Pectin modified with gallol derivative and its use

Abstract

The present application provides a hydrogel that exhibits the same or better effects by utilizing pectin, a naturally occurring substance. Specifically, the application provides a hydrogel or patch that is stable, has strong adhesive strength, is favorable for drug delivery, allows for sustained treatment, has extremely low cytotoxicity, and is unlikely to induce an immune response, and can be used in many body sites, especially the oral cavity. The application also provides a hydrogel patch that employs a freezing method before crosslinking and exhibits superior adhesive strength compared to a freezing method after crosslinking.

Description

[Technical field]

[0001] The present invention relates to a pectin modified with a gallol derivative and uses thereof. [Background technology]

[0002] Globally, the market for functional medical material technology for medical procedures such as drug delivery, cell transplantation, hemostasis, and suturing is growing at a rapid pace. According to statistics, the controlled release drug delivery market is expected to grow at an average annual rate of 13.8% until 2025 due to the increasing trend of research and development and the need for elderly and pediatric patients. The global stem cell market is expected to grow rapidly from the level of 62.8 billion dollars in 2017 to 394.4 billion dollars in 2025.

[0003] Due to these prospects, the demand for materials for drug delivery and cell transplantation is expected to soar.In addition, the market for hemostasis and suture materials is estimated to grow to KRW 4.5 trillion by 2024, and is expected to grow further in the future.

[0004] Due to the need for biomaterials that have adhesive properties to the surfaces of various materials or biological tissues in stable drug delivery and cell transplantation into the body, as well as in stopping bleeding at bleeding sites and suturing wound tissue, various adhesive hydrogel technologies have been developed, some of which are also applied in clinical practice.

[0005] The oral cavity generally refers to the area from the lips to the beginning of the pharynx, and is composed of mucous membranes. The mucous membranes in the oral cavity maintain the homeostasis of the human body, play a role in the primary defense against external infections, and act as a passageway for the absorption of nutrients and drugs. If the mucous membrane is damaged, bacterial infection and loss of organ function due to drying of the underlying tissue of the mucous membrane are induced. Diseases that occur in the oral cavity include oral cancer, stomatitis, leukoplakia, hand, foot and mouth disease, oral tuberculosis, oral candidiasis, and periodontitis, and oral mucositis has recently emerged as the most serious side effect in the treatment of cancer patients. In addition, infections that occur in association with oral mucositis can induce systemic sepsis in a state where the immune system is weakened by anti-cancer treatment, which can lead to death. Such intractable oral ulcers reduce the quality of life of patients by inducing pain, reducing nutritional intake, and increasing the need for intravenous nutrition. In particular, neutropenia acts as a high risk factor for sepsis in cancer patients, and 10% of patients with severe mucositis die as a result. In addition, the recurrence rate of cancer is increasing due to reductions in the planned doses of anti-cancer and radiation therapy, or the cessation of treatment. Furthermore, if oral mucositis occurs, additional treatments and procedures such as prescription of narcotic painkillers to control pain, prescription of antibiotics to prevent infection, and intravenous nutrition are required, so if oral ulcers develop during anti-cancer treatment, total medical costs will increase by more than double. Over the past six years, the number of oral mucositis patients in Korea has increased by more than 30%, and the medical fee payment rate has increased by about 70%.

[0006] As described above, there are many different diseases that can occur in the oral cavity, including stomatitis, and the incidence of these diseases is also increasing, but the flow of saliva in the oral cavity makes it difficult to maintain drugs for a long period of time, and the oral cavity is also prone to bending, making it difficult to introduce drugs using a patch, etc. Therefore, in order to improve the efficiency of treating a wide range of oral diseases, it is urgently necessary to develop a mucoadhesive patch that can effectively adhere to the curved mucosal surface in the oral cavity and continuously release drugs for a long period of time.

[0007] To solve these problems, Korean Patent Registration Publication No. 10-1942220 provides a chitosan membrane with catechol groups and a manufacturing method thereof. This invention has the characteristics of being able to effectively adhere to the curved mucosal surface in the oral cavity, capture various drugs, and release the drugs continuously for a long period of time.

[0008] In addition, many prior art documents, such as Korean Patent Publication No. 10-2018-0127634, use a freeze-drying method after crosslinking to produce hydrogels, but this has the problem of reducing adhesive strength. Summary of the Invention [Problem to be solved by the invention]

[0009] In comparison with Korean Patent Registration Publication No. 10-1942220 and Korean Patent Publication No. 10-2018-0127634, the present application provides a hydrogel that exhibits the same or better effects by utilizing pectin, a naturally occurring substance. Specifically, the present application provides a hydrogel or patch that is stable, has strong adhesive strength, is favorable for drug delivery, allows for sustained treatment, has extremely low cytotoxicity, is unlikely to induce immune responses, and can be used in many body sites, especially the oral cavity. In addition, the present application provides a hydrogel patch that employs a freezing method before crosslinking and exhibits superior adhesive strength compared to a freezing method after crosslinking.

[0010] However, the subject of the present application is not limited to the above, and should be construed as including all subjects within the scope of what a person skilled in the art can understand. [Means for solving the problem]

[0011] In order to solve the above-mentioned problems, a first aspect of the present application provides a pectin derivative (Pec-PG) modified with a gallol group derivative.

[0012] A second aspect of the present application provides a hydrogel prepared by oxidizing the pectin derivative.

[0013] A third aspect of the present application provides a patch comprising the pectin derivative.

[0014] A fourth aspect of the present application provides a method for producing pectin modified with a gallol group derivative, the method comprising the step of reacting pectin, EDC, NHS and a gallol group derivative in a predetermined ratio.

