KR102817601B1 - Preparation method of zinc ion-generating gelatin based bioactive tissue adhesive hydrogels and biomedical use thereof - Google Patents

Abstract

The present invention relates to a method for producing an in situ cross-linked tissue adhesive hydrogel with a sustained release of zinc ions using zinc peroxide and to biomedical uses thereof. More specifically, the present invention relates to a novel type of hydrogel capable of producing a hydrogel by inducing a thiol-bonded gelatin derivative (GtnSH) with disulfide bonds (-S-S-) mediated by zinc peroxide and a thiol-ene reaction between a thiol-bonded gelatin derivative (GtnSH) and a maleimide-bonded gelatin derivative (GtnMI), and capable of releasing zinc ions for a long period of time.

Description

{PREPARATION METHOD OF ZINC ION-GENERATING GELATIN BASED BIOACTIVE TISSUE ADHESIVE HYDROGELS AND BIOMEDICAL USE THEREOF}

The present invention relates to a method for producing a gelatin-based zinc ion-releasing bioactive tissue adhesive hydrogel using zinc peroxide and its biomedical use.

Polymeric hydrogels are composed of three-dimensional networks of hydrophilic polymers and have been widely used in various biomedical applications due to their excellent biocompatibility, high water content, excellent permeability of nutrients and metabolites, and structural and functional similarity to living tissues.

In particular, in situ cross-linked hydrogels can be injected into the wound with minimal invasion, so they do not cause additional damage to the surrounding tissues compared to wound closure using sutures or staplers, and they maintain the wound environment moist to absorb exudates from the wound. In addition, they can be used as delivery vehicles because they can easily carry various therapeutic factors (cells, growth factors, genes, drugs, etc.). These in situ cross-linked hydrogels can be manufactured using natural and synthetic polymers, and hydrogels can be formed through various chemical and physical cross-linking.

Bioadhesives are materials that adhere to tissue surfaces through polymerization or crosslinking, and include tissue adhesives, hemostatic agents, and tissue sealants. They are being studied in various biomedical applications for purposes such as wound closure and treatment, drug delivery, and medical device attachment. For various clinical applications, ideal bioadhesive materials should have 1) strong adhesive strength on tissue-like or moist surfaces, 2) mechanical strength similar to tissue, 3) biocompatibility, and 4) biodegradability.

Among them, bioactive adhesives refer to tissue adhesives of materials that can influence the surrounding environment by stimulating biological responses through interaction with tissues upon contact, and have high compatibility with biological cells. In general, bioactive substances are derived naturally and exhibit excellent biofunctionality, biocompatibility, and biodegradation properties. By manufacturing hydrogels using natural/synthetic polymers, efforts are being made to manufacture bioactive adhesives with improved tissue adhesion and excellent biocompatibility than existing commercialized products. Such tissue adhesive hydrogels can be manufactured using natural and synthetic polymers, and can be formed through various chemical and physical crosslinking.

Zinc ion (Zn ²⁺ ) is one of the essential nutrients in the body and plays an important role in regulating immune function, wound healing, and hemostasis in the body. Deficiency of zinc ion can delay wound healing and cause abnormal thrombosis and long-term bleeding, and supply of zinc ion enhances cell proliferation and reduces the expression of anti-inflammatory factors. In addition, it is known to promote wound healing and regeneration by helping tissue regeneration processes such as promoting neovascularization and hair follicle regeneration, and promote hemostasis by inducing platelet aggregation and fibrin clot formation. Recently, various materials that release zinc ions are being developed.

For example, this includes the technology for producing hydrogels through ionic cross-linking between alginate and zinc chloride (ZnCl ₂ ) or metal ion coordinates between zinc ions and thiol-based polymers. In addition, the use of carriers such as hydrogels loaded with zinc oxide (ZnO) nanoparticles or zinc-based metal-organic frameworks also belongs to this category. Although zinc ion-releasing hydrogels can be produced through these methods, they have limitations such as a complicated production process, the need for the introduction of additional materials for cross-linking, and the difficulty in continuously releasing zinc ions.

There is a growing need to develop an in situ tissue adhesive hydrogel that can overcome the limitations of existing tissue adhesive biomaterials by 1) releasing zinc ions in a sustained manner for a long period of time, 2) simultaneously cross-linking, 3) exhibiting excellent biocompatibility, and 4) having enhanced tissue adhesiveness and hemostatic effects.

Korean Patent Publication No. 10-2016-0031624

The present invention is to satisfy the above-mentioned conventional needs, and to produce a new type of in situ cross-linked polymer adhesive hydrogel that improves tissue adhesiveness and long-term sustained release of zinc ions simultaneously with cross-linking without the introduction of additional substances through a thiol group, a maleimide group, and zinc peroxide. The technical task of the present invention is to provide a method for producing a hydrogel whose physical/chemical/biological properties can be easily controlled by changing the concentration of hydrogel components, and various biomedical uses thereof.

To achieve the above technical task, the present invention used a natural/synthetic polymer having a gelatin-based thiol (-SH) reactive group and a maleimide reactive group and zinc peroxide.

