HYDROGEL COMPRISING HYALURONIC ACID MODIFIED BY SEROTONIN AND USES THEREOF

Abstract

The present disclosure relates to a hydrogel comprising hyaluronic acid modified by serotonin, a use thereof, and a method of preparing the same.

Claims

1. A hydrogel including hyaluronic acid modified with serotonin.

2. The hydrogel of claim 1, wherein the modified hyaluronic acid includes a repeating unit structure represented by Chemical Formula 1: ##STR00003##

3. The hydrogel of claim 1, wherein the hydrogel is one in which the modified hyaluronic acid is cross-linked by chemical bonding between 5-hydroxyindole moieties of the serotonin.

4. The hydrogel of claim 1, wherein the hydrogel is adhesive or biodegradable.

5. A hemostatic agent composition including the hydrogel of claim 1.

6. The hemostatic agent composition of claim 5, wherein the hemostatic agent has a tissue adhesion inhibiting effect.

7. The hemostatic agent composition of claim 5, wherein the hemostatic agent is in the form of an adhesive patch or a film.

8. A tissue adhesive composition including the hydrogel of claim 1.

9. The tissue adhesive composition of claim 8, wherein the tissue adhesive has a tissue adhesion inhibiting effect.

10. A composition for promoting cell differentiation including the hydrogel of claim 1.

11. The composition for promoting cell differentiation of claim 10, wherein the cell is stem cell, fetal stem cell, induced pluripotent stem cell, embryonic stem cell or adult stem cell.

12. A composition for cell culture including the hydrogel of claim 1.

13. The composition for cell culture of claim 12, wherein the culture is a three-dimensional culture.

14. A composition for drug delivery including the hydrogel of claim 1.

15. The composition for drug delivery of claim 14, wherein the drug is encapsulated in the hydrogel.

16. The composition for drug delivery of claim 14, wherein the drug is selected from the group consisting of an immune cell activator, an anticancer agent, a therapeutic antibody, an antibiotic, an antibacterial agent, an antiviral agent, an anti-inflammatory agent, a contrast medium, a protein drug, a growth factor, a cytokine, a peptide drug, a hair growth solution and combinations thereof.

17. The composition for drug delivery of claim 14, wherein the pharmaceutical composition is in the form of an adhesive patch or a film.

18. A method of preparing a hydrogel including hyaluronic acid modified with serotonin, comprising: (a) a process of preparing a solution containing hyaluronic acid modified with serotonin by substituting a hydroxyl group (—OH) in hyaluronic acid with serotonin; and (b) a process of cross-linking the modified hyaluronic acid in the solution to form a hydrogel.

19. The method of preparing a hydrogel of claim 18, wherein the process (a) is a process of substituting a hydroxyl group (—OH) located at R1 in the hyaluronic acid including a repeating unit structure represented by Chemical Formula 2 with ##STR00004##

20. The method of preparing a hydrogel of claim 14, wherein the cross-linking in the process (b) is carried out through an oxidation reaction using one or more oxidizing agents selected from the group consisting of hydrogen peroxide (H.sub.2O.sub.2), peroxidase and NaIO4 (sodium periodate).

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0059] FIG. 1 is a schematic diagram showing a synthesis process of a serotonin-modified hyaluronic acid derivative.

[0060] FIG. 2 shows the results of .sup.1H-NMR analysis to check whether serotonin-modified hyaluronic acid is correctly synthesized ((1): a hydrogen proton of a methyl group in a hyaluronic acid skeleton, (2): a hydrogen proton of an aromatic ring of serotonin).

[0061] FIG. 3 shows a chemical structure during a process in which serotonin-modified hyaluronic acid derivatives are cross-linked to form a hydrogel.

[0062] FIG. 4 shows the results of checking cross-linking of a serotonin-modified hyaluronic acid hydrogel by comparing spectra before oxidation and after oxidation through photoelectron analysis (XPS analysis).

[0063] FIG. 5 shows the results of checking cross-linking of serotonin-modified hyaluronic acid hydrogel by comparing spectra before oxidation and after oxidation through Fourier transform infrared spectroscopy (FTIR).