[0015] This is merely one example of a means for solving the problem, and should be interpreted as including any means for solving the problem that would be understandable to a person of ordinary skill in the art. Effect of the Invention

[0016] The pectin modified with the gallol derivative of the present invention can adjust the degree of swelling and decomposition, and can control the delivery rate of the loaded drug by forming a strong bond between the mucosal tissue and the gallol group. It has low cytotoxicity and excellent biocompatibility, is unlikely to induce an immune response in the body, and is stable as a hydrogel, so it can be applied to various body parts, especially the oral cavity, where adhesion is difficult. [Brief description of the drawings]

[0017] [Figure 1a] FIG. 1 shows an example of a process for producing gallol group-modified pectin. [Figure 1b] FIG. 1 shows the results of an experiment that confirmed whether or not Pec-PG hydrogels could be formed depending on the concentration. [Figure 1c] FIG. 13 is a diagram showing the experimental results in which a concentration suitable for spray molding was confirmed. [Figure 2a] FIG. 13 is a diagram showing the results of an analysis of the kinetics of hydrogel formation upon crosslinking depending on the source of pectin in Example 2. [Figure 2b] FIG. 13 is a diagram showing the results of an analysis of the kinetics of hydrogel formation upon crosslinking depending on the source of pectin in Example 2. [Figure 3a]FIG. 13 shows the results of analysis of the physical properties of the gallol group-modified pectin hydrogel, including (a) the swelling degree and (b) the decomposition degree, according to Example 3. [Figure 3b] FIG. 13 shows the results of analysis of the physical properties of the gallol group-modified pectin hydrogel, including (a) the swelling degree and (b) the decomposition degree, according to Example 3. [Figure 4a] FIG. 1 shows (a) the internal structure of the hydrogel as determined through scanning electron microscope (SEM) analysis, Hematoxylin & Eosin (H&E) staining, and Toluidine blue (TB) staining in Example 4, and (b) the pore size in the hydrogel as determined through Porosimeter analysis. [Figure 4b] FIG. 1 shows (a) the internal structure of the hydrogel as determined through scanning electron microscope (SEM) analysis, Hematoxylin & Eosin (H&E) staining, and Toluidine blue (TB) staining in Example 4, and (b) the pore size in the hydrogel as determined through Porosimeter analysis. [Figure 5a] FIG. 13 shows (a) the results of Fourier-transform infrared spectroscopy (FT-IR) analysis and (b) the results of UV-vis spectrophotometry analysis in Example 5. [Figure 5b] FIG. 13 shows (a) the results of Fourier-transform infrared spectroscopy (FT-IR) analysis and (b) the results of UV-vis spectrophotometry analysis in Example 5. [Figure 6a] FIG. 13 shows the results of measuring the elastic modulus of a hydrogel in frequency sweep mode using a rheological analyzer according to Example 6. [Figure 6b] FIG. 13 shows the results of measuring the elastic modulus of a hydrogel in frequency sweep mode using a rheological analyzer according to Example 6. [Figure 7a]FIG. 13 shows the results of an atomic force microscopy (AFM) analysis performed in Example 7 to analyze the manner in which Pec-PG reacts with mucin on the surface of the mucosal layer (a, b), the results of a UV-vis spectrophotometry analysis (c), and the results of an analysis of the zeta potential of a mixture of Pec-PG and mucin (d). [Figure 7b] FIG. 13 shows the results of an atomic force microscopy (AFM) analysis performed in Example 7 to analyze the manner in which Pec-PG reacts with mucin on the surface of the mucosal layer (a, b), the results of a UV-vis spectrophotometry analysis (c), and the results of an analysis of the zeta potential of a mixture of Pec-PG and mucin (d). [Figure 7c] FIG. 13 shows the results of an atomic force microscopy (AFM) analysis performed in Example 7 to analyze the manner in which Pec-PG reacts with mucin on the surface of the mucosal layer (a, b), the results of a UV-vis spectrophotometry analysis (c), and the results of an analysis of the zeta potential of a mixture of Pec-PG and mucin (d). [Figure 7d] FIG. 13 shows the results of an atomic force microscopy (AFM) analysis performed in Example 7 to analyze the manner in which Pec-PG reacts with mucin on the surface of the mucosal layer (a, b), the results of a UV-vis spectrophotometry analysis (c), and the results of an analysis of the zeta potential of a mixture of Pec-PG and mucin (d). [Figure 8] FIG. 11 is a diagram showing the results of comparing the release patterns of the protein BSA (bovine serum albumin) in Example 8. [Figure 9a] FIG. 13 shows the results of Example 9, in which (a) cell viability was confirmed using the Live / Dead staining method, and (b) the proliferation rate of stem cells cultured in Pec-PG hydrogel was confirmed using the MTT assay. [Figure 9b]FIG. 13 shows the results of Example 9, in which (a) cell viability was confirmed using the Live / Dead staining method, and (b) the proliferation rate of stem cells cultured in Pec-PG hydrogel was confirmed using the MTT assay. [Figure 10a] FIG. 10 shows the results of comparing the amount of TNF-α secreted between the Pec-PG hydrogel of the present application and the control group (a), and the results of confirming the immune response using mice through H&E staining (b) and TB staining (c) in accordance with Example 10. [Figure 10b] FIG. 10 shows the results of comparing the amount of TNF-α secreted between the Pec-PG hydrogel of the present application and the control group (a), and the results of confirming the immune response using mice through H&E staining (b) and TB staining (c) in accordance with Example 10. [Figure 10c] FIG. 10 shows the results of comparing the amount of TNF-α secreted between the Pec-PG hydrogel of the present application and the control group (a), and the results of confirming the immune response using mice through H&E staining (b) and TB staining (c) in accordance with Example 10. [Figure 11a] FIG. 11 shows the results of comparing the weights of the major organs of rats with those of a control group in an oral administration experiment to evaluate the safety of the hydrogel of the present application (Example 11) (a), and the results of histological analysis through H&E staining (b). [Figure 11b] FIG. 11 shows the results of comparing the weights of the major organs of rats with those of a control group in an oral administration experiment to evaluate the safety of the hydrogel of the present application (Example 11) (a), and the results of histological analysis through H&E staining (b). [Figure 12a] FIG. 12 shows the results of confirming the tissue adhesive strength of the hydrogel of the present application using pig tongue tissue (a), the results of confirming the strength using tongue tissue from a living rat (b), and the results of confirming the strength when a hydrogel mixed with a fluorescent substance was applied to rat tongue tissue (c) from Example 12. [Figure 12b] FIG. 12 shows the results of confirming the tissue adhesive strength of the hydrogel of the present application using pig tongue tissue (a), the results of confirming the strength using tongue tissue from a living rat (b), and the results of confirming the strength when a hydrogel mixed with a fluorescent substance was applied to rat tongue tissue (c) from Example 12. [Figure 12c] FIG. 12 shows the results of confirming the tissue adhesive strength of the hydrogel of the present application using pig tongue tissue (a), the results of confirming the strength using tongue tissue from a living rat (b), and the results of confirming the strength when a hydrogel mixed with a fluorescent substance was applied to rat tongue tissue (c) from Example 12. [Figure 13a] FIG. 13 shows the experimental