1) A gelatin derivative (GtnSH) bound to thiol can be synthesized by EDC/NHS reaction to increase the thiol content compared to when synthesized using a conventional method.

2) By introducing a natural/synthetic polymer having a maleimide reactive group, the mechanical strength and phase transition time can be improved by inducing a thiol-ene reaction between a thiol (-SH) reactive group and a natural/synthetic polymer having a maleimide reactive group.

3) By introducing natural/synthetic polymers with maleimide reactive groups, various chemical bonds such as hydrogen bonding, Michael type addition, and imine bonding with the tissue surface can be induced, enabling rapid and strong attachment of hydrogel-containing materials to various tissues.

4) An in situ cross-linked polymer hydrogel is produced through the formation of disulfide bonds (-S-S-) of natural/synthetic polymers having a thiol (-SH) reactive group by hydrogen peroxide (intermediate) generated by the decomposition of zinc peroxide.

5) A novel in situ cross-linked polymer hydrogel is provided that releases zinc ions generated by the decomposition of zinc peroxide in a sustained manner through the introduction of a maleimide reactive group and a high thiol content in the polymer chain.

According to one embodiment of the present invention, a natural/synthetic polymer having a thiol reactive group (average 141.02 to 184.84 μmol/g (polymer)) can be produced through an Ethyl(dimethylaminopropyl) carbodiimide (EDC)/N-Hydroxysuccinimide (NHS) reaction using gelatin and cystamine dihydrochloride.

In addition, natural/synthetic polymers having maleimide reactive groups (average 85.76–116.18 μmol/g (polymer)) can be prepared through the Ethyl(dimethylaminopropyl) carbodiimide (EDC)/N-Hydroxysuccinimide (NHS) reaction using gelatin and 6-Maleimidohexanoic acid (MHA).

In addition, the present invention provides a method for producing a hydrogel, which can control physical/chemical/biological properties such as 1) phase transition time of the hydrogel, 2) mechanical strength, 3) hydrogen peroxide and zinc ion release behavior, 4) biodegradability, 5) cell and tissue compatibility, 6) tissue adhesiveness, and 7) hemostatic effect, depending on the increase in thiol content, introduction of a maleimide reactor, and concentration of zinc peroxide.

In addition, the present invention provides a carrier for tissue regeneration and drug delivery comprising an in situ cross-linked sustained-release zinc ion-releasing hydrogel.

In addition, the present invention provides a material for tissue adhesion and hemostasis comprising an in situ cross-linked sustained-release zinc ion-releasing hydrogel.

Specifically, according to one embodiment of the present invention, there is provided a method for producing a polymer, comprising: a) synthesizing a polymer having a thiol group introduced into a main chain through an EDC/NHS synthesis method; b) preparing a polymer having a maleimide reactive group by bonding a carboxyl group of 6-maleimidohexanoic acid (MHA) to a primary amine group; And c) a step of dissolving a polymer having a thiol group introduced and a polymer having a maleimide reactive group in a solvent, and then mixing and reacting zinc peroxide (ZnO ₂ ) to form a hydrogel; wherein step c) comprises inducing disulfide bonds (-SS-) between polymer main chains having a thiol group introduced by decomposition of zinc peroxide (ZnO ₂ ), and in situ crosslinking through a thiol-ene reaction between the polymer main chain having a thiol reactive group and the polymer main chain having a maleimide reactive group. The present invention provides a method for producing a gelatin-based zinc ion-releasing bioactive tissue adhesive hydrogel using zinc peroxide.

It is characterized in that zinc ions generated by the decomposition of the above zinc peroxide are released in a sustained manner from the hydrogel.

The zinc peroxide of the above step c) is characterized in that it is used in an amount of 0.125 to 0.5 wt% of the reaction solution. The zinc peroxide of the above step c) can be used in an amount of, for example, 0.125 to 0.5 wt%, preferably 0.25 to 0.5 wt%, more preferably 0.5 wt% of the reaction solution.

The EDC/NHS synthesis method in step a) is characterized in that it is performed by adding cystamine dihydrochloride, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), N-hydroxysuccinimide (NHS), and DL-dithiothreitol (DTT) to the polymer.

The polymers of steps a) and b) above may be at least one of natural polymers and multi-polymers composed of gelatin, chitosan, heparin, cellulose, dextran, alginate, collagen, and hyaluronic acid, respectively.

The above multi-arm polymer may be any one or more of multi-arm polyethylene glycol selected from 3-arm PEG, 4-arm PEG, 6-arm PEG or 8-arm PEG and any one or more of polymers selected from the group consisting of Tetronic series (4arm-PPO-PEO), but is not necessarily limited thereto.

The solvent in step c) is characterized in that it is DPBS (Dulbecco's phosphate buffered saline; DPBS).

The concentration ratio of the polymer having the thiol group introduced and the polymer having the maleimide reactive group may be 2:8 to 8:2. The concentration ratio of the polymer having the thiol group introduced and the polymer having the maleimide reactive group may be, for example, 3:7 to 7:3, preferably 4:6 to 6:4, more preferably 5:5.

