11895-11897

Disclosure of the Invention The present invention has been conceived to solve the above-mentioned problems, and it is an object of the present invention to provide an osteoarthritic device capable of being used as an injectable carrier for proteins and stem cells, It is an object of the present invention to provide a medical material having moisture permeability.

According to an embodiment of the present invention, it is possible to prevent infiltration of bacteria, germs, foreign substances and the like from the outside, to provide a wet environment suitable for wound, and to provide a medical material capable of promoting wound healing such as wound, And to provide the above-mentioned objects.

According to an embodiment of the present invention, there is provided a medical material which can minimize scars remaining after wound healing and prevent adhesion of organs.

According to one embodiment of the present invention, there is provided a medical material which can be applied to various medical fields such as otorhinolaryngology, ophthalmology, dentistry, and neurosurgery by having an adhesive force, biocompatibility and water resistance in an aqueous solution.

A first object of the present invention is to provide a crosslinked hydrogel comprising a compound having a catechol-based functional group as an active ingredient, which comprises: i) a compound having a catechol-based functional group, Or iii) a living / synthetic polymer having a catechol-based functional group and a living / synthetic polymer having an -NH2 or -SH group at the end, or iv) a catechol-based functional group And a crosslinked hydrogel formed by cross-linking a living / synthetic polymer having -NH 2 or -SH group and a living / synthetic polymer having -NH 2 or -SH group at the terminal.

Also, the compound having a catechol-based functional group may be selected from the group consisting of 2-chloro-3 ', 4'-dihydroxyacetophenone, dopa, dopamine, cupric acid, gallic acid, chlorogenic acid, (-) - epicatechin, (-) - epialocatechin-3-gallate, (-) - epigallocatechin, epicatechin gallate, melanin, tannin, flavonoids, cyanidin, dihydroquercetin, oron, dihydroimylcetin, delphinidin, mirucetin, quercetin, Catechin, catechin, (-) - epigallocatechin gallate, catechin, and luteolin.

The biocompatible polymer may be selected from the group consisting of hyaluronic acid, chitosan, chitosan derivatives (collagen), collagen, fibronectin, collagen, gelatin, alginic acid, (PLGA), poly (ε-caprolactone) (PCL), poly (lactic acid) (PLA), poly (glycolic acid) And may be at least one selected from the group consisting of polyanhydride, polyester, lysine, polyethyleneimine (PEI) and glutathione (GSH).

The biocomplex / synthetic polymer having a catechol-based functional group may be any one or more of the compounds represented by the following general formulas (1) to (4).

[Chemical Formula 1]

In Formula 1,

x + y is an integer from 200 to 6,000, y is an integer from 1 to 50,

(2)

In Formula 2,

A is SH, NH2 functional group, n is 1 to 3, x + y is an integer of 200 to 6,000, y and z are an integer of 1 to 50,

(3)

In Formula 3,

Z is a compound having a catechol functional group, n + m is an integer of 10 to 700,

n is an integer from 0 to 699, m is an integer from 1 to 700, and x is an integer from 1 to 700

Lt; / RTI >

[Chemical Formula 4]

In Formula 4, Z is a compound having a catechol-based functional group,

n + m is an integer of 10 to 700, n and m are each an integer of 1 to 700,

The biomedical / synthetic polymer having -NH2 or -SH groups may be selected from the group consisting of chitosan, chitosan derivatives (chitosan), aminfluronic acid, catecholamine, thiolfluoronic acid, amine hyaluronic acid, thiol hyaluronic acid, lysine, , Polyethyleneimine (PEI), and glutathione (GSH).

In addition, the biomedical / synthetic polymer having -NH 2 or -SH group may be any one or more of the compounds represented by the following general formulas (6) to (8).

[Chemical Formula 5]

In Formula 5, n + m is an integer of 10 to 700, n is an integer of 1 to 700,

[Chemical Formula 6]

In Formula 6, x is an integer of 1 to 12, n is an integer of 78 to 100, m is an integer of 30 to 65,

(7)

In the above formula (7), x is an integer of 1 to 12, n is an integer of 78 to 100, and m is an integer of 30 to 65.

In addition, the biomolecule / synthetic polymer having the catechol-based functional group may be formed by conjugating the compound having the catechol-based functional group with the biomolecule / synthetic polymer.

In the cross-linked hydrogel of iii), the compound having the catechol-based functional group is contained in an amount of 0.1 to 99.9% by weight, and the bio / synthetic polymer having the NH 2 or SH-functional group is contained in an amount of 0.1 to 99.9% .

In addition, the biological / synthetic polymer having a catechol-based functional group and an NH2 or-SH group is formed by conjugating the compound having a catechol-based functional group with a living / synthetic polymer having NH2 or -SH group.

In the cross-linked hydrogel of (iv), the compound having the catechol-based functional group and the NH 2 or SH functional group is contained in an amount of 0.1 to 98% by weight, and the bio / synthetic polymer having NH 2 or -SH group is contained in an amount of 0.1 to 98% , And the bio / synthetic polymer having -NH 2 or -SH group at the terminal is contained in an amount of 1 to 50% by weight.

The crosslinked hydrogel according to the first aspect of the present invention may be characterized in that it is formed at 20 to 80 ° C.

A second object of the present invention is to provide a method for producing a cross-linked hydrogel comprising a compound having a catechol-based functional group as an active ingredient, wherein a solution in which a biosynthetic polymer having a catechol-based functional group is dissolved in a solvent, Or a solution obtained by dissolving a living body / synthetic polymer having -SH group in a solvent; maintaining at pH 8.5 or higher; And a step 3 for removing impurities by centrifugation. The cross-linked hydrogel can be obtained by the following method.