results of Example 13, which confirmed the water evaporation rate and moisturizing function for a Petri dish (a), a sponge (b), and the tongue tissue of a living rat (c and d). [Figure 13b] FIG. 13 shows the experimental results of Example 13, which confirmed the water evaporation rate and moisturizing function for a Petri dish (a), a sponge (b), and the tongue tissue of a living rat (c and d). [Figure 13c] FIG. 13 shows the experimental results of Example 13, which confirmed the water evaporation rate and moisturizing function for a Petri dish (a), a sponge (b), and the tongue tissue of a living rat (c and d). [Figure 13d] FIG. 13 shows the experimental results of Example 13, which confirmed the water evaporation rate and moisturizing function for a Petri dish (a), a sponge (b), and the tongue tissue of a living rat (c and d). [Figure 14a] FIG. 15 shows the results of analysis of the swelling degree (b) and decomposition degree (c) of a patch according to the present application, according to Example 14. [Figure 14b] FIG. 15 shows the results of analysis of the swelling degree (b) and decomposition degree (c) of a patch according to the present application, according to Example 14. [Figure 14c] FIG. 15 shows the results of analysis of the swelling degree (b) and decomposition degree (c) of a patch according to the present application, according to Example 14. [Figure 15a] FIG. 15 shows the results of confirming the pore size of the patch by Porosimeter analysis (a) and the results of comparing the pore size between the hydrogel and the patch (b). [Figure 15b]FIG. 15 shows the results of confirming the pore size of the patch by Porosimeter analysis (a) and the results of comparing the pore size between the hydrogel and the patch (b). [Figure 16a] FIG. 16 shows the results of measuring the elastic modulus of a hydrogel patch using a rheology analyzer according to Example 16. [Figure 16b] FIG. 16 shows the results of measuring the elastic modulus of a hydrogel patch using a rheology analyzer according to Example 16. [Figure 17a] FIG. 17 shows the results of comparing the tissue adhesive strength of hydrogel patches through a Tack test analysis according to Example 17. [Figure 17b] FIG. 17 shows the results of comparing the tissue adhesive strength of hydrogel patches through a Tack test analysis according to Example 17. [Figure 18] FIG. 13 shows the results of confirming the drug release profile through HPLC analysis in Example 18. [Figure 19a] FIG. 13 shows the results of analyzing antioxidant function in Example 19. [Figure 19b] FIG. 13 shows the results of analyzing antioxidant function in Example 19. [Figure 19c] FIG. 13 shows the results of analyzing antioxidant function in Example 19. [Figure 20a] FIG. 2 shows the results of confirming the effect of gum regeneration and inflammation treatment in rats in Example 20 (a); the results of confirming the area of ​​the damaged area and the effect of tissue regeneration (b, c); the results of H&E staining, immunostaining for epithelial tissue markers (cytokeratin 5; CK5, cytokeratin 13; CK13) and quantitative analysis based on the stained images (d to f); and the result of confirming the number of inflammatory cells through immunostaining for leukocyte marker CD11b (g). [Figure 20b]FIG. 2 shows the results of confirming the effect of gum regeneration and inflammation treatment in rats in Example 20 (a); the results of confirming the area of ​​the damaged area and the effect of tissue regeneration (b, c); the results of H&E staining, immunostaining for epithelial tissue markers (cytokeratin 5; CK5, cytokeratin 13; CK13) and quantitative analysis based on the stained images (d to f); and the result of confirming the number of inflammatory cells through immunostaining for leukocyte marker CD11b (g). [Figure 20c] FIG. 2 shows the results of confirming the effect of gum regeneration and inflammation treatment in rats in Example 20 (a); the results of confirming the area of ​​the damaged area and the effect of tissue regeneration (b, c); the results of H&E staining, immunostaining for epithelial tissue markers (cytokeratin 5; CK5, cytokeratin 13; CK13) and quantitative analysis based on the stained images (d to f); and the result of confirming the number of inflammatory cells through immunostaining for leukocyte marker CD11b (g). [Figure 20d] FIG. 2 shows the results of confirming the effect of gum regeneration and inflammation treatment in rats in Example 20 (a); the results of confirming the area of ​​the damaged area and the effect of tissue regeneration (b, c); the results of H&E staining, immunostaining for epithelial tissue markers (cytokeratin 5; CK5, cytokeratin 13; CK13) and quantitative analysis based on the stained images (d to f); and the result of confirming the number of inflammatory cells through immunostaining for leukocyte marker CD11b (g). [Figure 20e] FIG. 2 shows the results of confirming the effect of gum regeneration and inflammation treatment in rats in Example 20 (a); the results of confirming the area of ​​the damaged area and the effect of tissue regeneration (b, c); the results of H&E staining, immunostaining for epithelial tissue markers (cytokeratin 5; CK5, cytokeratin 13; CK13) and quantitative analysis based on the stained images (d to f); and the result of confirming the number of inflammatory cells through immunostaining for leukocyte marker CD11b (g). [Fig. 20f]FIG. 2 shows the results of confirming the effect of gum regeneration and inflammation treatment in rats in Example 20 (a); the results of confirming the area of ​​the damaged area and the effect of tissue regeneration (b, c); the results of H&E staining, immunostaining for epithelial tissue markers (cytokeratin 5; CK5, cytokeratin 13; CK13) and quantitative analysis based on the stained images (d to f); and the result of confirming the number of inflammatory cells through immunostaining for leukocyte marker CD11b (g). [Figure 20g] FIG. 2 shows the results of confirming the effect of gum regeneration and inflammation treatment in rats in Example 20 (a); the results of confirming the area of ​​the damaged area and the effect of tissue regeneration (b, c); the results of H&E staining, immunostaining for epithelial tissue markers (cytokeratin 5; CK5, cytokeratin 13; CK13) and quantitative analysis based on the stained images (d to f); and the result of confirming the number of inflammatory cells through immunostaining for leukocyte marker CD11b (g). [Figure 21a] FIG. 1 shows the results of analyzing the reactivity and interaction between Mucin protein and each of its derivatives by atomic force microscopy (AFM) according to Comparative Example 1. [Figure 21b] FIG. 1 shows the results of analyzing the reactivity and interaction between Mucin protein and each of its derivatives by atomic force microscopy (AFM) according to Comparative Example 1. [Figure 21c] 21a and 21b show the results of comparing the mucoadhesion of a Pec-PG hydrogel patch, an HA-PG hydrogel patch, and a Chi-CA hydrogel patch to pig gingival mucosa tissue through an actual Tack test analysis. [Figure 22] FIG. 2 shows the results of comparing the manufacturing methods of a gallol group-modified pectin hydrogel patch with those of the prior art in Comparative Example 2. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] Hereinafter, the present application will be described in detail with reference to the accompanying drawings so that a person having ordinary skill in the art to which the present application pertains can easily carry out the present application. However, the present application may be embodied in various different forms and is not limited to the embodiments described herein. In the drawings, in order to clearly explain the present application, parts that are not related to the description are omitted, and similar parts are designated by similar reference numerals throughout the specification.

