DOUBLE-CROSSLINKED SELF-HEALING HYDROGEL

Abstract

Disclosed is a double-crosslinked self-healing hydrogel having excellent mechanical properties and stability and self-healing properties. More particularly, the hydrogel can be injected into the body due to excellent mechanical properties and stability thereof, and thus, can be used as a hydrogel for drug and cell delivery. In addition, the hydrogel can be usefully used as a composition for 3D bioprinters due to self-healing properties thereof

Claims

1. A double-crosslinkable hydrogel composition, comprising: oxidized hyaluronate, glycol chitosan, adipic acid dihydrazide and an alginic acid-grafted hyaluronate modifier, wherein the oxidized hyaluronate and the glycol chitosan form an imine bond through Schiff base reaction, the alginic acid-grafted hyaluronate modifier is a structure wherein alginic acid is covalently bonded to a hyaluronate chain, the covalent bond being formed through a linker that allows a covalent bond between a carboxyl group of alginic acid and a carboxyl group of hyaluronate, and the alginic acid-grafted hyaluronate modifier forms ionic crosslinking.

2. The double-crosslinkable hydrogel composition according to claim 1, wherein a weight ratio of oxidized hyaluronate to glycol chitosan to adipic acid dihydrazide to an alginic acid-grafted hyaluronate modifier is 1:1:0.1:0.1 to 5:1:1.5:1.5.

3. The double-crosslinkable hydrogel composition according to claim 1, wherein an oxidation degree of the oxidized hyaluronate is 20% to 80%.

4. The double-crosslinkable hydrogel composition according to claim 1, wherein a weight ratio of alginic acid to hyaluronate in the alginic acid-grafted hyaluronate modifier is 10:1 to 1:10.

5. The double-crosslinkable hydrogel composition according to claim 1, wherein the linker of the alginic acid-grafted hyaluronate modifier is selected from the group consisting of adipic acid dihydrazide, diamine, divinyl sulfone, 1,4-butanediol diglycidyl ether (BDDE), glutaraldehyde, carbodiimide, hydroxysuccinimide, imidoester, maleimide, haloacetyl, disulfide, hydroazide and alkoxyamine.

6. The double-crosslinkable hydrogel composition according to claim 1, wherein the hydrogel composition further comprises an ionic crosslinking agent.

7. The double-crosslinkable hydrogel composition according to claim 6, wherein the ionic crosslinking agent is a divalent cation or a salt thereof.

8. The double-crosslinkable hydrogel composition according to claim 7, wherein the divalent cation is selected from the group consisting of calcium, barium, copper, iron and magnesium ions.

9. A composition for three-dimensional bioprinting, comprising the double crosslinkable hydrogel composition according to claim 1.

10. A method of preparing a double crosslinked hydrogel, the method comprising: mixing an oxidized hyaluronate solution, a glycol chitosan solution, an adipic acid dihydrazide solution and an alginic acid-grafted hyaluronate modifier solution to prepare a hydrogel; and treating the hydrogel with a divalent cation or a salt thereof, wherein the alginic acid-grafted hyaluronate modifier is a structure wherein alginic acid is covalently bonded to a hyaluronate chain, the covalent bond being formed through a linker that allows a covalent bond between a carboxyl group of alginic acid and a carboxyl group of hyaluronate.

11. The method according to claim 10, wherein a weight ratio of the oxidized hyaluronate to the glycol chitosan to the adipic acid dihydrazide to the alginic acid-grafted hyaluronate modifier in the hydrogel of the mixing is 1:1:0.1:0.1 to 5:1:1.5:1.5.

12. The method according to claim 10, wherein treatment with the divalent cation or the salt is carried out for 20 seconds to 300 seconds.

13. The method according to claim 12, wherein the divalent cation is selected from the group consisting of calcium ions, barium ions, and magnesium ions.

14. A double crosslinked hydrogel prepared by the method of claim 10.

15. A drug delivery system comprising the double crosslinked hydrogel of claim 14.

16. The drug delivery system according to claim 15, wherein the drug is selected from the group consisting of compounds, proteins, peptides, nucleic acids, saccharides, and cells.

Description

MODES OF THE INVENTION

[0053] Hereinafter, one or more embodiments are described in more detail through examples. However, these examples are for illustrative purposes only and the scope of the present disclosure is not limited to these examples.