[0064] FIG. 6 shows the results of checking cross-linking of serotonin-modified hyaluronic acid hydrogel through UV-vis (Ultraviolet-visible spectroscopy).

[0065] FIG. 7 shows a serotonin-modified hyaluronic acid hydrogel prepared by a cross-linking method through oxidation.

[0066] FIG. 8 shows the results of comparing the cross-linking rate depending on the oxidation conditions in a process of preparing a serotonin-modified hyaluronic acid hydrogel.

[0067] FIG. 9 shows the results of comparing the elasticity depending on the oxidation conditions in a process of preparing a serotonin-modified hyaluronic acid hydrogel.

[0068] FIG. 10 shows a swelling behavior depending on the oxidation conditions in a process of preparing a serotonin-modified hyaluronic acid hydrogel.

[0069] FIG. 11A shows the results of measuring a change in adhesiveness of a hydrogel depending on the concentrations of HRP.

[0070] FIG. 11B shows the results of measuring a change in adhesiveness of a hydrogel depending on the concentrations of hydrogen peroxide.

[0071] FIG. 12A shows the results of checking the bioadhesiveness of a serotonin-modified hyaluronic acid hydrogel to the tissue (tissue adhesiveness check).

[0072] FIG. 12B shows the results of checking the bioadhesiveness of a serotonin-modified hyaluronic acid hydrogel to the tissue by H&E tissue staining.

[0073] FIG. 13A shows the results of checking a tissue adhesion inhibiting effect of a serotonin-modified hyaluronic acid hydrogel (photos of tissue adhesion check).

[0074] FIG. 13B shows the results of checking a tissue adhesion inhibiting effect of a serotonin-modified hyaluronic acid hydrogel (graph measuring the degree of physical adhesion).

[0075] FIG. 14A shows the results of checking a blood clotting promoting effect of a serotonin-modified hyaluronic acid hydrogel in vitro (graph measuring platelet factor 4).

[0076] FIG. 14B shows the results of checking a blood clotting promoting effect of a serotonin-modified hyaluronic acid hydrogel in vitro (graph measuring factor V).

[0077] FIG. 14C shows the results of checking a blood clotting promoting effect of a serotonin-modified hyaluronic acid hydrogel in vitro (graph measuring the amount of blood clotting in the tissue).

[0078] FIG. 15A shows the results of checking a hemostatic effect of a serotonin-modified hyaluronic acid hydrogel in a liver bleeding mouse model (experimental schematic diagram).

[0079] FIG. 15B shows the results of checking a hemostatic effect of a serotonin-modified hyaluronic acid hydrogel in a liver bleeding mouse model (graph measuring the amount of bleeding).

[0080] FIG. 15C shows the results of checking a hemostatic effect of a serotonin-modified hyaluronic acid hydrogel in a liver bleeding mouse model (measurement of blood clotting time).

[0081] FIG. 16A shows the results of checking a hemostatic effect of a serotonin-modified hyaluronic acid hydrogel patch in a liver bleeding mouse model (photos of the tissue for hemostatic effect check).

[0082] FIG. 16B shows the results of checking a hemostatic effect of a serotonin-modified hyaluronic acid hydrogel patch in a liver bleeding mouse model (measurement of blood clotting time).

[0083] FIG. 16C shows the results of checking a hemostatic effect of a serotonin-modified hyaluronic acid hydrogel patch in a liver bleeding mouse model (graphs measuring the amount of bleeding).

[0084] FIG. 16D shows the results of checking a hemostatic effect of a serotonin-modified hyaluronic acid hydrogel patch in a liver bleeding mouse model (graphs measuring the amount of bleeding).

[0085] FIG. 17A shows the results of checking a hemostatic effect of a serotonin-modified hyaluronic acid hydrogel in a liver bleeding mouse model with hemophilia (photos of the tissue for hemostatic effect check).

[0086] FIG. 17B shows the results of checking a hemostatic effect of a serotonin-modified hyaluronic acid hydrogel in a liver bleeding mouse model with hemophilia (graph measuring the amount of bleeding).