[0019] Throughout the specification of this application, when a member is said to be located "on" another member, this does not only include the case where one member is in contact with the other member, but also the case where there is another member between the two members.

[0020] Throughout the specification of this application, when a part is said to "comprise" a certain element, this does not mean that it excludes other elements, but that it may further include other elements, unless specifically stated to the contrary.

[0021] The terms "about," "substantially," and the like, used throughout the specification of this application, when the manufacturing and material tolerances inherent in the recited meaning are presented, are used to mean at or near the numerical value, and are used to prevent unscrupulous infringers from unfairly using the disclosure in which precise or absolute numerical values ​​are recited to aid in the understanding of this application. The terms "step of" or "step of" used throughout the specification of this application do not mean "step for".

[0022] Throughout the specification of this application, the term "combinations thereof" contained in a Markush form expression means a mixture or combination of one or more selected from the group of elements set forth in the Markush form expression, and is meant to include one or more selected from the group of elements.

[0023] Throughout the specification of this application, the statement "A and / or B" means "A or B, or A and B."

[0024] Throughout this specification, any references to a substance or the like are by way of example only and are not meant to limit the substance to that particular reference.

[0025] Throughout this specification, "pectin" refers to a polysaccharide that is a hydrated gel surrounding the cellulose-hemicellulose network and is composed mainly of galacturonic acid, an oxide of galactose. It acts as a hydrophilic filler that prevents the network from building up and collapsing, and is responsible for determining the porosity of the cell wall for polymers. Its main portion is composed of α-1,4 bonds of D-galacturonic acid units.

[0026] Throughout the present specification, the term "gallol derivative" refers to pyrogallol, which is represented as C6H3(OH)3, or a compound having the structure of Formula 1.

[0027] [ka]

[0028] Throughout the specification of this application, "stomatitis" refers to inflammation occurring inside the mouth, and is a general term for inflammatory diseases that generally occur in the mucous membranes in the oral cavity (such as the tongue, gums, lips, and inside the cheeks). Causes include bacterial, viral, and fungal infections, and in severe cases of inflammation, blisters, ulcers, and the like may occur. Examples of stomatitis include oral mucositis, oral cancer, stomatitis, leukoplakia, hand, foot, and mouth disease, oral tuberculosis, oral candidiasis, mucosal wounds, periodontitis, aphthous ulcers, Behcet's syndrome, lichen planus, and herpes stomatitis, and are interpreted to include any inflammation occurring inside the oral cavity.

[0029] Throughout the specification of this application, the term "patch" refers to a membrane that can be attached to the mucosal surface in the oral cavity, and which can contain an additional therapeutic drug and release the drug over an extended period of time, and can be used to treat disease, but can also be used in treatment by attaching it to the surface of a wound in tissue without containing any additional drug, thereby suppressing further infection at the wound site, etc.

[0030] Throughout the present specification, "adhesion" may refer primarily to the binding between the mucosal tissue and the phenolic derivatives. Possible binding may be a variety of physical non-covalent bonds, such as covalent bonds, hydrogen bonds, hydrophobic interactions, and pi-pi interactions.

[0031] The first aspect of the present application provides a pectin derivative (Pec-PG) modified with a gallol group derivative. Due to the excellent tissue adhesiveness of the gallol group, the Pec-PG derivative is expected to be applicable to various medical and biomedical engineering fields, and is particularly useful as an artificial saliva or oral disease drug delivery medium, where adhesion to mucous membranes is important. In addition, due to the spontaneous oxidation ability of the gallol group, Pec-PG in a solution state can be sprayed or applied to the oral cavity to coat or adhere to areas of oral disease that are difficult to reach.