According to another embodiment of the present invention, a gelatin-based zinc ion-releasing bioactive tissue adhesive hydrogel using zinc peroxide is provided, manufactured according to the method for manufacturing a gelatin-based zinc ion-releasing bioactive tissue adhesive hydrogel using zinc peroxide.

The above hydrogel is characterized as being an injectable hydrogel.

In addition, the present invention provides a support for tissue regeneration and tissue engineering including a gelatin-based zinc ion-releasing bioactive tissue adhesive hydrogel using the zinc peroxide.

In addition, a material for tissue adhesion, wound healing or hemostasis is provided, which comprises a gelatin-based zinc ion-releasing bioactive tissue adhesive hydrogel using zinc peroxide.

In addition, a carrier for delivery of a bioactive substance or drug is provided, including a gelatin-based zinc ion-releasing bioactive tissue adhesive hydrogel using zinc peroxide.

In addition, a filling implant material is provided, which includes a gelatin-based zinc ion-releasing bioactive tissue adhesive hydrogel using zinc peroxide.

The novel in situ cross-linking tissue adhesive hydrogel according to the present invention has the following characteristics: 1) increase in thiol content in the polymer chain through the EDC/NHS synthesis method, 2) control of hydrogel formation and physical/chemical properties by controlling the composition of the hydrogel (thiol content, introduction of maleimide groups, zinc peroxide), 3) improvement of mechanical strength and tissue adhesiveness and control of phase transition time by introduction of maleimide reactive groups, and 4) control of the sustained release behavior of zinc ions generated inside the hydrogel by increasing thiol content.

In addition, according to one embodiment of the present invention, a polymer having a higher thiol content was prepared through an EDC/NHS synthesis method compared to a thiol group-containing polymer synthesized by a conventional method, and the mechanical strength can be improved and the phase transition time can be controlled according to an increase in the thiol content.

In addition, the tissue adhesiveness is improved by introducing a maleimide group and using zinc peroxide, and it is characterized by the sustained release of zinc ions due to the high thiol content in the polymer chain and the decomposition of zinc peroxide.

In addition, compared to existing tissue adhesive materials, the present invention can be manufactured by a simple method, and the phase transition time, mechanical strength, tissue adhesiveness, and hemostatic effect of the hydrogel can be controlled depending on the high thiol content in the hydrogel, the ratio of thiol groups and maleimide groups, and the concentration of zinc peroxide.

The fabricated hydrogel can control zinc release and biodegradation behavior depending on the concentration of zinc peroxide. It has excellent cell and tissue compatibility, and it was confirmed that it has excellent tissue adhesiveness and hemostatic ability that can adhere to various tissues in animal models.

That is, the limitations of existing tissue adhesive hydrogels can be overcome through the present invention, and based on excellent biocompatibility, it can be applied to various tissue regeneration, artificial tissue manufacturing, wound healing materials, tissue adhesive and hemostatic materials, drug delivery vehicles, etc.

Figure 1 is a schematic diagram of the synthesis of thiolated-gelatin (GtnSH).
Figure 2 is a schematic diagram of the synthesis of maleimide-conjugated gelatin (GtnMI).
Figure 3 is a schematic diagram of GtnSH/GtnMI hydrogel using zinc peroxide.
Figure 4 is a graph showing the phase transition time of a hydrogel according to the thiol content in a polymer chain (GtnSH) having a thiol group (-SH, thiol), the ratio of GtnSH to GtnMI, the concentration of Tris-HCl, and the concentration of zinc peroxide.
Figure 5 is a graph showing the mechanical strength of the hydrogel.
Figure 6 is a graph showing the hydrogen peroxide and zinc ion release behavior of the hydrogel.
Figure 7 is a graph showing the degree of enzymatic degradation of hydrogels.
Figure 8 is a graph showing the cell compatibility of the hydrogel.
Figure 9 is a drawing showing the tissue compatibility of the hydrogel.
Figure 10 is a graph showing the tissue adhesiveness of the hydrogel.
Figure 11 is a graph showing the hemostatic ability of the hydrogel.

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

As a result of repeated studies, the inventors of the present invention have fabricated a new type of in situ tissue adhesive hydrogel by increasing the thiol content in the polymer chain and introducing a maleimide group, and confirmed that it releases zinc ions for a long period of time (for 28 days) through the decomposition of zinc peroxide.

In addition, it was revealed that a polymer hydrogel can be simply manufactured by mixing a polymer and a zinc peroxide solution, and that the mechanical strength and tissue adhesiveness can be improved by introducing a maleimide reactor, and that the physical/chemical/biological properties can be easily controlled by adjusting the concentration and composition of the material.

The present invention induces a thiol-ene reaction by introducing a natural polymer having a maleimide reactive group into a natural polymer having a high content of thiol reactive groups, and induces a disulfide bond by applying zinc peroxide. Through this, a tissue adhesive in situ hydrogel that releases zinc ions in a sustained manner is manufactured.