EXAMPLE 1

Preparation of Double-Crosslinked Self-Healing Hydrogel

[0054] 1-1. Preparation of Oxidized Hyaluronate and Glycol Chitosan

[0055] Sodium hyaluronate (MW 2,500,000) was purchased from Lifecore, and glycol chitosan (GC; MW 50,000, Sigma Aldrich) was provided from Wako.

[0056] 1 g of hyaluronate (HA) was dissolved in 90 ml of distilled water. 0.26735 g of sodium periodate was dissolved in 10 ml of distilled water, followed by stirring the same. The sodium periodate solution was added to an HA solution under dark conditions. Next, the mixture was stirred for 24 hours. This solution was purified through dialysis using distilled water containing sodium chloride for 3 days. After dialysis, the solution was treated with activated carbon, followed by filtration (pore size: 0.22 μm). This filtrate was freeze-dried to obtain an oxidized hyaluronate (hereinafter referred to as OHA) having an oxidation degree of 50%. 1 g of glycol chitosan (GC) was dissolved in 100 ml of distilled water, and purified in the same manner as in the method described above.

[0057] 1-2. Preparation of Alginic Acid-Grafted Hyaluronate Modifier

[0058] For introduction of an amine group, 1-ethyl-3-(dimethylaminopropyl)carbodiimide (EDC; Sigma Aldrich), N-hydroxysulfosuccinimide (Sulfo-NHS; Thermo) and adipic acid dihydrazide were added to 1 g of hyaluronate (molecular weight 1,000,000; Humedix), followed by actively stirring the same for 20 hours to synthesize NH.sub.2-hyaluronate. Next, the resultant solution was freeze-dried.

[0059] Next, alginic acid (molecular weight 200,000-300,000; FMC Biopolymer) was combined with NH.sub.2-hyaluronate through carbodiimide chemistry to obtain an alginic acid-grafted hyaluronate modifier (hyaluronate-g-alginate; hereinafter referred to as HAH).

[0060] 1-3. Preparation of Self-Healing Double-Crosslinkable Hydrogel

[0061] 1% by weight of glycol chitosan (GC) (relative to the total weight of hydrogel) and 0.3% by weight of adipic acid dihydrazide (ADH; Sigma-Aldrich) were dissolved in distilled water, and, separately, 2% by weight of oxidized hyaluronate (OHA) and 0.3% by weight of HAH were dissolved in distilled water. Next, both the solutions were mixed to prepare a hydrogel. This hydrogel preparation method was adopted because oxidized hyaluronate (OHA) can immediately react with GC&ADH to form a gel, whereas HAH can form an ionic bond with glycol chitosan (GC). Next, CaSO.sub.4 slurry was added in an amount (in an amount wherein 0.42 g of CaSO.sub.4 was mixed with 1 g of alginic acid) of being dependent upon the content of alginic acid in the hydrogel to the hydrogel, thereby preparing a double crosslinked self-healing hydrogel (OHA-GC-ADH-HAH). Calcium ions serve to induce crosslinking among HAH.

[0062] As comparative examples, an existing self-healing hydrogel (OHA-GC-ADH), composed of glycol chitosan (GC), adipic acid dihydrazide (ADH) and an oxidized hyaluronate (OHA), and hydrogel (OHA-GC-ADH-ALG), to which alginic acid (0.3% by weight) and calcium ions were added, were respectively prepared.

[0063] FIG. 1 schematically illustrates a process of preparing a double-crosslinked self-healing hydrogel of the present disclosure.

EXAMPLE 2

Property Confirmation of Double Crosslinkable Self-Healing Hydrogel

[0064] 2-1. Confirmation of Change in Volume and Weight of Double-Crosslinked Self-Healing Hydrogel

[0065] The double-crosslinked self-healing hydrogel prepared in Example 1-3 was manufactured in a circular shape. Changes in the weight and diameter of the hydrogel were observed over time while storing at room temperature.

[0066] As a result, changes in the weight and size of the double-crosslinked self-healing hydrogel (OHA-GC-ADH-HAH and OHA-GC-ADH-ALG) over time were little, compared to the existing self-healing hydrogel (OHA-GC-ADH). This result indicates that the stability of hydrogel significantly increases due to double crosslinking (FIG. 2).