[0032] Here, the gallol group derivatives include 5-hydroxydopamine, tannic acid, gallic acid, epigallocatechin, epicatechin gallate, 2,3,4-trihydroxybenzaldehyde, 2,3,4-trihydroxybenzoic acid, The compound may be any one selected from the group consisting of 3,4,5-trihydroxybenzaldehyde, 3,4,5-trihydroxybenzamide, 5-tert-butylpyrogallol, and 5-methylpyrogallol.

[0033] The second aspect of the present application provides a hydrogel prepared by oxidizing the pectin derivative of the present application. The hydrogel of the present application has all the characteristics of the first aspect, and in particular, since the hydrogel of the present application is capable of sustained release of the loaded drug through excellent reactivity with various nucleophilic functional groups of the oxidized gallol group, it can be applied to various diseases and sites depending on the hydrogel formulation.

[0034] The oxidation of the hydrogel may be by the addition of an oxidizing agent, an enzyme, or a pH adjusting agent. The oxidizing agent may be periodate salt or hydrogen peroxide, the enzyme may be any one selected from the group consisting of peroxidase, horseradish peroxidase, and tyrosinase, and the pH adjusting agent may be any one selected from the group consisting of sodium hydroxide, lithium hydroxide, potassium hydroxide, rubidium hydroxide, cesium hydroxide, magnesium hydroxide, calcium hydroxide, strontium hydroxide, and barium hydroxide.

[0035] Hydrogel oxidation can also occur naturally, which means that Pec-PG in solution form can be sprayed or applied to the oral cavity, coating or adhering to areas of the oral cavity that are difficult to reach.

[0036] The application site of the hydrogel according to the present application may include the oral mucosa, nasal mucosa, ocular mucosa, gastric mucosa, intestinal mucosa, bronchial mucosa, heart, lungs, or urinary tract, and thus can be used to treat mucosal tissue diseases, particularly oral mucositis, oral cancer, stomatitis, leukoplakia, hand, foot, and mouth disease, oral tuberculosis, oral candidiasis, mucosal wounds, periodontitis, aphthous ulcers, Behcet's syndrome, lichen planus, or herpetic stomatitis.

[0037] In addition, the hydrogel according to the present application can be used to regulate and deliver drugs in vivo, where the drug can be encapsulated or carried in the hydrogel, and the drug can include at least one selected from the group consisting of DNA, mRNA, siRNA, miRNA, antisense oligonucleotide, antihistamines, proteins (growth factors), corticosteroid-based steroids, immune cell activators, anticancer drugs, therapeutic antibodies, antibiotics, antibacterial agents, antiviral agents, anti-inflammatory agents, protein drugs, growth factors, cytokines, peptide drugs, and anesthetics.

[0038] The anticancer drugs include anthracyclines such as doxorubicin, daunomycin, epirubicin, and idarubicin; taxanes such as paclitaxel, docetaxel, cabazitaxel, and tesetaxel; alkaloids such as cocumin, campothecin, berberine, evodiamine, matrine, piperine, sanguinarine, tetrandrine, thalicarpine, and ellipticine; vinca alkaloids such as vinblastine, vincristine, vindesine, and vinorelbine; alkaloids; platinums such as cisplatin, caboplatin, and oxaliplatin; antimetabolites such as 5-fluorouracil, capecitabine, methotrexate, and gemcitabine; topoisomerase inhibitors such as irinotecan, topotecan, etoposide, teniposide, etoposide, and amsacrine; antitumor antibiotics such as bleomycin, actinomycin, mitomycin, and mitoxantrone; alkylating agents such as cyclophosphamide, mechlorethamine, chlorambucil, melphalan, and nitrosourea; azacitidine, azathioprine, cytarabine, and doxifluridine.

[0039] A third aspect of the present application provides a patch containing the pectin derivative according to the present application. The patch according to the present application can be manufactured by thinly freezing a Pec-PG solution and then freeze-drying it to form a patch, and a desired drug can be pre-loaded in a hydrogel or patch to deliver the drug. In particular, existing prior art documents such as Korean Patent Publication No. 10-2018-0127634 mainly manufacture patches by freeze-drying after cross-linking, but the present application adopts a freeze-drying method before cross-linking. Due to this difference in method, the patch of the present application can have significantly better adhesive strength than existing patches.

[0040] Furthermore, the drug-preloaded Pec-PG hydrogel patch is very convenient for users and can be easily applied in vivo.

[0041] A fourth aspect of the present application provides a method for producing pectin modified with a gallol group derivative.

[0042] The above-mentioned preparation method may include a step of reacting pectin, EDC, NHS, and a gallol group derivative in a predetermined ratio, which may be, but is not limited to, pectin:EDC:NHS:PG=1:2:2:2.

[0043] In the first to fourth aspects of the present application, any common parts in each aspect are applicable to all of them.

[0044] Hereinafter, the present application will be described in detail with reference to the accompanying drawings, in which: However, the present application is not limited to the following embodiments and examples.

[0045] Example 1. Synthesis of gallol-modified pectin derivatives, preparation of hydrogels, and confirmation of suitable concentrations

[0046] Pectin (Pec), which is widely used as a food additive and food supplement, was modified with a gallol group (PG) to synthesize a pectin derivative, and Pec-PG hydrogel was produced based on this.

[0047] The gallol group was 5-hydroxydopamine, and a pectin derivative (Pec-PG) was synthesized using the 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) / N-Hydroxysuccinimide (NHS) chemical reaction in the molar ratio Pectin:EDC:NHS:PG=1:2:2:2 (see Figure 1a).

[0048] In the case of Pec-PG concentration, Pec-PG with a concentration of less than 1% (w / v) did not form a hydrogel when treated with 4.5 mg / ml NaIO4 oxidizing agent, and at a concentration of 1%, a hydrogel with very weak physical properties was formed, with the structure easily collapsing even with slight vibration. Since a stable hydrogel was formed from Pec-PG with a concentration of 2% (w / v), Pec-PG with a concentration of 2% (w / v) or more was used in the examples of this application (see Figure 1b). However, this does not mean that the concentration of Pec-PG in this application is limited.