The hydrogel of the present invention has the advantages of high mechanical strength, fast phase transition time, and easy control of sustained ion release by using a polymer chain having a higher thiol content than existing hydrogels through the EDC/NHS reaction.

In addition, there is an advantage in that the physical/chemical properties such as mechanical strength and tissue adhesiveness can be improved by inducing a thiol-ene reaction through the introduction of a polymer main chain having a maleimide reactive group, and various chemical bonds such as disulfide bonds, hydrogen bonds, Michael type additions, and imine bonds between the polymer and tissue can be regulated to control tissue adhesiveness.

A tissue-adhesive in situ hydrogel is fabricated by inducing disulfide bonds between thiol groups through the application of zinc peroxide and releasing zinc ions in a sustained manner.

Referring to FIG. 1, cystamine dihydrochloride, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), N-hydroxysuccinimide (NHS), and DL-dithiothreitol (DTT) are added to gelatin to bond the primary amine group of cystamine dihydrochloride to the carboxyl group of gelatin, thereby producing a polymer main chain having a thiol reactive group. The above polymer may be a natural polymer or a multi-polymer such as gelatin, chitosan, heparin, cellulose, dextran, alginate, collagen, and hyaluronic acid, but is not necessarily limited thereto. The thiol-containing polymer chain produced by the EDC/NHS synthetic method has the advantage of being able to flexibly control the thiol reactive group content according to various synthetic conditions.

Referring to FIG. 2, 6-maleimidohexanoic acid (MHA), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), and N-hydroxysuccinimide (NHS) are added to gelatin to bond the carboxyl group of MHA to the primary amine group of gelatin, thereby producing a polymer backbone having a maleimide reactive group. The polymer may be a natural polymer or a multi-polymer such as gelatin, chitosan, heparin, cellulose, dextran, alginate, collagen, and hyaluronic acid, but is not necessarily limited thereto.

Referring to Figure 3, when a polymer backbone having a thiol reactive group and a zinc peroxide solution are mixed, the released hydrogen peroxide induces disulfide bonds. An in situ cross-linked tissue adhesive hydrogel can be manufactured through a thiol-ene reaction between a polymer backbone having a thiol reactive group and a polymer backbone having a maleimide reactive group. It can be attached to tissue through imine bonds, hydrogen bonds, etc. between the maleimide reactive group and tissue.

The hydrogel produced by the present invention has the advantage of being able to easily control physicochemical properties such as phase transition time, mechanical strength, biodegradability via enzymes, and release of hydrogen peroxide and zinc ions by controlling the concentration of zinc peroxide, and has excellent cytocompatibility.

In addition, the present invention provides a material for tissue adhesion and hemostasis, comprising the zinc ion-releasing in situ cross-linked hydrogel.

The above hemostatic material may be applied to any one selected from the group consisting of, but not necessarily limited to, neurosurgery including vascular surgery, orthopedic surgery including bone fusion, hemostasis in patients with lacerations, suturing of the femoral artery, cataract incision suture, cartilage healing, skin adhesion, hemostasis of incisions of organs/gland, gastrointestinal ligation, and tendon/ligament healing.

Hereinafter, the present invention will be described in more detail through examples and experimental examples. However, these examples are only intended to help understand the present invention and the scope of the present invention is not limited to these examples in any way.

< Example 1> Preparation of materials

Gelatin (type A from porcine skin, >300 bloom), cystamine dihydrochloride (TR), 6-maleimidohexanoic acid (MHA), zinc peroxide (ZnO ₂ ), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), N-hydroxysuccinimide (NHS), DL-dithiothreitol (DTT), collagenase type Ⅱ, and zinc assay kit were purchased from Sigma Aldrich (St. Louis, MO, USA), and 1 M Tris HCl (UltraPure ^TM 1 M Tris-HCI Buffer) solution was purchased from Invitrogen. I bought it.

Dulbecco's Modified Eagle's Medium (DMEM), penicillin-streptomycin (P/S), trypsin/EDTA, and Dulbecco's phosphate buffered saline (DPBS) solution were purchased from Gibco (Grand Island, NY, USA), fetal bovine serum (FBS) was purchased from Research and Diagnostic Technology, and EGM-2 Single quots medium (Endothelial Cell Growth Medium; EGM) was purchased from Lonza. In addition, Cell Proliferation reagent WST-1 was purchased from Roche Diagnostics, and Live/Dead Viability/Cytotoxicity Kit was purchased from Life science.

Other chemicals and solvents were used without further purification.

<Example 2> Synthesis and structural analysis of gelatin derivatives containing thiol (Figures 1 and 2)

After dissolving 250 mg of gelatin in 125 mL of deionized water (DIW), the solution was mixed with a solution of 450.5 mg of cystamine dihydrochloride in 2.5 mL of DIW for 10 minutes. After mixing a solution of 192 mg of EDC in 2.5 mL of DIW with the gelatin/cystamine dihydrochloride mixture, a solution of 116 mg of NHS in 2.5 mL of DIW was additionally added and mixed. At this time, the reaction temperature was 37°C, and the reaction was carried out for 2 hours.