The biological / synthetic polymer having a catechol-based functional group can be obtained by adding a compound having a catechol-based functional group to a solution in which a living body / synthetic polymer is dissolved in a solvent, reacting the solution in a nitrogen atmosphere under an acidic condition of pH 5 or less, and dialyzing under acidic conditions of pH 4 or less to form a catechol group conjugated to the living / synthetic polymer.

This step 2 can be characterized in that it is maintained at a temperature of 20 to 80 캜 for 1 to 24 hours.

Step 3 may further comprise the step of preparing a scaffold and lyophilizing it.

As described above, according to one embodiment of the present invention, bioavailability of a drug such as protein is maximized by using an injectable hydrogel, and it can be used as a matrix for differentiation of stem cells. Also, it has excellent osmotic pressure, absorption ability, water repellency, moisture permeability, and can prevent invasion of bacteria, bacteria, foreign substances from the outside, and provides a wet environment suitable for wound, It is possible not only to promote wound healing but also to minimize the scars remaining after wound healing and to prevent adhesion of organs.

In addition, according to one embodiment of the present invention, it is possible to use as an anti-adhesion agent, a replica of wound epithelium, an anti-wrinkle agent or a medical adhesive because of its excellent adhesive force and coating power. Especially it has adhesiveness, biocompatibility, It can be applied to various medical fields such as ophthalmology, dentistry, and neurosurgery.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a graph showing the results of measurement of strength of cross-linked hydrogel of catechol hyaluronic acid (HA-C) and chitosan derivative (chitosan) according to an embodiment of the present invention.
FIG. 2 is a graph showing the results of measurement of strength of cross-linked hydrogel of catechol hyaluronic acid (HA-C) and chitosan derivative (chitosan) according to one embodiment of the present invention.
FIG. 3 shows the results of measurement of swelling (%) of crosslinked hydrogel of catechol hyaluronic acid (HA-C) and chitosan derivative (chitosan) according to one embodiment of the present invention.
FIG. 4 is a graph showing the result of strength measurement after swelling the cross-linked hydrogel of catechol hyaluronic acid (HA-C) and chitosan derivative (chitosan) according to one embodiment of the present invention with time.
FIG. 5 is a graph showing a result of measuring the strength of catechol hyaluronic acid and an amine pluronic crosslinked hydrogel according to an embodiment of the present invention.
FIG. 6 is a graph showing the results of measurement of strength of catechol hyaluronic acid and a thiolcloronic crosslinked hydrogel according to an embodiment of the present invention.
FIG. 7 is a graph showing the results of measurement of strength of a catechol-keying and an amine-pluronic crosslinked hydrogel according to an embodiment of the present invention.
FIG. 8 is a graph showing the results of measurement of strength of a catechol-keyed hydrogel and a thiolcloronic crosslinked hydrogel according to an embodiment of the present invention.
9 is a graph showing the results of strength measurement of crosslinked hydrogels according to one embodiment of the present invention.
FIG. 10 is a photograph showing particle diameters of crosslinked hydrogels for drug delivery according to one embodiment of the present invention. FIG.

Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings. However, the detailed description of known functions and configurations incorporated herein will be omitted when it may unnecessarily obscure the subject matter of the present invention.

The same reference numerals are used for portions having similar functions and functions throughout the drawings. Throughout the specification, when a part is connected to another part, it includes not only a case where it is directly connected but also a case where the other part is indirectly connected with another part in between. In addition, the inclusion of an element does not exclude other elements, but may include other elements, unless specifically stated otherwise.

Hereinafter, the constitution, manufacturing method and experimental example of the crosslinked hydrogel for drug delivery according to one embodiment of the present invention will be described in detail.

The crosslinked hydrogel according to one embodiment of the present invention comprises a compound having a catechol-based functional group as an active ingredient, and is characterized in that i) a crosslinking between a compound having a catechol-based functional group and a compound having a functional group (SH and NH2) (Ii) a biopolymer having a catechol-based functional group and an SH or NH2 functional group and a compound having a functional group (SH and NH2).

Hereinafter, the cross-linked hydrogel of the present invention will be described in detail as follows.

According to one embodiment of the present invention, crosslinked hydrogels can be formed by cross-linking of compounds having a catechol-based functional group.

The compound having a catechol-based functional group is a compound having an adhesive catechol group and is a compound having an adhering power, such as dopa, dopamine, cupric acid, gallic acid, chlorogenic acid, (-) - epicatechin, (-) - epicatechin gallate, melanin, tannin, (-) - epicalocatechin-3-gallate, catechin, luteolin, and the like can be used as the antioxidant.

 According to another embodiment of the present invention, a compound having a catechol-based functional group may be conjugated to a biocompatible polymer to form a crosslinked product.

The compounds having the catechol-based functional groups are the same as those described above, so that a detailed description thereof will be omitted.

The compound having a catechol functional group is preferably contained in the crosslinked product in an amount of 0.1 to 99.9% by weight, more preferably 1 to 90% by weight. When the content is within the above range, the crosslinking of the catechol system is better.

The biocompatible polymer is a polymer that is chemically decomposed in the body. Any biocompatible polymer that is used in the art can be used without limitation. Examples of natural polymers include hyaluronic acid, chitosan, chitosan derivatives Collagen, fibronectin, gelatin, alginic acid, albumin, lysine, glutathione, and the like having a -NH 2 or -SH group at the terminal thereof, collagen, (PLGA), poly (glycolic acid) (PGA), poly (D, L-lactide-co-glycolide) (PLGA) caprolactone (PCL), polyanhydride, polyester, and polyethyleneimine (PEI). The hyaluronic acid preferably has a molecular weight of 100 to 2,000 kDa and the chitosan has a molecular weight of 1 to 220 kDa.