[0049] In addition, to confirm the appropriate concentration for molding for spraying, an experiment was conducted in which Pec-PG solutions of various concentrations were sprayed through the nozzle of a sprayer. The higher the concentration of Pec-PG, the more force was required to spray (see Figure 1c; left).

[0050] As the concentration of Pec-PG increased, there was a significant difference in the amount of sprayed each time it was sprayed. In particular, in the case of 4% Pec-PG, the amount sprayed decreased significantly after three or more sprays. This suggests that the ease of spraying decreases as the concentration of Pec-PG increases (see Figure 1c; right).

[0051] Therefore, it was confirmed that 2% Pec-PG is suitable for spray molding. This does not mean that other concentrations of Pec-PG cannot be used, but simply that 2% is suitable for spray molding.

[0052] Example 2. Analysis of hydrogel formation kinetics upon crosslinking depending on the source of pectin

[0053] The hydrogel formation kinetics during crosslinking was analyzed depending on the origin of pectin. Citrus peel-derived pectin and apple-derived pectin were modified with gallol groups (PG) via EDC / NHS reaction, and the gelation kinetics were compared when the synthesized pectin derivatives were crosslinked to form hydrogels.

[0054] The process of hydrogel formation was analyzed when 2% (w / v) citrus Pec-PG pre-gel solution and 2% (w / v) apple Pec-PG pre-gel solution were mixed with 4.5 mg / ml NaIO4 oxidant to induce crosslinking.

[0055] In the 2% (w / v) citrus Pec-PG pre-gel solution, the loss modulus (G'') was higher than the storage modulus (G'), but when an oxidizing agent was added for cross-linking, it was confirmed that G' became higher than G''. This means that Pec-PG derived from citrus peel can stably form a hydrogel (see Figure 2a).

[0056] In the 2% (w / v) apple Pec-PG pre-gel solution, the loss modulus (G'') remained higher than the storage modulus (G'), and no change occurred even when an oxidizing agent was added for cross-linking. This means that apple-derived Pec-PG does not easily form hydrogel (see Figure 2b).

[0057] In other words, it was confirmed that because the structure of pectin differs depending on the origin, hydrogel may not be formed even if the gallol group is modified. In addition, it was confirmed that the degree of esterification of pectin and the amount of galacturonic acid, which has a functional group (COOH group) that can be modified, can affect the synthesis of Pec-PG derivatives. In fact, pectin derived from citrus peel contains more than 70% galacturonic acid, so there are sufficient functional groups for gallol group modification and there are no problems with the synthesis of Pec-PG derivatives and the formation of hydrogel. On the other hand, pectin derived from apple has a relatively low ratio of galacturonic acid and is highly esterified, making it difficult to synthesize Pec-PG derivatives and form hydrogel.

[0058] Example 3. Analysis of physical properties of gallol-modified pectin hydrogels

[0059] Two commonly used concentrations of Pec-PG pre-gel solutions (2% (w / v) and 4% (w / v)) were mixed with 4.5 mg / ml NaIO4 oxidant to induce crosslinking and prepare hydrogels, and the swelling ratio of each hydrogel was then measured.

[0060] When kept under physiological conditions (37°C in PBS buffer) for 7 days, the 2% (w / v) hydrogel showed a slightly higher swelling degree than the 4% (w / v) hydrogel by the fourth day (see Figure 3a), suggesting that the lower concentration resulted in a lower degree of polymer cross-linking and therefore a higher swelling degree.

[0061] To compare the degradation patterns of Pec-PG hydrogels, treatment with pectin-degrading enzyme was performed. When treated with the same concentration of pectin-degrading enzyme (1 U / sample), it was confirmed that the hydrogel decomposition occurred more slowly at higher concentrations because the ratio of cross-linked gallol groups in the 4% (w / v) hydrogel was higher (see Figure 3b).

[0062] In other words, it was confirmed that the physical properties such as the swelling degree and decomposition behavior of Pec-PG hydrogels can be controlled by adjusting the concentration of Pec-PG.

[0063] Example 4. Analysis of the internal structure of gallol-modified pectin hydrogel

[0064] Hydrogels were prepared by mixing 2% (w / v) Pec-PG pre-gel solution with 4.5 mg / ml NaIO4 oxidant.

[0065] The internal structure of the hydrogel was examined using a scanning electron microscope (SEM), hematoxylin & eosin (H&E) staining, and toluidine blue (TB) staining, and it was found to have a dense porous structure at the micro level (see Figure 4a).

[0066] The pore size in the hydrogel was confirmed through porosimeter analysis, and it was found that many pores with a size of about 100 μm were distributed (see FIG. 4b).

[0067] Since the hydrogel according to the present application has such a porous structure, it can provide a favorable environment for cell attachment and proliferation, and can also be effective in loading and releasing drugs.

[0068] Example 5. Analysis of the chemical properties of gallol-modified pectin hydrogels

[0069] Through Fourier-transform infrared spectroscopy (FT-IR) analysis, the chemical structure of Pec-PG (1 mg / ml) before (Pec-PG) and after (Pec-PG+NaIO4) oxidation was analyzed (see Figure 5a).

[0070] ~3435cm indicating OH group -1 All peaks were observed.

[0071] ~2925cm, showing CH stretching group -1 All peaks were observed.

[0072] Ester carbonyl stretching group ~1743cm -1 All peaks were observed.

[0073] Carboxylate ion stretching group ~1620cm -1 peak and ~1440cm -1 All peaks were observed.

[0074] Galacturonan backbone ~1100cm -1 peak and ~1020cm -1 All peaks were observed.

[0075] 784cm showing the formation of biphenol and biphenyl ether -1 The peak was observed only in oxidized Pec-PG.