The mixture reacted for 2 hours was mixed with a solution containing 617 mg of DTT dissolved in 5 mL of DIW. At this time, the reaction temperature was 37°C, and the reaction was carried out for 24 hours.

The above reacted mixture was placed in a dialysis tube with a molecular weight cut-off (MWCO) of 3.5 kD, and the solution was replaced with a 5 mM hydrogen chloride (HCl) solution and a 1 mM HCl solution every day for 2 days at 37°C. After dialysis, the dialysate was vacuum filtered, and the reacted mixture was frozen at -80°C for more than one day.

Thiol-conjugated gelatin derivative (GtnSH) polymer was obtained through freeze-drying. The amount of thiol introduced into GtnSH was qualitatively and quantitatively measured through ¹ H-NMR and Ellman's assay.

After dissolving 6 mg of GtnSH in 600 μl of _D2O , the solution was placed in an NMR tube and measured. The measurement results confirmed that a thiol group was introduced into gelatin through a decrease in the primary amine peak at 3.0 ppm and an increase in the cystamine peaks at 2.8 ppm and 3.5 ppm.

L-cysteine was used to display the standard curve. 100 μl of cysteine (L-cystein) solutions at various concentrations and 1 mg/ml GtnSH solution were mixed with 100 μl of Ellman's reagent solution, blocked from light, and reacted at room temperature for 20 minutes. The absorbance was measured at 405 nm using an absorbance detector, and the thiol content in GtnSH was calculated through the cysteine standard curve. As a result of the measurement, it was confirmed that the thiol content in GtnSH was 141.02 to 184.84 μmol/g (polymer) on average.

<Example 2> Synthesis and structural analysis of gelatin derivatives containing maleimide (Figure 2)

After dissolving 250 mg of gelatin in 50 ml of DPBS, it was mixed with a solution of 211.2 mg of MHA dissolved in 120 ml of DPBS for 10 minutes. A solution of 230.1 mg of EDC dissolved in 5 mL of DPBS and a solution of 161.1 mg of NHS dissolved in 5 mL of DPBS were mixed, and then added to the gelatin and MHA mixture solution and mixed. At this time, the reaction temperature was 37 to 40°C, and the reaction was carried out for 2 hours in a light-blocking state.

The above reacted mixture was placed in a dialysis tube with a molecular weight cut-off (MWCO) of 3.5 kDa and dialyzed against deionized water (DIW) solution at 37 to 40°C for 3 days, replacing the solution three times each time, at 37°C in a light-blocking state. After dialysis, the dialysate was vacuum filtered and the reacted mixture was frozen at -80°C for more than one day.

A maleimide gelatin derivative (GtnMI) polymer was obtained through freeze-drying.

The amount of maleimide introduced into GtnMI was qualitatively and quantitatively measured using ¹ H-NMR and Ellman's assay.

After dissolving 6 mg of GtnMI in 600 μl of _D2O , the solution was placed in an NMR tube and measured. The measurement results confirmed that a maleimide group was introduced into gelatin through a decrease in the primary amine peak at 3.0 ppm and an increase in the maleimide peak at 6.8 ppm.

For the standard curve, cysteine (L-cysteine) and 6-maleimidohexanoic acid (6-maleimidohexanoic acid) were used. 6-maleimidohexanoic acid solutions and 1 mg/mL GtnMI solution were mixed with 50 μl of 0.04 mg/mL cysteine solution, blocked from light, and reacted at room temperature for 10 minutes. The reacted solution was mixed with 50 μl of Ellman's reagent solution, blocked from light, and reacted at room temperature for 20 minutes. The absorbance was measured at 405 nm using an absorbance detector, and the maleimide content in GtnMI was calculated using the maleimide standard curve. As a result of the measurement, it was confirmed that the maleimide content in GtnMI was 85.76 to 116.18 μmol/g (polymer) on average.

<Example 3> Hydrogel formation (Figure 3)

GtnSH polymer and GtnMI polymer were each dissolved in DPBS at 37°C. A zinc peroxide solution (0–0.5 wt%) was mixed into the GtnMI polymer solution, and then mixed into the GtnSH polymer solution to prepare a hydrogel.

<Experimental Example 1> Analysis of the phase transition time of hydrogel according to the polymer concentration ratio, concentration of Tris-HCl solution, concentration of zinc peroxide, and thiol content (Figure 4)

The vial tilting method was used to analyze the phase transition time of the hydrogel according to the polymer ratio, the concentration of Tris-HCl solution, and the concentration of zinc peroxide. 1) The gelatin concentration was fixed at 10 wt% and the zinc peroxide concentration at 0.25 wt%, and the polymer concentration ratios of GtnSH:GtnMI were varied to 8:2, 6:4, 5:5, 4:6, and 2:8 for the measurement. 2) The polymer concentration ratio of GtnSH:GtnMI was fixed at 5:5 and the zinc peroxide concentration was 0%, and the concentration of Tris-HCl was added at 0 to 1 M. 3) The concentration of Tris-HCl was fixed at 0.01 M and the zinc peroxide concentration was added at 0 to 0.5%. 4) The polymer concentration ratio was fixed at 5:5, the zinc peroxide concentration at 0.25%, and the Tris-HCl concentration at 0.01 M, and the thiol group-containing polymer chains synthesized using the existing Traut's reagent and the polymer chains with increased thiol content through the EDC/NHS synthesis method were compared and measured. More detailed hydrogel manufacturing conditions are shown in Table 1.