The biocompatible polymer is preferably contained in the crosslinked product in an amount of 0.1 to 99.9% by weight, more preferably 10 to 99% by weight. When the content is within the above range, the crosslinking of the catechol system is better.

According to another embodiment of the present invention, a compound having a catechol-based functional group is conjugated with a living body / synthetic polymer to form a biosynthetic polymer having a catechol-based functional group, and a living body / The crosslinked hydrogel may be formed by cross-linking with a synthetic polymer or a biocompatible polymer.

The compound having the catechol-based functional group is the same as described above, and it is preferably contained in the crosslinked hydrogel in an amount of 0.1 to 98% by weight, more preferably 1 to 70% by weight. When the content is within the above range, the crosslinking of the catechol system is better.

The biocompatible polymer is also the same as described above, and is preferably contained in the crosslinked hydrogel in an amount of 0.1 to 98% by weight, more preferably 1 to 50% by weight. When the content is within the above range, the crosslinking is better.

The compound having a catechol-based functional group is conjugated to a biocompatible polymer. Specifically, a biosynthetic polymer is dissolved in a solvent (a solution at pH 7.4), and a compound having a catechol-based functional group is added to this solution to adjust the pH The reaction is carried out at room temperature under an acidic condition of 5 or less and then dialyzed under an acidic condition of pH 4 or less to obtain a final compound conjugated with a catechol group to the biocompatible polymer.

The bio / synthetic polymer having a catechol-based functional group formed by conjugation of a compound having a catechol-based functional group and a bio / synthetic polymer may be a compound represented by the following general formulas (1) to (4).

[Chemical Formula 1]

In Formula 1,

x + y is an integer from 200 to 6,000, and y is an integer from 1 to 100.

(2)

In Formula 2,

A is SH, NH2 functional group, n is 1 to 3

x + y is an integer from 200 to 6,000, y and z are integers from 1 to 50,

(3)

In Formula 3,

Z is a compound having a catechol functional group,

n + m is from 10 to 700,

n is an integer of 0 to 699, m is an integer of 1 to 700, and x is an integer of 0 to 700. [

[Chemical Formula 4]

In Formula 4,

Z is a compound having a catechol functional group,

n is an integer of 1 to 100 and m is an integer of 1 to 100, respectively.

In the above formulas (3) and (4), Z is a compound having a catechol-based functional group and specifically includes dopa, dopamine, cupric acid, gallic acid, chlorogenic acid, (-) - epicatechin, (-) - epicatechin gallate, melanin, tannin (-) - epigallocatechin, (-) - epigallocatechin-3-gallate, (-) - epigallocatechin, dicycloheptadienine, - epigallocatechin gallate, catechin or luteolin.

As described above, a biocompatible polymer having a catechol-based functional group formed by conjugating a compound having a catechol-based functional group with a biocompatible polymer is cross-linked with a living / synthetic polymer having a -NH2 or -SH group at the end or a biocompatible polymer.

The biomedical / synthetic polymer having the -NH2 or -SH group at the terminal may be selected from the group consisting of chitosan, chitosan derivative (chitosan), amine fluroonic, catecholamine, thiolfluoronic acid, amine hyaluronic acid, thiolhyaluronic acid, collagen collagen, lysine, polyethyleneimine (PEI), glutathione (GSH), and the like. In this case, the chitosan has a molecular weight of 1 to 220 kDa, the pluronic has F68 having a weight average molecular weight of 8,700 Da, F127 having 12,600 Da, and hyaluronic acid having a weight average molecular weight of 230 to 2,000 kDa.

In particular, as the living body / synthetic polymer having -NH 2 or -SH group at the terminal, compounds represented by the following formulas (5) to (7) can be used.

[Chemical Formula 5]

In Formula 5,

n + m is an integer of 10 to 700, and n is an integer of 0 to 700. [

[Chemical Formula 6]

In Formula 6,

x is an integer of 1 to 12, n is an integer of 78 to 100, and m is an integer of 30 to 65.

(7)

In Formula 7,

x is an integer of 1 to 12, n is an integer of 78 to 100, and m is an integer of 30 to 65.

The biocompatible polymer capable of crosslinking with the biocompatible polymer having the catechol-based functional group may be of the same kind as described above.

The bio / synthetic polymer or the biocompatible polymer is preferably contained in the crosslinked hydrogel in an amount of 0.1 to 98% by weight, more preferably 1 to 50% by weight. When the content is within the above range, the crosslinking is better.

The biocompatible polymer having a catechol-based functional group is then crosslinked with a biosynthetic polymer having a -NH 2 or -SH group at its end or with a biocompatible polymer. The biocompatible polymer having a catechol-based functional group is dissolved in TBS (pH 7.4) And the biomedical / synthetic polymer or biocompatible polymer having -NH 2 or -SH group at the terminal thereof is dissolved in a solvent of TBS (pH 8.5). After mixing the two solutions, the pH is adjusted to 8.5 or more, The mixed solution is maintained at a temperature of 0 to 80 캜 for 1 to 24 hours. The crosslinked hydrogel is then centrifuged to remove impurities and made into a scaffold and lyophilized to give the final crosslinked hydrogel.

Hereinafter, the present invention will be described in more detail with reference to examples. These embodiments are for purposes of illustration only and are not intended to limit the scope of protection of the present invention.