[0076] Through this, cross-linking between oxidized gallol groups (PG) was confirmed only in Pec-PG treated with an oxidizing agent.

[0077] Through UV-vis spectrophotometry analysis, we added an oxidant (NaIO4) to the Pec-PG solution and analyzed the chemical reactions that occurred after oxidation (see Figure 5b).

[0078] After the oxidizing agent was added, the peaks at 280nm and 350nm increased, confirming that the gallol group (PG) was oxidized to form semi-quinone and phenoxyl radical, through which cross-linking occurred.

[0079] Example 6. Analysis of mechanical properties of gallol-modified pectin hydrogel

[0080] Hydrogels were fabricated by mixing two different concentrations (2% (w / v), 4% (w / v)) of Pec-PG pre-gel solutions with 4.5 mg / ml NaIO4 oxidant.

[0081] The elastic modulus of the hydrogel was measured using a rheological analyzer in frequency sweep mode (see Figure 6a).

[0082] The storage modulus (G') was consistently higher than the loss modulus (G'') for both 2% (w / v) and 4% (w / v) Pec-PG hydrogels, confirming the formation of a stable polymer network structure inside the hydrogel.

[0083] The average elastic modulus of the 2% (w / v) Pec-PG hydrogel was approximately 1.7 kPa, whereas the 4% (w / v) Pec-PG hydrogel had an average elastic modulus of approximately 4.9 kPa, confirming an increase in the elastic modulus relative to the concentration of Pec-PG (see Figure 6 b).

[0084] In other words, it was confirmed that the mechanical properties of the Pec-PG hydrogel could be adjusted by adjusting the concentration of Pec-PG.

[0085] Example 7. Analysis of adhesion of gallol-modified pectin to mucosal surfaces

[0086] Atomic force microscopy (AFM) analysis was performed to analyze the manner in which Pec-PG reacts with mucin on the surface of the mucosal layer.

[0087] When mucin itself or mucin was mixed with pectin (2mg / ml pectin), the formation of small aggregates was observed on the surface, and when mucin was mixed with 2mg / ml Pec-PG, it was confirmed that significant large aggregates were formed in layers on the surface (see Figures 7a and 7b). This adhesion occurs through strong binding reactions and interactions between the gallol groups of Pec-PG and the nucleophilic functional groups of mucin present in the mucosal tissue.

[0088] The Pec-PG solution was mixed with mucin present on the surface of the mucous layer, and the chemical reaction occurring between mucin and gallol groups (PG) was analyzed through UV-vis spectrophotometry. When 0.5mg / ml Pec-PG was mixed with 0.5mg / ml mucin, the peaks at ~280nm and ~350nm increased. This confirmed the occurrence of strong covalent bonds between the gallol groups and the nucleophilic functional groups of mucin present in the mucosal tissue (see Figure 7c).

[0089] A zeta potential analysis was performed to analyze the reaction of Pec-PG with mucin on the surface of the mucous layer over time. Mucin was mixed with 2% (w / v) Pec-PG, and it was confirmed that as time passed, the zeta potential of the reactant gradually became more negatively charged due to the strong binding reaction and interaction between the gallol group of Pec-PG and the nucleophilic functional group of mucin. This means that a strong covalent bond was formed between the gallol group of Pec-PG and the nucleophilic functional group of mucin (see Figure 7d).

[0090] Therefore, the Pec-PG of the present application can be stably adhered to mucosal tissue.

[0091] Example 8. Analysis of drug release profile of gallol-modified pectin hydrogel

[0092] To verify the sustained-release drug delivery function of the Pec-PG hydrogel, the release profile of the protein BSA (bovine serum albumin) was compared under physiological conditions (37°C in PBS buffer) for the following two groups (see Figure 8).

[0093] 1) BSA loaded onto 2% (w / v) Pec-PG hydrogel (Pec-PG2%)

[0094] 2) BSA loaded onto 4% (w / v) Pec-PG hydrogel (Pec-PG4%)

[0095] The drug release profile was confirmed through BCA assay analysis, and it was observed that Pec-PG hydrogel exhibited sustained drug release behavior due to the strong binding between the gallol group and various nucleophilic functional groups of the protein.

[0096] The degradation rate of the 2% Pec-PG hydrogel was slightly faster, and BSA was released more quickly. The 4% Pec-PG hydrogel released BSA over a longer period of time than the 2% Pec-PG hydrogel.

[0097] Therefore, it is expected that effective drug delivery can be achieved by using drug-loaded Pec-PG hydrogel in areas where effective delivery is difficult, such as areas with oral diseases, and that the concentration can be adjusted as needed to induce sustained treatment for each tissue.

[0098] Example 9. Evaluation of cytotoxicity of gallol-modified pectin hydrogel

[0099] Human adipose-derived stem cells (hADSCs) were encapsulated in a Pec-PG pre-gel solution, and 4.5 mg / ml NaIO4 oxidant was added to induce crosslinking, after which the stem cells were three-dimensionally cultured in the Pec-PG hydrogel.

[0100] The cell viability was confirmed by live / dead staining after culturing for 7 days. The cell viability was consistently over 94% throughout the culture period, confirming that the Pec-PG hydrogel has no cytotoxicity and excellent biocompatibility (see Figure 9a).

[0101] The proliferation rate of stem cells cultured in the Pec-PG hydrogel was confirmed through MTT analysis, and it was confirmed that the stem cells proliferated normally during the 7-day culture period (see Figure 9b).

[0102] Therefore, Pec-PG hydrogel does not exhibit cytotoxicity and has excellent biocompatibility.

[0103] Example 10. Evaluation of immune response of gallol group-modified pectin hydrogel

[0104] The degree of induction of immune response was confirmed by measuring the infectious cytokine TNF-α (tumor necrosis factor-α) secreted from macrophages through blank culture of macrophages and Pec-PG hydrogel using ELISA analysis.