Experimental results: 1) When the polymer ratio GtnSH:GtnMI was 5:5, the phase transition time was reduced to less than 10 seconds. This is because the thiol-ene reaction is promoted depending on the ratio of thiol groups and maleimide groups, which shortens the gel formation time.

2) As the concentration of Tris-HCl increased, the phase transition time decreased. This is because the formation of the thiol-ene reaction was promoted as the thiol-ene reaction rate increased as the pH decreased, shortening the gel formation time.

3) As the concentration of zinc peroxide increased, the phase transition time decreased. This is because the formation of disulfide bonds is promoted as the amount of hydrogen peroxide generated when zinc peroxide dissociates increases, shortening the gel formation time.

4) As the thiol content in the polymer chain increased, the phase transition time decreased. This is because the formation of disulfide bonds between thiol groups and the thiol-ene reaction between thiol groups and maleimide was promoted, which shortened the gel formation time.

< Experimental Example 2> Analysis of mechanical strength of hydrogel according to concentration of zinc peroxide (Fig. 5)

A rheometer was used to analyze the mechanical strength of the hydrogel. 1) The change in hydrogel properties according to the change in thiol content in the polymer chain (GtnSH(TR), GtnSH(EDC/NHS)) was measured. 2) The change in hydrogel properties according to the change in zinc peroxide concentration was measured.

The concentration of zinc peroxide was varied from 0 wt%, 0.125 wt%, 0.25 wt%, and 0.5 wt% under constant compositions of GtnSH (5 wt%) and GtnMI (5 wt%).

Experimental results: 1) When the hydrogel was fabricated with a polymer chain (GtnSH (EDC/NHS)) with increased thiol content through the EDC/NHS synthesis method, it had high mechanical strength. 2) It had a mechanical strength of over 500 Pa at all zinc peroxide concentrations. This is because the introduction of the polymer chain (GtnMI) having a maleimide group formed a thiol-ene reaction between the thiol group and the maleimide group, and the formation of disulfide bonds was promoted due to the increase in hydrogen peroxide according to the zinc peroxide concentration, which increased the crosslinking degree of the hydrogel.

<Experimental Example 3> Measurement of hydrogen peroxide and zinc ion release behavior according to zinc peroxide concentration (Figure 6)

The release behavior of hydrogen peroxide and zinc ions from the hydrogel was measured using Cu(II)-neocuproine spectrometric assay and zinc-ligand binding assay.

1) The thiol-containing polymer chains synthesized by the existing Traut's reagent under a constant composition of GtnSH (5 wt%), GtnMI (5 wt%) and 0.25 wt% of zinc peroxide were compared with the polymer chains having increased thiol content through the EDC/NHS synthesis method. 2) The zinc peroxide concentrations were changed to 0 wt%, 0.125 wt%, 0.25 wt%, and 0.5 wt% under a constant composition of GtnSH (5 wt%) and GtnMI (5 wt%).

Experimental results: 1) It was confirmed that the hydrogel produced through a polymer chain with increased thiol content via the EDC/NHS synthesis method had a lower initial zinc ion release amount than the hydrogel produced by the conventional synthesis method, and that it was released after 3 days or more. The amount of hydrogen peroxide released by the hydrogel without zinc peroxide up to 7 days was 200 μM. This was generated when the thiol group was oxidized by oxygen to form a disulfide bond and release hydrogen peroxide. It was confirmed that there was no difference in the amount of hydrogen peroxide released as the concentration of zinc peroxide increased, but in the case of a zinc peroxide concentration of 0.5 wt%, it was confirmed that an excessive amount of hydrogen peroxide was released after the 21st day. In addition, it was confirmed that the release concentration of zinc ions (more than 200 μM) and time (more than 28 days) increased. Through this, it was confirmed that the produced hydrogel can control the initial zinc ion release in a sustained manner depending on the thiol content in the polymer chain, and that there is a difference in the concentration of hydrogen peroxide and zinc ions released depending on the introduction of zinc peroxide.

<Experimental Example 4> Measurement of biodegradability of hydrogel (Fig. 7)

To evaluate the biodegradability of hydrogels by enzymes, hydrogels were prepared in 1 ml syringes.

The concentration of zinc peroxide was varied from 0 wt%, 0.125 wt%, 0.25 wt%, and 0.5 wt% under constant compositions of GtnSH (5 wt%) and GtnMI (5 wt%).