[0087] FIG. 17C shows the results of checking a hemostatic effect of a serotonin-modified hyaluronic acid hydrogel in a liver bleeding mouse model with hemophilia (measurement of blood clotting time).

[0088] FIG. 17D shows the results of checking a hemostatic effect of a serotonin-modified hyaluronic acid hydrogel in a liver bleeding mouse model with hemophilia (H&E tissue staining result).

[0089] FIG. 18 shows the results of checking a tissue adhesion inhibiting effect in vivo.

[0090] FIG. 19 shows the results of checking the degree of penetration of macrophages at a hemostasis site through toluidine blue staining.

[0091] FIG. 20 shows the results of checking the degree of degradation of a serotonin-modified hyaluronic acid hydrogel over time.

[0092] FIG. 21A shows the results of confirming that a serotonin-modified hyaluronic acid hydrogel has no cytotoxicity to HepG2 cell.

[0093] FIG. 21B shows the results of confirming that a serotonin-modified hyaluronic acid hydrogel has no cytotoxicity to hADSC.

[0094] FIG. 21C shows the results of confirming that a serotonin-modified hyaluronic acid hydrogel has no cytotoxicity to hiPSC-derived hepatocyte.

[0095] FIG. 21D shows the results of confirming that a serotonin-modified hyaluronic acid hydrogel has no cytotoxicity to hNSC.

[0096] FIG. 22 shows the results of checking a cell differentiation promoting effect of a serotonin-modified hyaluronic acid hydrogel.

[0097] FIG. 23 shows the results of checking a cell culture effect of a serotonin-modified hyaluronic acid hydrogel.

[0098] FIG. 24 shows the results of checking a drug delivery effect and a slow release effect of a serotonin-modified hyaluronic acid hydrogel.

BEST MODE FOR CARRYING OUT THE INVENTION

[0099] Hereafter, the present disclosure will be described in detail with reference to Examples. However, the following Examples are illustrative only for better understanding of the present disclosure but do not limit the present disclosure.

Example 1. Synthesis of Serotonin-Modified Hyaluronic Acid Derivative (HA-ST)

[0100] To synthesize a serotonin-modified hyaluronic acid derivative (HA-ST), hyaluronic acid having a molecular weight of 200 kDa (Lifecore Biomedical, Ill., USA) was dissolved in tertiary distilled water (TDW) to a concentration of 1 mg/ml.

[0101] Into the solution, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC, Thermo Fisher Scientific, Waltham, Mass., USA) and N-hydroxysuccinimide (NHS, Sigma-Aldrich, St. Louis, Mo., USA) were added in the same molar ratio as hyaluronic acid, and stirred for 30 minutes at pH 5.5 to 6.0.

[0102] Serotonin hydrochloride was added into the solution in a molar ratio of 1:1 with hyaluronic acid, and stirred overnight at room temperature at pH 5.5 to 6.0 to obtain serotonin-modified hyaluronic acid (FIG. 1).

[0103] Reaction by-products and unreacted chemicals were removed in a PBS buffer solution of pH 5.0 by using a Cellu Sep T2 dialysis membrane. The product synthesized in the above process was freeze-dried and kept at 4° C.

[0104] To check whether the serotonin-modified hyaluronic acid is correctly synthesized through the above process, .sup.1H-NMR (300 MHz, Bruker, Billerica, Mass., USA) analysis was performed. A schematic diagram of the synthesis process is as shown in FIG. 1. As a result of the synthesis, it was confirmed that serotonin was bound to hyaluronic acid to have respective peak values as shown in FIG. 2.

Example 2. Preparation of Serotonin-Modified Hyaluronic Acid Hydrogel and Patch Thereof

[0105] A hyaluronic acid hydrogel was prepared by cross-linking the serotonin-modified hyaluronic acid derivative of Example 1. Specifically, to this end, a hydrogel was prepared by inducing cross-linking through an oxidation reaction using hydrogen peroxide (H.sub.2O.sub.2) and peroxidase (Horseradish peroxidase, HRP).

[0106] As shown in FIG. 3, it was confirmed that the cross-linking of the hyaluronic acid derivative was achieved by chemical bonding between 5-hydroxyindole moieties of serotonin, and the obtained hydrogel had a pale yellow color.