Example  1. Catechol system  A compound having a functional group is bonded to a biocompatible polymer By conjugation  Formed Bridge Hydrogel  Produce

(Manufactured by Catechol hyaluronic acid (HA-C))

1 wt% of hyaluronic acid having a molecular weight of about 230 kDa was completely dissolved in 2x PBS (phosphate buffered saline, pH 7.4) buffer solution. At this time, the buffer solution was deoxygenated and filled with nitrogen. To this solution was added 0.4% by weight of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC) and 0.3% by weight of N-hydroxysuccinimide (NHS) Was adjusted to about 3-4. Next, 0.4% by weight of dopamine was added, the pH was maintained at about 3 to 4, and the mixed solution was stirred at room temperature for 24 hours. The mixed solution was sealed with a pre-washed dialysis membrane (molecular weight cut-off of 6-8 kDa) and dialyzed in 2x PBS solution of pH 3 or less for 48 hours to remove residual EDC, NHS and dopamine. Then, lyophilization was performed to obtain catechol hyaluronic acid (HA-C) (see the following reaction formula 1).

[Reaction Scheme 1]

In the above Reaction Scheme 1, x + y is an integer of 200 to 6,000, and y is an integer of 1 to 50.

(Catechol hyaluronic acid crosslinked product)

The prepared catechol hyaluronic acid (HA-C) was completely dissolved in a TBS (pH 8.5) buffer solution at a concentration of 60% by weight, and the mixed solution was maintained at room temperature for 24 hours for complete crosslinking reaction. Then, the prepared catechol hyaluronic acid crosslinked product was subjected to centrifugal separation three times to remove impurities, and was made into a scaffold and lyophilized.

Example  2. Cathecol hyaluronic acid ( HA -C) and chitosan derivatives ( Kick out ) Bridge Hydrogel  Produce

(Chitosan derivative (manufactured by Kidakaku))

2% by weight of chitosan having a molecular weight of about 5 kDa was completely dissolved in a 1% acetic acid (pH 4.5) solution, and an amount of methanol equal to 1% acetic acid was added thereto. Then, 4.5% by weight of lactose and 2% by weight of sodium cyanoborohydride were dissolved in a mixed solvent of 1% acetic acid and methanol in the same proportions, and then added to the solution in which the chitosan was dissolved. The mixed solution was stirred at room temperature for 24 hours, sealed with a pre-washed dialysis membrane (molecular weight cut-off of 6-8 kDa), and dialyzed in secondary distilled water for 24 hours to obtain residual chitosan, lactose and sodium cyanoborohydride . Then, it was lyophilized to obtain a chitosan derivative (key-fall) (see Reaction Scheme 2 below).

[Reaction Scheme 2]

In the above Reaction Scheme 2, n + m is an integer of 10 to 700, and n is an integer of 1 to 700.

(Crosslinked hydrogel of catechol hyaluronic acid and chitosan derivative)

The catechol hyaluronic acid (HA-C) prepared in Example 1 was completely dissolved in a TBS (pH 7.4) buffer solution at a concentration of 4 (w / w)%, A chitosan derivative was dissolved in TBS (pH 8.5) at a concentration of 2 (w / w)%, and the pH of the solution was adjusted to 8.5 after mixing the two solutions. The mixed solution was kept at room temperature for 24 hours for a complete crosslinking reaction of the catechol hyaluronic acid and the chitosan derivative crosslinked hydrogel. Then, the prepared catechol hyaluronic acid and the chitosan derivative crosslinked hydrogel were centrifuged three times to remove impurities, and then made into a scaffold and lyophilized.

Experimental Example  1. Catechol hyaluronic acid and chitosan derivatives Bridge Hydrogel  Strength measurement

The cross-linked hydrogel of catechol hyaluronic acid and chitosan derivative prepared in Example 2 was centrifuged to remove the supernatant, and the viscoelasticity of the hydrogel was measured by a Rheometer to confirm its strength. The results are shown in FIG. 1 . At this time, as a control group, a cross-linked hydrogel of hyaluronic acid and chitosan derivative (chitosan) was used.

As shown in FIG. 1, it was confirmed that the crosslinked hydrogel of catechol hyaluronic acid and chitosan derivative prepared according to the present invention had a larger strength than the cross-linked hydrogel of hyaluronic acid and the chitosan derivative cross-linked.

Experimental Example  2. Catechol hyaluronic acid and chitosan derivatives Bridge Hydrogel  Measure strength over time

The strength of the catechol hyaluronic acid and chitosan derivative crosslinked hydrogel prepared in Example 2 over time was measured with a rheometer, and the results are shown in Fig.

As shown in FIG. 2, it was confirmed that the cross-linked hydrogel of catechol hyaluronic acid and chitosan derivative prepared according to the present invention changed from a sol state to a gel state with time, And it was confirmed that it maintained a large strength.

Experimental Example  3. Catechol hyaluronic acid and chitosan derivatives Bridge Hydrogel  Swelling degree measurement

The catechol hyaluronic acid and the chitosan derivative crosslinked hydrogel prepared in Example 2 were immersed in distilled water and stored in an incubator at 37 ° C. Then, the surface of the cross-linked hydrogel was wiped off and the weight was measured. The weight of the catechol hyaluronic acid and chitosan derivative crosslinked hydrogel was measured before the immersion. At this time, the degree of swelling of the crosslinked hydrogel was calculated by the following equation (1), and the percentage swelling by the degree of swelling was calculated by the following equation (2).

[Equation 1]

&Quot; (2) "

Experimental Results As shown in FIG. 3, it was found that the cross-linked hydrogel of catechol hyaluronic acid (HA-C) and chitosan derivative (chitosan) prepared according to the present invention absorbs water by the degree of swelling .