[0067] 2-2. Confirmation of Mechanical Strength of Self-Healing Double-Crosslinkable Hydrogel

[0068] Double-crosslinked self-healing hydrogels having different HAH concentrations were prepared, and the mechanical strength thereof was measured. For the measurement, a rotary flowmeter equipped with a cone and a plate fixture (plate diameter: 20 mm, cone angle: 4°) was used, and the temperature was kept constant at 25° C.

[0069] As measurement results, it was confirmed that the mechanical strength of a self-healing hydrogel (OHA-GC-ADH) increased in proportion to the concentration of HAH when HAH was added to the hydrogel, and the mechanical strength increased by about two times when HAH was added in an amount of 0.3% by weight. In addition, it was confirmed that mechanical strength was further improved due to double crosslinking when calcium ions were added (FIG. 3).

[0070] 2-3. Optimization of Self-Healing Double Crosslinked Hydrogel

[0071] Experiments were carried out as in Example 1-3, except that double-crosslinked self-healing hydrogels were prepared while varying a treatment time with calcium ions. The prepared hydrogels were subjected to mechanical strength measurement. As measurement results, it was confirmed that there was no significant difference in mechanical strength of the hydrogels when a treatment time with calcium ions exceeded 1 minute (FIG. 4). An optimal treatment time with calcium ions was determined to be 1 minute because a non-uniform hydrogel may be rather formed due to internal penetration of calcium, not hydrogel surface coating effect, with increasing treatment time with calcium ions.

[0072] In addition, double-crosslinked self-healing hydrogel disks containing HAH at various concentrations were manufactured and cut, followed by bonding for 30 minutes. These double-crosslinked self-healing hydrogel disks were shaken with a mixer (300 RPM, 15 seconds) to verify self-healing. As results, it was confirmed that self-healing properties of the hydrogel were decreased when the concentration of HAH included in the hydrogel was 0.4% by weight or more (FIG. 5). Thus, the concentration of HAH was determined to be 0.3% by weight at which the self-healing properties of the hydrogel were not affected while increasing the mechanical properties thereof.

[0073] 2-4. Confirmation of Self-Healing Properties of Double Crosslinkable Hydrogel

[0074] The self-healing properties of the self-healing hydrogel prepared in Example 1-3 were evaluated while alternating a strain value to 1% and 300% using a rotary flowmeter. Here, the strain value was applied for 1 minute at each of 1% and 300%. As evaluation results, it was confirmed that, when the structure of the self-healing double-crosslinkable hydrogel (OHA-GC-ADH-HAH, [OHA]=2% by weight, [GC]=1% by weight, [ADH]=0.3% by weight and [HAH]=0.3% by weight) was deformed and destroyed, and then the applied force was removed, the original properties of the self-healing double crosslinked hydrogel were recovered (FIG. 6).

[0075] In addition, two different self-healing double-crosslinkable hydrogel disks (OHA-GC-ADH-HAH, [OHA]=2% by weight, [GC]=1% by weight, [ADH]=0.3% by weight and [HAH]=0.3% by weight) with or without a pigment (rhodamine B) were manufactured. Each of the disks was cut into two pieces and optionally fitted together. After 30 minutes, it was visually checked whether the disks were bonded. As results, it was confirmed that an interface of each of the disks disappeared and rhodamine B moved, indicating that the pieces of each of the cut disks were bonded to each other (FIG. 7).

Example 3

Use of Double-Crosslinked Self-Healing Hydrogel

[0076] 3-1. Ink for 3D Bioprinters

[0077] The hydrogel solution excluding calcium ions of Example 1-3 was printed into various structure shapes using an ink for 3D bioprinters. The printed structures were immersed in a solution containing calcium ions for 1 minute to be crosslinked. Next, the structure was washed with PBS. As results, it was confirmed that the hydrogel solution satisfactorily served as a bio-ink to be satisfactorily printed in a designed structure shape (FIG. 8).

[0078] 3-2. Cell Delivery System

[0079] ATDC5 cells were mixed at a concentration of 10.sup.7/ml with a hydrogel solution excluding calcium ions. The mixture was used as an ink for bioprinters and printed in the shape of a structure. The printed structure was immersed in a solution containing calcium ions for 1 minute to be crosslinked, and was washed with PBS. Next, the survival rate of cells included in the structure was investigated using Live/DEAD Viability/Cytotoxicity Kit (Invitrogen).

[0080] As results, it was confirmed that cell viability was not significantly affected by the secondary crosslinking by calcium ions, and the 3D bioprinting, indicating that the above processes hardly affected cells.