[0107] To specifically confirm the cross-linking of the hyaluronic acid hydrogel, spectra before oxidation and after oxidation were analyzed through photoelectron analysis (XPS analysis), Fourier transform infrared spectroscopy (FTIR) and UV-vis (Ultraviolet-visible spectroscopy).

[0108] As a result, through XPS analysis and FTIR analysis, it was confirmed that the peaks corresponding to C—C bonding and C—O—C bonding, which are bonds generated by the cross-linking reaction, increased (FIG. 4 and FIG. 5), and through UV-vis analysis, it was confirmed that a peak (.sup.˜400 nm) corresponding to quinone increased (FIG. 6). Thus, it was confirmed that a chemical bond between 5-hydroxyindole moieties of serotonin was generated.

[0109] Further, it was confirmed that a serotonin-modified hyaluronic acid hydrogel was prepared by the above-described cross-linking method through oxidation (FIG. 7).

[0110] Also, a 2% serotonin-modified hyaluronic acid solution was mixed with 24 U/ml of HRP and water at a ratio of 8:1:1 and poured in an appropriate volume (3 to 4 ml based on a 35 pie dish) to sufficiently fill the bottom of a petri dish and then frozen and freeze-dried to prepare a patch.

Example 3. Check of Change in Cross-Linking Rate Depending on Oxidation Conditions

[0111] In the process of preparing the hydrogel according to Example 2, the concentration of the serotonin-modified hyaluronic acid solution was fixed to 2% by weight and the molar ratio of hydrogen peroxide (H.sub.2O.sub.2) and HRP was changed to measure the gel-sol transition time and gelation completion time of the serotonin-modified hyaluronic acid derivative.

[0112] As a result, it was confirmed that the formation rate of the serotonin-modified hyaluronic acid hydrogel was not significantly affected by the concentration of hydrogen peroxide, but was highly dependent on the concentration of HRP.

[0113] Specifically, as shown in FIG. 8, when the concentration of HRP was set to 12, 18 and 24 U/ml, the gelation time was less than 30 seconds. However, when the concentration of HRP was set to 6 U/ml, it took 50 seconds to complete gelation, whereas the rate depending on the concentration of hydrogen peroxide did not change much.

Example 4. Check of Change in Elasticity of Hydrogel Depending on Oxidation Conditions

[0114] The viscoelastic modulus of the serotonin-modified hyaluronic acid hydrogel was analyzed by measuring a storage modulus G′ and a loss modulus G″ stored in a frequency sweep mode in a frequency range of 0.1 to 1 Hz using a rheometer (MCR 102 rheometer, Anton Paar, Ashland, Va., USA). The elasticity of the hydrogel was expressed by calculating the average storage modulus at 1 Hz (n=3).

[0115] As shown in FIG. 9, the elastic modulus indicating the strength of the hydrogel increased as the concentration of hydrogen peroxide increased in the oxidation condition. Thus, it was confirmed that the strength and elasticity of the hydrogel can be regulated by regulating the concentrations of hydrogen peroxide and HRP.

Example 5. Check of Swelling Behavior of Hydrogel Depending on Oxidation Conditions

[0116] It was confirmed that a swelling behavior was contrary depending on the oxidation conditions. Swelling characteristics were evaluated by measuring the weight of the hydrogel remaining at a specific time point (at days 0, 1, 3, 5 and 7). Specifically, the swelling behavior of the hydrogel formed by varying only the concentration of the serotonin-modified hyaluronic acid solution to 1% by weight or 2% by weight under 1 mM hydrogen peroxide and 12 U/ml HRP conditions was checked.

[0117] As a result, as shown in FIG. 10, when a serotonin-modified hyaluronic acid solution of 2% by weight was used, the hydrogel swelled, whereas when a serotonin-modified hyaluronic acid solution of 1% by weight was used, the hydrogel shrank. Thus, it was confirmed that the swelling behavior of the hydrogel can be regulated depending on the concentration of the serotonin-modified hyaluronic acid solution.