Experimental Example  4. Cathecol hyaluronic acid and chitosan derivatives Bridge Hydrogel  Measure gel strength in swelling state

The degree of swelling with time was measured using the catechol hyaluronic acid and the chitosan derivative crosslinked hydrogel prepared in Example 2, and the gel strength at that time was measured using a rheometer. The results are shown in FIG.

Experimental Results As shown in FIG. 4, it was confirmed that the catechol hyaluronic acid and the chitosan derivative crosslinked hydrogel prepared according to the present invention maintained a gel strength similar to that of the initial stage even after 20 days.

Example  3. Catechol hyaluronic acid ( HA -C) and amines Pluronic (F127- EDA ) Bridge Hydrogel  Produce

(Manufactured by Amine Fluoronic (F127-EDA))

Aminfluronic (F127-EDA) in which both ends of the pluronic were substituted with amines was synthesized. 11% by weight of pluronic with a molecular weight of about 12 kDa, 0.5% by weight of 4-dimethylaminopyridine (DMAP) and 0.5% by weight of triethylamine (TEA) were completely dissolved in 1,4-dioxane and then succinic anhydride ) Was added. The mixed solution was stirred at room temperature for 24 hours, the solvent was removed, and the solution was precipitated in cold diethyl ether. The resulting precipitate was filtered and dried to obtain a white precipitate. The resulting precipitate was dissolved in N, N-dimethylformamide (DMF), and then 0.4% by weight of N, N'-dicyclohexylcarbodiimide (DCC) and 0.2% by weight of N-hydroxysuccinimide (NHS) . Then, 0.2 wt% of ethylenediamine and N, N-dimethylformamide (DMF) were slowly added to the mixed solution. The mixed solution was stirred at 30 DEG C for 24 hours, the solvent was removed, and the solution was precipitated in cold diethyl ether. The precipitate thus formed was filtered and dried to obtain a white precipitate. This precipitate was amine pluronic (F127-EDA) having a weight average molecular weight of about 12,600 Da (see Reaction Scheme 3 below).

[Reaction Scheme 3]

In the above Reaction Scheme 3, x is an integer of 1 to 12, n is an integer of 78 to 100, and m is an integer of 30 to 65.

(Catechol hyaluronic acid and aminuclear crosslinked hydrogel)

Catechol hyaluronic acid (HA-C) having a molecular weight of about 230 kDa prepared in Example 1 was completely dissolved in a TBS (pH 7.4) buffer solution at a concentration of 5 (w / w)% and crosslinked by reaction with an amine (F127-EDA) was dissolved in TBS (pH 8.5) at various concentrations ranging from 15 to 25 (w / w)%, and the two solutions were mixed and adjusted to pH 8.5. The mixed solution was maintained at 4 DEG C for 24 hours for a complete crosslinking reaction of catechol hyaluronic acid and an amine fluroonic crosslinked hydrogel. Then, the prepared catechol hyaluronic acid and aminuclear cross-linked hydrogel were centrifuged three times to remove impurities, and then made into a scaffold and lyophilized.

Experimental Example  5. Cathecol hyaluronic acid and amine Pluronic Bridge Hydrogel  Strength measurement

The strength of the hydrogel was measured by using a catechol hyaluronic acid and an amine flouronic crosslinked hydrogel prepared in Example 3 over time according to a rheometer. The results are shown in FIG.

As a result of the experiment, as shown in Fig. 5, the catechol hyaluronic acid and the amine fl uoronic crosslinked hydrogel prepared according to the present invention had a solubility at the intersection of G 'and G "

gel phase change was observed. It was confirmed that catechol hyaluronic acid and aminuclear bridged hydrogels showed high strength.

Example  4. Hyaluronic acid ( HA ) And catecholamine ( EDA -C) Bridge Hydrogel

(Catecholamine (EDA-C))

A catechol group was synthesized at both ends of ethynedianamine. 0.5% by weight of ethylenediamine was dissolved in N, N-dimethylformamide (DMF), and then 0.2% by weight of N, N'-dicyclohexylcarbodiimide (DCC) and 0.2% by weight of N-hydroxysuccinimide (NHS) % By weight. Then, 0.2% by weight of hydrocuppedic acid was added to the mixed solution. The mixed solution was stirred at room temperature for 24 hours, the solvent was removed, and the solution was precipitated in cold diethyl ether. The resulting precipitate was filtered and dried to obtain a white precipitate. This precipitate was catecholamine (EDA-C).

(Hyaluronic acid and catecholamine crosslinked hydrogel)

After dissolving hyaluronic acid having a molecular weight of about 230 kDa in a concentration of 5 (w / w)% completely in TBS (pH 7.4) buffer solution, the above-prepared catecholamine (EDA-C) Was dissolved in TBS (pH 8.5) at various concentrations ranging from 15 to 25 (w / w)%, and the pH was adjusted to 8.5 after mixing the two solutions. The mixed solution was maintained at 4 DEG C for 24 hours for a complete crosslinking reaction of hyaluronic acid (HA) and catecholamine (EDA-C) crosslinked hydrogel. Then, the prepared hyaluronic acid (HA) and catecholamine (EDA-C) cross-linked hydrogel were centrifuged three times to remove impurities, followed by preparing a scaffold and lyophilization.