After stabilizing the hydrogel in a carbon dioxide incubator for 30 minutes, the hydrogel was transferred to a microtube and the weight of the gel was measured. Afterwards, the tube was treated with DPBS or collagenase (0.005 mg/ml) enzyme and cultured.

At specified time intervals, the treatment solution was removed, the weight of the hydrogel was measured, and then 200 μl of new medium was added.

The degree of biodegradability was calculated using the following mathematical formula 1.

[Mathematical Formula 1]

(Here, Wd and Wi represent the weight of the hydrogel over time and the initial hydrogel, respectively.)

As a result of the experiment, it was confirmed that the sample treated with DPBS did not decompose the hydrogel over time, whereas the group treated with collagenase decomposed within 24 hours. This confirmed that the gelatin introduced with thiol groups and maleimide has the ability to be decomposed by enzymes. In addition, it was confirmed that the biodegradability of the hydrogel releasing zinc ions at a zinc peroxide concentration of 0.125 wt% decreased after 3 hours, which is because the zinc ions exhibit an inhibitory effect on collagenase.

<Experimental Example 5> Evaluation of cell compatibility of hydrogel (Fig. 8)

To evaluate the cytotoxicity of the hydrogel, 20 μL of the hydrogel was placed in a 96-well plate and human dermal fibroblasts (HDFs) were cultured to perform a cytotoxicity evaluation.

The concentration of cells used in the experiment was 1.5 X 10 ⁴ cells/wells, and after culturing for 24 hours at 37°C and 5% CO ₂ atmosphere, cell activity was evaluated through WST-1 assay and Live and dead staining.

Live/Dead analysis is a method to evaluate the presence or absence of cytotoxicity by indicating cells killed by cytotoxicity in red and living cells in green.

As a result of the experiment, the zinc ion-releasing hydrogel showed a cell viability of over 100% compared to the control group, and the hydrogel showed 80-90% living cells depending on the concentration of zinc peroxide in the Live/Dead assay. It was confirmed that the hydrogel had excellent cell compatibility.

<Experimental Example 6> Evaluation of tissue compatibility of hydrogel (Fig. 9)

To evaluate the tissue compatibility of the hydrogel, a biocompatibility test was conducted. 200 μL hydrogel was implanted subcutaneously in C57BL/6 female mice (6 weeks old), and major organs (heart, liver, spleen, lungs, and kidneys) were obtained after the hydrogel was degraded (after 8 weeks).

Paraffin blocks were prepared for each organ and sectioned at 4 μm to obtain sections for each organ. Structural differences were compared with normal tissues that were not treated with hydrogel through H&E staining. As a result of the experiment, no inflammatory response or structural changes were observed in the tissue, confirming that the hydrogel has excellent tissue compatibility.

<Experimental Example 7> Evaluation of tissue adhesiveness of hydrogel (Fig. 10)

The tissue adhesive strength of hydrogels on porcine skin was measured using a hydraulic universal testing machine (UTM) based on the ASTM F2255-03 (“Test Method for Strength Properties of Tissue Adhesives in Lap-Shear by Tension Loading”) evaluation method.

The size of the porcine skin was 1 cm in diameter, and after decellularization using sodium dodecyl sulfate (SDS), it was freeze-dried and used. All parts of the porcine skin were attached to a rectangular silicon substrate (1 cm x 4 cm) using ethyl cyanoacrylate glue.

Before the experiment, the skin surface was wetted with 150 μL of DPBS, and 50 μL of adhesive hydrogel was applied to the area of the pig skin.

The above adhesive was maintained for 30 minutes at room temperature in a humid environment with a force of 100 g applied.

Subsequently, the sample was loaded into the transfer chamber at a crosshead speed of 1 mm/min.

The maximum strength for displacement was measured, and the shear stress at failure (ultimate bond strength) was used to characterize the bond strength of each sample.

At least three samples were used for measurements.

The tissue adhesion was evaluated using pig skin, and the introduction of maleimide group-containing polymer chains resulted in improved adhesion strengths of more than 30 kPa at all zinc peroxide concentrations.

When the concentration of zinc peroxide was changed to 0 wt%, 0.125 wt%, 0.25 wt%, and 0.5 wt% under a constant composition of GtnSH (5 wt%) and GtnMI (5 wt%), the adhesive strength could be controlled from 34 to 93 kPa, and in particular, the highest adhesive strength was observed when it was 0.125 wt%.

<Experimental Example 8> Evaluation of tissue adhesion of hydrogel on various biosurfaces (Figure 10)

To determine whether the hydrogel maintains tissue adhesion on various tissue surfaces, tissue adhesion was evaluated using mouse organs (heart, lung, kidney, and spleen). The extracted organs were washed in DPBS (1% P/S) for 1 hour.

The concentration of zinc peroxide was changed to 0 wt% and 0.125 wt% under a constant composition of GtnSH (5 wt%) and GtnMI (5 wt%).

To evaluate the tissue adhesion of the hydrogel, the extracted organs were cut in half, 20 μL of hydrogel was applied to the organ tissue surface, and each surface was adhered to each other.