Example 6. Analysis on Adhesiveness of Hydrogel

[0118] To evaluate the adhesiveness of the hydrogel prepared in the present disclosure, the hydrogel was cross-linked between a probe and a plate of a rheometer (MCR 102 rheometer, Anton Paar, Ashland, Va., USA), and a force applied thereto was measured while increasing the space between the plates to 10 μm/s (n=3).

[0119] To check a difference in adhesiveness depending on the oxidation conditions, the adhesiveness of hydrogels formed by fixing the concentration of hydrogen peroxide to 1 mM and varying only the concentration of HRP to 12, 18 and 24 U/ml was measured. As a result, as shown in FIG. 11A, it was confirmed that the adhesiveness of the hydrogel was highest at the HRP concentration of 12 U/ml under the hydrogen peroxide concentration of 1 mM.

[0120] Further, the adhesiveness of hydrogels formed by fixing the concentration of HRP to 12 U/ml and varying the concentration of hydrogen peroxide to 1, 2, 3 and 4 mM was measured. As a result, as shown in FIG. 11B, it was confirmed that the adhesiveness of the hydrogel was highest at the hydrogen peroxide concentration of 1 mM under the HRP concentration of 12 U/ml.

[0121] The above results indicate that the adhesiveness of the hydrogel can be regulated by regulating the concentrations of hydrogen peroxide and HRP, and, thus, a desired adhesiveness can be obtained.

Example 7. Check of Adhesiveness of Hydrogel to Biological Tissue

[0122] It was checked whether the serotonin-modified hyaluronic acid hydrogel of the present disclosure has a sufficient adhesiveness to the tissue in the body.

[0123] Specifically, after the hydrogel was applied to the peritoneal tissue and then pulled to check whether the hydrogel was well adhered to the tissue. As a result, as shown in FIG. 12A, it was confirmed that the hydrogel was well adhered to the tissue with high adhesiveness.

[0124] Further, it was checked whether the hydrogel was adhered to the tissue through histological staining (H&E staining) on the peritoneal tissue. As a result, as shown in FIG. 12B, it was confirmed that the serotonin-modified hyaluronic acid hydrogel was firmly adhered to the tissue.

[0125] This is because the serotonin-modified hyaluronic acid hydrogel is bound to the protein on the tissue surface due to a chemical reaction that occurs when serotonin molecules are oxidized, so that the serotonin-modified hyaluronic acid hydrogel has tissue adhesiveness. It was confirmed that a hydrogel film can be formed on the tissue surface and the serotonin-modified hyaluronic acid hydrogel can have a sufficient adhesiveness to be adhered to the tissue in the body and maintained well.

Example 8. Check of Tissue Adhesion Inhibiting Effect of Hydrogel

[0126] It was checked whether the serotonin-modified hyaluronic acid hydrogel of the present disclosure can inhibit abnormal adhesion between tissues. An experiment was conducted using a peritoneal injury mouse model. Specifically, adhesion between the damaged tissue surface and other tissues of the peritoneal injury mouse model was induced, and a difference depending on the presence or absence of treatment with the hydrogel of the present disclosure was checked.

[0127] As a result, as shown in FIG. 13A, when adhesion between the damaged tissue surface and other tissues of the peritoneal injury mouse model was induced, abnormal tissue adhesion occurred in almost all animals in the absence of treatment (NT), whereas in the presence of treatment with the serotonin-modified hyaluronic acid hydrogel of the present disclosure (HA-ST), tissue adhesion did not occur in all animals.

[0128] Further, the degree of adhesion as described above was also checked quantitatively, and as shown in FIG. 13B, it was also checked numerically that adhesion did not occur at all in the presence of treatment with the serotonin-modified hyaluronic acid hydrogel of the present disclosure.

[0129] These results indicate that the hyaluronic acid-based hydrogel thin film formed on the tissue surface has an adhesion inhibiting effect according to the bioadhesion inhibiting mechanism of hyaluronic acid.

Example 9. Check of Blood Clotting Promoting Effect In Vitro

[0130] To check a hemostatic effect of the serotonin-modified hyaluronic acid hydrogel, ELISA analysis was performed for protein quantification by separating supernatants of samples obtained by mixing and reacting platelet-rich plasma (PRP) isolated from blood with a control PBS, a serotonin solution Serotonin, a hyaluronic acid solution HA and a serotonin-modified hyaluronic acid solution HA-ST, and the amounts of platelet factor 4 and factor V, which are the main blood clotting factors secreted from platelets, were measured.