Example   5. Cathecol hyaluronic acid ( HA -C) and Cyol Pluronic (F127- CA ) Bridge Hydrogel  Produce

(Manufactured by Thyol Pluronic (F127-CA)),

(F127-CA) substituted with thiol at both ends of the pluronic were synthesized. (SA) was prepared by completely dissolving 11 wt% of pluronic, 0.5 wt% of 4-dimethylaminopyridine (DMAP) and 0.5 wt% of triethylamine (TEA) in 1,4- 0.5% by weight. The mixed solution was stirred at room temperature for 24 hours, the solvent was removed, and the solution was precipitated in cold diethyl ether. The resulting precipitate was filtered and dried to obtain a white precipitate. The resulting precipitate was dissolved in N, N-dimethylformamide (DMF), and then 0.4% by weight of N, N'-dicyclohexylcarbodiimide (DCC) and 0.2% by weight of N-hydroxysuccinimide (NHS) . Then, 0.2 wt% of cysteamine was added to the mixed solution. The mixed solution was stirred at 30 DEG C for 24 hours, the solvent was removed, and the solution was precipitated in cold diethyl ether. The resulting precipitate was filtered and dried to obtain a white precipitate. This precipitate was thionoluronic (F127-CA) having a weight average molecular weight of 12,600 Da (see Reaction Scheme 4 below).

[Reaction Scheme 4]

In the above Reaction Scheme 4, n is an integer of 78 to 100, and m is an integer of 30 to 65.

(Preparation of catechol hyaluronic acid and thiolcloronic crosslinked hydrogel)

Catechol hyaluronic acid (HA-C) having a molecular weight of about 230 kDa prepared in Example 1 was completely dissolved in a TBS (pH 7.4) buffer solution at a concentration of 5 (w / w)%, (F127-CA) prepared above was dissolved in TBS (pH 8.5) at various concentrations ranging from 15 to 25 (w / w)%. After mixing the two solutions, the pH was adjusted to 8.5 Respectively. The mixed solution was kept at 4 캜 for 24 hours for a complete crosslinking reaction of catechol hyaluronic acid and thiol cyclic hydrogel. Then, the produced catechol hyaluronic acid and thyroid pluronic crosslinked hydrogel were subjected to centrifugation three times to remove impurities, and were made into a scaffold and lyophilized.

Experimental Example  6. Cathecol with hyaluronic acid Cyol Pluronic Bridge Hydrogel  Strength measurement

The hydrogel strength was measured with a catechol hyaluronic acid and a thiol pluronic crosslinked hydrogel prepared in Example 6 over time using a rheometer, and the results are shown in FIG.

As shown in FIG. 6, the catechol hyaluronic acid and thiol pluronic crosslinked hydrogel prepared according to the present invention had a sol at the intersection of G 'and G "

It was confirmed that the phase change occurred in the standing gel, and they showed high strength.

Example  6. Catechol Chitlac-C and  Amine Pluronic (F127- EDA ) Bridge Hydrogel  Produce

(Manufactured by chitlac-C)

0.5% by weight of the chitosan derivative (Kidulac) prepared in Example 2 was completely dissolved in the second distilled water (pH 5.5). 0.2% by weight of hydrocappeak exase and 0.2% by weight of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC) were completely dissolved in a mixed solution of secondary distilled water and ethanol in the same ratio, Was added to the dissolved mixed solution. The mixed solution was stirred at room temperature for 12 hours while maintaining a pH of 5.5 with 1M HCl. Then, the membrane was sealed with a pre-washed dialysis membrane (molecular weight cut-off of 3 kDa), and dialyzed with secondary distilled water (pH 5.5) for 2 days to obtain a residual hydrocapped ether and 1-ethyl-3- Carbodiimide (EDC) was removed. Then, lyophilization was performed to obtain catechol-chitlac-C (see Scheme 5 below).

[Reaction Scheme 5]

In the above Reaction Scheme 5, Z is a compound having a catechol-based functional group, n + m is an integer of 10 to 700, n is an integer of 1 to 699, and x is an integer of 1 to 700.

(Preparation of catechol ketals and aminuclear crosslinked hydrogel)

The prepared chitlac-C having a molecular weight of about 12 kDa was completely dissolved in a TBS (pH 7.4) buffer solution at a concentration of 0.5 (w / w)%, Amine fluoronics (F127-EDA) prepared in Example 3 was dissolved in TBS (pH 8.5) at various concentrations ranging from 15 to 25 (w / w)%, and the pH was adjusted to 8.5 after mixing the two solutions . The mixed solution was maintained at 4 캜 for 24 hours for a complete crosslinking reaction of the catechol ketals and the amine-pluronic crosslinked hydrogel. Then, the produced catechol ketals and aminuclear bridged hydrogel were centrifuged three times to remove impurities, and then made into a scaffold and lyophilized.

Experimental Example  7. Catechol With the key dropping  Amine Pluronic Bridge Hydrogel  Strength measurement

The strength of the hydrogel was measured by a rheometer over time using the catechol ketals and aminfluronic crosslinked hydrogel prepared in Example 6, and the results are shown in FIG.

As a result, as shown in FIG. 7, it was confirmed that the catechol ketals and aminuclear crosslinked hydrogels prepared according to the present invention had a phase change from the sol to the gel at the intersection of G 'and G " , It can be confirmed that they exhibit a large strength.

Example  7. Catechol Chitlac-C and  Amine hyaluronic acid ( HA - EDA ) Bridge Hydrogel  Produce

(Amine hyaluronic acid (HA-EDA))

0.5% by weight of hyaluronic acid having a molecular weight of about 230 kDa was completely dissolved in 2x PBS (phosphate buffered saline, pH 7.4) buffer solution. At this time, the buffer solution was deoxygenated and filled with nitrogen. 0.2% by weight of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC) and 0.2% by weight of N-hydroxysuccinimide (NHS) Was added and the mixed solution was stirred at room temperature for 24 hours. The mixed solution was sealed with a pre-washed dialysis membrane (molecular weight cut-off of 6-8 kDa) and dialyzed in 2x PBS solution for 48 hours to remove residual EDC, NHS and ethylene diamine. Then, (HA-EDA).