The experimental results confirmed that all hydrogels had excellent adhesiveness on the tissue surface.

<Experimental Example 9> Evaluation of hemostatic ability of hydrogel (Figure 11)

To determine whether the hydrogel can stop bleeding on the organ surface, the hemostatic ability was evaluated using the liver of a mouse. The liver of a C57BL/6 female mouse (6 weeks old) was wounded by piercing it with an 18 gauge needle. 100 μL of hydrogel was immediately applied to the wounded area, and the measurement was made for 3 minutes by tilting it at a constant angle (60 degrees). To evaluate the hemostatic ability of the hydrogel, filter paper was used, and the bleeding amount was analyzed by measuring the weight of the filter paper stained with blood. It was compared with the normal bleeding amount not treated with the hydrogel, and the hemostatic ability was calculated using the following mathematical equation 2.

[Mathematical formula 2]

(Here, W _A and W _B represent the weight of the blood-stained filter paper and the initial filter paper, respectively.)

As a result of the experiment, when the concentration of zinc peroxide was changed to 0 wt% and 0.125 wt% under a constant composition of GtnSH (5 wt%) and GtnMI (5 wt%), the bleeding amount could be controlled from 35 to 60.7 mg. This is because zinc ions promote hemostasis by inducing platelet aggregation and fibrin clot formation. Through this, it was confirmed that the fabricated hydrogel had an excellent hemostatic effect depending on the introduction of zinc peroxide.

As described above, although the present invention has been described by means of limited embodiments and drawings, the present invention is not limited thereto, and it is obvious that various modifications and variations are possible within the scope of the technical idea of the present invention and the equivalent scope of the claims to be described below by a person skilled in the art to which the present invention pertains.

Claims (13)

a) A step of synthesizing gelatin having a thiol group introduced into the main chain using an EDC/NHS synthesis method;
b) a step of preparing gelatin having a maleimide reactive group by bonding a carboxyl group of 6-maleimidohexanoic acid (MHA) to a primary amine group; and
c) a step of dissolving gelatin having a thiol group introduced and gelatin having a maleimide reaction group in a solvent and then mixing and reacting with zinc peroxide (ZnO ₂ ) to form a hydrogel;
The zinc peroxide of step c) above is used in an amount of 0.125 to 0.5 wt% in the reaction solution,
The step c) above is characterized in that disulfide bonds (-SS-) are induced between gelatin main chains to which thiol groups are introduced by hydrogen peroxide (intermediate) generated by the decomposition of zinc peroxide (ZnO ₂ ), and crosslinking is in situ through a thiol-ene reaction between a gelatin main chain having a thiol reactive group and a gelatin main chain having a maleimide reactive group.
Method for preparing a gelatin-based zinc ion-releasing bioactive tissue adhesive hydrogel using zinc peroxide.
In the first paragraph,
It is characterized in that zinc ions generated by the decomposition of the zinc peroxide are released in a sustained manner from the hydrogel.
Method for preparing a gelatin-based zinc ion-releasing bioactive tissue adhesive hydrogel using zinc peroxide.
delete In the first paragraph,
The EDC/NHS synthesis method in step a) is characterized in that it is performed by adding cystamine dihydrochloride, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), N-hydroxysuccinimide (NHS) and DL-dithiothreitol (DTT) to gelatin.
Method for preparing a gelatin-based zinc ion-releasing bioactive tissue adhesive hydrogel using zinc peroxide.
delete In the first paragraph,
The solvent of the above step c) is characterized in that it is DPBS (Dulbecco's phosphate buffered saline; DPBS).
Method for preparing a gelatin-based zinc ion-releasing bioactive tissue adhesive hydrogel using zinc peroxide.
In the first paragraph,
The concentration ratio of the gelatin having the above thiol group introduced and the gelatin having the maleimide reactive group is characterized in that it is 2:8 to 8:2 in weight %.
Method for preparing a gelatin-based zinc ion-releasing bioactive tissue adhesive hydrogel using zinc peroxide.
Manufactured according to the method of any one of claims 1 to 2, 4, and 6 to 7,
Gelatin-based zinc ion-releasing bioactive tissue adhesive hydrogel using zinc peroxide.
In Article 8,
The above hydrogel is characterized in that it is an injectable hydrogel.
Gelatin-based zinc ion-releasing bioactive tissue adhesive hydrogel using zinc peroxide.
A gelatin-based zinc ion-releasing bioactive tissue adhesive hydrogel comprising zinc peroxide according to Article 8.
Scaffolds for tissue regeneration and tissue engineering.
A gelatin-based zinc ion-releasing bioactive tissue adhesive hydrogel comprising zinc peroxide according to Article 8.
Materials for tissue adhesion or hemostasis.
A gelatin-based zinc ion-releasing bioactive tissue adhesive hydrogel comprising zinc peroxide according to Article 8.
A carrier for delivering a biologically active substance or drug.
A gelatin-based zinc ion-releasing bioactive tissue adhesive hydrogel comprising zinc peroxide according to Article 8.
Implant materials for filling.