[0131] As a result, as shown in FIG. 14A, in the presence of treatment with the serotonin-modified hyaluronic acid hydrogel, it was confirmed that platelet factor 4 was secreted at a significantly higher level than that of all other groups. Also, as shown in FIG. 14B, in the presence of treatment with the serotonin-modified hyaluronic acid hydrogel, it was confirmed that a secretion amount of factor V was about twice or more compared to that in the presence of treatment only with a hydrogel.

[0132] Further, after blood was mixed and reacted with each sample (the control PBS, the serotonin solution Serotonin, the hyaluronic acid solution HA, the serotonin-modified hyaluronic acid solution HA-ST) on a substrate, non-clotted blood was washed off. Then, the weight of the solidified residue was measured. As a result, it was confirmed that blood clotting occurred in the largest amount in the presence of treatment with the serotonin-modified hyaluronic acid hydrogel as shown in FIG. 14C.

[0133] The above results indicate that the serotonin-modified hyaluronic acid of the present disclosure promotes blood clotting by promoting serotonin-mediated platelet activity.

Example 10. Check of Hemostatic Effect In Vivo

[0134] To check the hemostatic effect of the serotonin-modified hyaluronic acid hydrogel (and the patch thereof) in vivo, liver bleeding models were constructed using 4-week-old female ICR mice (Orient Bio, Seongnam-si). Specifically, after anesthetizing each mouse and dissecting the abdomen, a sterilized filter paper was placed under the liver and bleeding was induced using an 18G needle, and the damaged area was immediately covered with the serotonin-modified hyaluronic acid hydrogel (and the patch thereof) or a fibrin adhesive (TISSEEL, Baxter, Vienna, Austria). Thereafter, the filter paper was replaced every 30 seconds until the observation was completed, and the amount of bleeding was measured by measuring the weight of the collected filter papers. After the evaluation of bleeding was completed, the peritoneum and the incision site were sutured with 6-0 Prolene sutures. An untreated mouse was used as a control. After 7 days of treatment, the mice were sacrificed and their physiological conditions were examined.

[0135] As shown in FIG. 15A, a control group NT, a commercial fibrin-based hemostatic agent treatment group Fibrin glue and a serotonin-modified hyaluronic acid hydrogel treatment group HA-ST were prepared with the liver bleeding mouse models. Then, the amount of bleeding was measured.

[0136] As a result, as shown in FIG. 15B, it was confirmed quantitatively that in the presence of treatment with the serotonin-modified hyaluronic acid hydrogel, the hemostatic effect was remarkably excellent compared to that in the presence of treatment with the conventional commercial fibrin-based hemostatic agent.

[0137] Further, photos of the filter paper for blood absorption used to check the amount of bleeding were examined in chronological order, and as a result, it was confirmed that bleeding stopped within a short time in the presence of treatment with the serotonin-modified hyaluronic acid hydrogel as shown in FIG. 15C.

[0138] Also, as shown in FIG. 16A, it was confirmed that in the presence of treatment with the serotonin-modified hyaluronic acid hydrogel patch HA-ST Patch, the hemostatic effect was excellent, and as shown in FIG. 16B, bleeding stopped within a significantly short time compared to the control group. Further, it was confirmed quantitatively that as shown in FIG. 16C and FIG. 16D, the hemostatic effect was remarkably excellent compared to that in the control group NT or that in the presence of treatment with the conventional commercial fibrin-based hemostatic agent Fibrin.

[0139] Furthermore, the hemostatic effect of in a liver bleeding mouse model with hemophilia was checked by constructing liver bleeding models in the same manner as the above method for 6-week-old hemophilic mice (B6; 129S-F8.sup.tm1Kaz/J, Jackson Laboratory, Bar Harbor, Me., USA).