(Preparation of crosslinked catechol ketone and amine hyaluronic acid hydrogel)

The catechol ketals with a molecular weight of about 12 kDa prepared in Example 6 were completely dissolved in a buffer solution of TBS (pH 7.4) at a concentration of 0.5 (w / w)%, and amine hyaluronic acid was added in an amount of 15 to 25 (w / % Solution in TBS (pH 8.5) and the pH was adjusted to 8.5 after mixing the two solutions. The mixed solution was maintained at room temperature for 24 hours for a complete crosslinking reaction of the catechol ketals and the amine-hyaluronic acid crosslinked hydrogel. Then, the prepared catechol ketals and amin hyaluronic acid cross-linked hydrogels were centrifuged three times to remove impurities, followed by making into scaffolds and lyophilization.

Example  8. Catechol Chitlac-C and Cyol Pluronic (F127- CA ) Bridge Hydrogel  Produce

The catechol ketal with a molecular weight of about 12 kDa prepared in Example 6 was completely dissolved in a TBS (pH 7.4) buffer solution at a concentration of 0.5 (w / w)% and then subjected to the above- The thyroid pluronic prepared in Example 5 was dissolved in TBS (pH 8.5) at various concentrations ranging from 15 to 25 (w / w)%, the pH of the solution was adjusted to 8.5 after mixing the two solutions. The mixed solution was maintained at 4 캜 for 24 hours for a complete crosslinking reaction of the catechol ketals and the thiolcloronic crosslinked hydrogel. Then, the produced catechol keyhole and the thiolcloronic crosslinked hydrogel were centrifuged three times to remove impurities, and were made into a scaffold and lyophilized.

Experimental Example  8. Catechol With the key dropping Cyol Pluronic Bridge Hydrogel  Strength measurement

The strength of the hydrogel was measured by a rheometer using the catechol ketals and thiol cyclic crosslinked hydrogel prepared in Example 8. The results are shown in FIG.

As a result, as shown in FIG. 8, it was confirmed that the catechol ketals and thiol cyclic hydrogels prepared according to the present invention had a phase change from the sol to the gel at the intersection of G 'and G'' , And they were confirmed to show a large strength.

Example  9. Cathechol chitosan ( chitosan -C) and amines Pluronic (F127- EDA ) Bridge Hydrogel  Produce

(Manufactured by chitosan-C)

0.5% by weight of chitosan was completely dissolved in the second distilled water (pH 5.5). After 0.2% by weight of hydrocappeak oxide and 0.2% by weight of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC) were completely dissolved in the same ratio of secondary distilled water and ethanol mixed solution, ≪ / RTI > The mixed solution was stirred at room temperature for 12 hours while maintaining a pH of 5.5 with 1M HCl. Then, the membrane was sealed with a pre-washed dialysis membrane (molecular weight cut-off of 3 kDa), and dialyzed with secondary distilled water (pH 5.5) for 2 days to obtain a residual hydrocapped ether and 1-ethyl-3- Carbodiimide (EDC) was removed. Then, lyophilized to obtain catechol-chitosan-C (see Scheme 6 below).

[Reaction Scheme 6]

In the above Reaction Scheme 6, Z is a compound having a catechol functional group, n is an integer of 10 to 700, and m is an integer of 1 to 699.

(Preparation of catechol chitosan and aminuclear bridged hydrogel)

The prepared catechol chitosan having a molecular weight of about 9 kDa was completely dissolved in a TBS (pH 7.4) buffer solution at a concentration of 0.5 (w / w)% and then the amine prepared in Example 3 Pluronic was dissolved in TBS (pH 8.5) at various concentrations ranging from 15 to 25 (w / w)%, the two solutions were mixed and the pH was adjusted to 8.5. The mixed solution was maintained at 4 캜 for 24 hours for a complete crosslinking reaction of the catechol chitosan and the amine flurotonic crosslinked hydrogel. Then, the produced catechol-chitosan and aminuclear cross-linked hydrogel were centrifuged three times to remove impurities, and then made into a scaffold and lyophilized.

Example  10. Cathechol chitosan ( chitosan -C) and amine hyaluronic acid ( HA - EDA ) Bridge Hydrogel  Produce

The catechol chitosan having a molecular weight of about 12 kDa prepared in Example 9 was completely dissolved in a TBS (pH 7.4) buffer solution at a concentration of 0.5 (w / w)% and then 15 to 25 (w / w)% of amine hyaluronic acid (PH 8.5) at various concentrations, and the pH was adjusted to 8.5 after mixing the two solutions. The mixed solution was kept at room temperature for 24 hours for a complete crosslinking reaction of catechol chitosan and amine hyaluronic acid crosslinked hydrogel. Then, the produced catechol-chitosan and amine-hyaluronic acid cross-linked hydrogel were centrifuged three times to remove impurities, and were made into a scaffold and lyophilized.

Example  11. Cathechol chitosan ( chitosan -C) and Cyol Pluronic  (F127- CA ) Bridge Hydrogel  Produce

The catechol chitosan having a molecular weight of about 9 kDa prepared in Example 9 was completely dissolved in a TBS (pH 7.4) buffer solution at a concentration of 0.5 (w / w)% and then crosslinked by reaction with thiol 5 was dissolved in TBS (pH 8.5) at various concentrations ranging from 15 to 25 (w / w)%. The pH of the solution was adjusted to 8.5 after mixing the two solutions. The mixed solution was maintained at 4 캜 for 24 hours for a complete crosslinking reaction of the catechol-chitosan and the thiolcloronic crosslinked hydrogel. Then, the catechol-chitosan and the thiolcloronic crosslinked hydrogel were centrifuged three times to remove impurities, and were made into a scaffold and lyophilized.

Example  12. Catechol Amine (EDA-C) and  Chitosan derivatives ( chitlac ) Bridge Hydrogel  Produce

The chitosan derivative (Ketalac) prepared in Example 2 was completely dissolved in a TBS (pH 8.5) buffer solution at a concentration of 5 (w / w)% and then crosslinked by reaction with amine The prepared catecholamine was dissolved in TBS (pH 7.4) buffer at various concentrations ranging from 15 to 25% (w / w), and the two solutions were mixed and adjusted to pH 8.5. The mixed solution was maintained at room temperature for 24 hours for a complete crosslinking reaction of catecholamine and chitosan derivative crosslinked hydrogel. Then, the prepared catecholamine and chitosan derivative crosslinked hydrogel were centrifuged three times to remove impurities, and then made into a scaffold and lyophilized.

Example  13. Catechol Amine (EDA-C) and  Chitosan ( chitosan ) Bridge Hydrogel  Produce

Chitosan was completely dissolved in a TBS (pH 8.5) buffer solution at a concentration of 5 (w / w)%, and then catecholamine prepared in Example 4 was dissolved in 15-25 (pH 7.4) buffer solution at various concentrations in the range of (w / w)%, and the pH was adjusted to 8.5 after mixing the two solutions. The mixed solution was kept at room temperature for 24 hours for a complete crosslinking reaction of catecholamine and chitosan crosslinked hydrogel. Then, the produced catecholamine and chitosan cross-linked hydrogel were subjected to centrifugation three times to remove impurities, and were made into a scaffold and lyophilized.

Experimental Example  9. Bridge Hydrogel  Strength measurement

The strengths of the crosslinked products and crosslinked hydrogels prepared in Examples 2 to 14 were measured, and the results are shown in FIG.

As a result, as shown in FIG. 9, the G 'value of each hydrogel was about 600 or more. From these results, it was found that the crosslinked hydrogels prepared in Examples 2 to 13 had strong strength .

Claims (15)

A crosslinked hydrogel comprising a compound having a catechol-based functional group as an active ingredient,
A biocomplex / synthetic polymer having a catechol-based functional group, and a living / synthetic polymer having a -NH2 functional group at the terminal,
The living body / synthetic polymer having the catechol-based functional group is formed by conjugating a compound having a catechol-based functional group with a living body / synthetic polymer,
The bio / synthetic polymer having the catechol-based functional group is contained in an amount of 0.1 to 99.9 wt%, and the bio / synthetic polymer having the -NH 2 functional group is contained in an amount of 0.1 to 99.9 wt%
The biocomplex / synthetic polymer having the catechol-based functional group is any one or more of compounds represented by the following general formulas (1) to (3)
Wherein the bio / synthetic polymer having the -NH2 functional group is any one or more of compounds represented by the following Chemical Formulas (4) to (5):
[Chemical Formula 1]

In Formula 1,
A is SH, NH2 functional group, n is 1 to 3
x + y is an integer from 200 to 6,000, y and z are integers from 1 to 50,
(2)

In Formula 2,
Z is a compound having a catechol functional group,
The compound having a catechol-based functional group may be selected from the group consisting of 2-chloro-3 ', 4'-dihydroxyacetophenone, dopa, dopamine, cupric acid, gallic acid, chlorogenic acid, (-) - epicatechin, (-) - epilacocatechin-3-gallate, (-) - epigallose, (-) - galactose, melanin, tannin, flavonoids, cyanidines, dihydroquercetin, oron, dihydroxylicillin, delphinidin, mirucetin, quercetin Catechin, (-) - epigallocatechin gallate, catechin and luteolin,
n + m is an integer of 10 to 700,
n is an integer from 0 to 699, m is an integer from 1 to 700, and x is an integer from 1 to 700
Lt; / RTI >
(3)

In Formula 3,
Z is a compound having a catechol functional group,
n + m is an integer of 10 to 700,
n and m are each an integer of 1 to 700,
[Chemical Formula 4]

In Formula 4,
n + m is an integer of 10 to 700, n is an integer of 1 to 700,
[Chemical Formula 5]

In Formula 5,
x is an integer of 1 to 12, n is an integer of 78 to 100, m is an integer of 30 to < RTI ID = 0.0 >
Lt; / RTI >
delete delete delete delete delete delete delete delete delete The method according to claim 1,
Wherein the cross-linked hydrogel is formed at 20 to < RTI ID = 0.0 > 80 C. < / RTI >
A method for producing a crosslinked hydrogel according to claim 1,
A step 1 of mixing a solution obtained by dissolving a biosynthetic / synthetic polymer having a catechol-based functional group in a solvent TBS and a solution prepared by dissolving a living / synthetic polymer having a -NH2 functional group at a terminal in a solvent TBS;
maintaining at pH 8.5 or higher; And
And removing the impurities by centrifugal separation
The biomolecule / synthetic polymer having the catechol-
A compound having a catechol-based functional group is added to a solution in which a living body / synthetic polymer is dissolved, reacted in an acidic condition of pH 5 or less under nitrogen atmosphere, and then dialyzed under an acidic condition of pH 4 or less, A caller is formed by conjugation,
The step (2)
Maintained at a temperature of 20 to 80 DEG C for 1 to 24 hours,
In step 3,
Preparing a scaffold, and lyophilizing the cross-linked hydrogel. delete delete delete

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