[0140] As a result, as shown in FIG. 17A, it was confirmed that even the hemophilic mouse model, which lacks a blood clotting factor and thus cannot stop bleeding well, showed an excellent hemostatic effect compared to the control group, and the amount of bleeding also significantly decreased as shown in FIG. 17B.

[0141] Also, as shown in FIG. 17C, it was confirmed that in the presence of treatment with the serotonin-modified hyaluronic acid hydrogel of the present disclosure, bleeding stopped within a significantly short time compared to the control group. Further, as shown in FIG. 17D, it was confirmed through H&E tissue staining that the serotonin-modified hyaluronic acid hemostatic agent induced thrombus formation at a bleeding site to enable effective hemostasis.

Example 11. Check of Tissue Adhesion Inhibiting Effect In Vivo

[0142] Conventional hemostatic agents have a side effect of causing adhesion to surrounding tissues due to a fibrin mass formed during the hemostatic process. Accordingly, to confirm whether the serotonin-modified hyaluronic acid hemostatic agent of the present disclosure has an adhesion inhibiting effect in addition to the hemostatic action, the degree of tissue adhesion in any three of a total of 9 liver bleeding muse models at days 1, 3 and 7 after treatment with the hemostatic agent was checked.

[0143] As a result, it was confirmed that the adhesion inhibiting effect of the serotonin-modified hyaluronic acid hydrogel hemostatic agent was excellent compared to the control group NT not treated with the hemostatic agent and the conventional commercial fibrin hemostatic agent Fibrin glue as shown in FIG. 18.

Example 12. Check of Biocompatibility of Hydrogel

[0144] In the hemostatic effect experiment, macrophages were stained through toluidine blue staining on liver tissue samples collected at day 3 after treatment with the hemostatic agent to check the degree of penetration of macrophages at a hemostasis site.

[0145] As a result, as shown in FIG. 19, the tissue area treated with the serotonin-modified hyaluronic acid hydrogel did not show any particular difference in the degree of penetration of macrophages indicated by the white arrow compared to the normal tissue, which means that no sensitive immune response occurs against the serotonin-modified hyaluronic acid hydrogel of the present disclosure. Thus, it was confirmed that the serotonin-modified hyaluronic acid hydrogel of the present disclosure possesses excellent biocompatibility.

[0146] Further, the enzymatic degradation behavior of the serotonin-modified hyaluronic acid hydrogel prepared by mixing a serotonin-modified hyaluronic acid solution of 2% by weight, 1 mM hydrogen peroxide, and 12 U/ml HRP at a volume ratio of 8:1:1 was checked. As a result, as shown in FIG. 20, it was confirmed that the serotonin-modified hyaluronic acid hydrogel of the present disclosure was degraded over several hours by a hyaluronidase. Thus, it was confirmed that the serotonin-modified hyaluronic acid hydrogel of the present disclosure can be degraded in the body. The above-described results indicate that since sensitive immune response did not occur against the hydrogel of the present disclosure and degradation occurred in the body over time, the hydrogel of the present disclosure is excellent in biocompatibility.

Example 13. Check of Cytotoxicity

[0147] To evaluate the cytotoxicity and biocompatibility of the serotonin-modified hyaluronic acid hydrogel of the present disclosure, HepG2 cells and human adipose-derived stem cells (hADSCs) (1.0×10.sup.6 cells/hydrogel 100 μl) were encapsulated in the serotonin-modified hyaluronic acid hydrogel cross-linked under 12 U/ml HRP and 1 mM hydrogen peroxide conditions and subjected to Live/Dead staining while being cultured in three dimensions. Specifically, cell survival was measured at days 0 and 7 with a Live/Dead viability/cytotoxicity kit (Invitrogen, Carlsbad, Calif., USA) according to the manufacturer's protocol. The stained cells were observed with a model IX73 fluorescence microscope (Olympus, Tokyo, Japan), and the ratio of viable cells indicated in green to dead cells indicated in red was quantified by manually counting from images (n=3) obtained by the microscope. HepG2 cells were cultured in high glucose Dulbecco's Modified Eagle's medium supplemented with 10% fetal bovine serum (Invitrogen) and 1% penicillin/streptomycin (Invitrogen), and hADSCs were cultured in MesenPRO-RS™ medium.

[0148] As a result of checking the toxicity, as shown in FIG. 21A and FIG. 21B, both HepG2 cells and hADSCs showed a survival rate of about 100% even 7 days later. Thus, it was confirmed that the serotonin-modified hyaluronic acid hydrogel of the present disclosure has little cytotoxicity.

[0149] Further, as a result of performing Live/Dead staining as described above while encapsulating hiPSC-derived hepatocytes differentiated from human induced pluripotent stem cells in the serotonin-modified hyaluronic acid hydrogel of the present disclosure and culturing them in three dimensions, few dead cells were observed as shown in FIG. 21C. Thus, it was confirmed that the serotonin-modified hyaluronic acid hydrogel of the present disclosure has no cytotoxicity.

[0150] Furthermore, as a result of performing Live/Dead staining as described above while encapsulating human neural stem cells (hNSCs) in the serotonin-modified hyaluronic acid hydrogel of the present disclosure and culturing them in three dimensions, even hNSCs which are difficult to be kept alive showed a survival rate of about 80% even 7 days later. Thus, it was confirmed that the serotonin-modified hyaluronic acid hydrogel of the present disclosure has no cytotoxicity as described above.

Example 14. Check of Cell Differentiation Promoting Effect

[0151] Immunostaining (ICC) was performed while encapsulating human neural stem cells (hNSCs) in the serotonin-modified hyaluronic acid hydrogel and culturing them in three dimensions. As a result, as shown in FIG. 22, it was confirmed that a neuron-specific marker TUJ1 had a high level of expression and neurites projected well. This indicates that the serotonin-modified hyaluronic acid hydrogel of the present disclosure is effective in maintaining and differentiating stem cells and can be used as a supporter for three-dimensional culture and transplantation of stem cells.

Example 15. Check of Cell Culture Effect

[0152] Human adipose-derived stem cells (hADSCs) and human umbilical vein endothelial cells (HUVECs) were simultaneously encapsulated in the serotonin-modified hyaluronic acid hydrogel to perform a three-dimensional co-culture, followed by immunostaining.

[0153] As a result, as shown in FIG. 23, it was confirmed that each cell maintained its unique morphology and induced vascular network formation through interaction with each other, and at the same time, it was confirmed that their corresponding cell markers were normally expressed.

[0154] From the above results, it was confirmed that a three-dimensional co-culture of various cells can be performed using the serotonin-modified hyaluronic acid hydrogel of the present disclosure.

Example 16. Check of Drug Delivery Effect

[0155] To check a drug delivery effect of the serotonin-modified hyaluronic acid hydrogel, a vascular endothelial growth factor (VEGF), which is one of growth factors as a drug, was loaded in the serotonin-modified hyaluronic acid hydrogel and then, a release pattern thereof in a hyaluronidase (0.5 U/ml) solution and a PBS solution at 37° C. was observed.

[0156] As a result, as shown in FIG. 24, it was confirmed that the growth factor was hardly released under conditions (PBS) in which the hydrogel was not degraded due to excellent binding ability to protein, but in the presence of the hyaluronidase, which is an environment where hyaluronic acid can be degraded like an in vivo environment, the loaded growth factor was gradually released as the hydrogel was biodegraded.

[0157] The above results indicate that the serotonin-modified hyaluronic acid hydrogel of the present disclosure can effectively carry a drug and can also control the drug to be gradually released in the body. This means that the serotonin-modified hyaluronic acid hydrogel of the present disclosure has a high applicability as a drug delivery system.

[0158] The above description of the present disclosure is provided for the purpose of illustration, and it would be understood by a person with ordinary skill in the art that various changes and modifications may be made without changing technical conception and essential features of the present disclosure. Thus, it is clear that the above-described embodiments are illustrative in all aspects and do not limit the present disclosure. For example, each component described to be of a single type can be implemented in a distributed manner. Likewise, components described to be distributed can be implemented in a combined manner.

[0159] The scope of the present disclosure is defined by the following claims rather than by the detailed description of the embodiment. It shall be understood that all modifications and embodiments conceived from the meaning and scope of the claims and their equivalents are included in the scope of the present disclosure.