[0105] Macrophages were cultured on the bottom of a well-plate, and a transwell was placed on top of it. A 2% (w / v) Pec-PG hydrogel was placed on top of the transwell, and an empty culture was performed in a state where the cells were only affected by the components eluted from the hydrogel without any direct contact with the cells. LPS (lipopolysaccharide) was used as a control.

[0106] As a result, when cultured with Pec-PG hydrogel, the amount of TNF-α secreted by macrophages was similar to that of the no-treatment group, showing a clear difference from the control group (see Figure 10a).

[0107] In another experiment, a 2% (w / v) Pec-PG pre-gel solution was injected subcutaneously into mice without treatment with an oxidizing agent, and crosslinking was induced through natural oxidation due to the oxidative environment present in the body. The tissues into which the hydrogel was injected were harvested on the 3rd, 7th, and 14th days for histological analysis.

[0108] As a result, H&E staining showed that the tissue into which the hydrogel was injected was not significantly different from normal tissue, and it was confirmed that no serious immune reaction had occurred, and that surrounding cells had flowed into the hydrogel (see Figure 10b).

[0109] TB staining confirmed that the tissue into which the hydrogel was injected had almost no mast cells, just like normal tissue, confirming that no immune reaction to the foreign substance had occurred (see Figure 10c).

[0110] Therefore, Pec-PG hydrogel has a very low potential to induce an immune response in tissues.

[0111] Example 11. Evaluation of safety after ingestion of gallol-modified pectin hydrogel

[0112] Due to the nature of the product being applied to the oral cavity, to evaluate the safety of ingested Pec-PG hydrogel, a 2% (w / v) Pec-PG pre-gel solution or PBS buffer was orally administered to rats daily for 28 days, and the rats' body weights and amounts of food ingested were periodically measured and compared during the feeding period (see Figure 11a).

[0113] As a result, no significant difference was observed in body weight and in the weight of food consumed between the group that received the PBS buffer and the group that received the Pec-PG solution.

[0114] In addition, after ingestion of the Pec-PG hydrogel, the major organs of the rats (heart, liver, spleen, lungs, and height) were removed and weighed on the 28th day, and histological analysis was performed through H&E staining (see Figure 11b).

[0115] As a result, there was no significant difference in organ weight between the groups that received PBS and those that received Pec-PG hydrogel (top figure), and the results of histological analysis showed that no tissue damage or immune reaction occurred (bottom figure).

[0116] Therefore, it is expected that when Pec-PG hydrogel is applied to humans, it can be applied without any major safety issues.

[0117] Example 12. Measurement of tissue adhesive strength of gallol group-modified pectin hydrogel

[0118] Through a tack test using pig tongue tissue, the tissue adhesive strength of 2% (w / v) and 4% (w / v) Pec-PG hydrogels was compared with commercially available artificial saliva products, Control 1 (product name: Xerova; Kolmar) and Control 2 (product name: Biotene; GSK) (see Figure 12a).

[0119] Pig tongue tissue was attached to the plate and probe of a rheological analyzer, and then Pec-PG hydrogel cross-linked through natural oxidation or a conventional artificial saliva product was applied between the tissues to adhere them to each other. The force at which the tissues were attached and detached was measured while pulling the tissue.

[0120] The adhesion strength of Control 1 and Control 2 was measured at about 0.2~0.5 kPa, confirming that the tissue adhesion strength was very low. In comparison, the Pec-PG hydrogel was confirmed to have excellent tissue adhesion strength at about 6~6.5 kPa.

[0121] The adhesive strength of 2% (w / v) and 4% (w / v) Pec-PG hydrogels was compared with commercially available artificial saliva products, Control 1 (product name Xerova; Kolmar) and Control 2 (product name Biotene; GSK), on tongue tissue from live rats.

[0122] A liquid Pec-PG solution was sprayed onto rat tongue tissue, and crosslinking was induced and adhered through natural oxidation in the body without any additional oxidizing agent treatment. After 30 minutes, the tissue was collected and subjected to histological analysis (see Figure 12b).

[0123] In the case of Control 1 and Control 2 products, none of the products remained on the tongue tissue; the 2% (w / v) Pec-PG hydrogel remained as a thin coating (arrow), and the 4% (w / v) Pec-PG hydrogel remained as an even thicker coating (arrow).

[0124] The hydrogel mixed with the fluorescent substance was applied to rat tongue tissue and washed with PBS buffer, and the adhesive strength was confirmed under conditions that mimicked the actual oral environment in which saliva is present (see Figure 12c).

[0125] In the Control 1 and Control 2 groups, it was confirmed that the fluorescent signal disappeared as the number of PBS washings increased, while the fluorescent signal was well maintained and observed even after three PBS washings for the 2% (w / v) and 4% (w / v) Pec-PG hydrogels.

[0126] Therefore, due to the excellent adhesive strength of the Pec-PG hydrogel, the hydrogel is able to adhere and remain attached for a longer period of time to mucosal tissue, especially in the stimulating oral environment caused by movements such as food intake and chewing, and it is expected that the oral cavity's moisture retention and drug delivery effects will be sustained for a longer period of time compared to existing materials.

[0127] Example 13. Analysis of moisturizing ability of gallol group-modified pectin hydrogel

[0128] To evaluate the moisturizing ability of Pec-PG hydrogel, the amount of water evaporation was compared between Pec-PG hydrogel and commercially available artificial saliva products, Control 1 (product name: Xerova; Kolmar) and Control 2 (product name: Biotene; GSK) (see Figure 13a). After applying Pec-PG hydrogel and the existing artificial saliva products to a Petri dish, the weight was measured over time to observe the amount of water evaporation.

[0129] During the observation period of 100 minutes in total, the water in Control 1 evaporated quickly and its weight decreased the fastest, while the 2% (w / v) Pec-PG, 4% (w / v) Pec-PG, and Control 2 products lost water at a similar rate.

[0130] In addition, to analyze the moisturizing ability of the Pec-PG hydrogel, the amount of water evaporation from the sponge was compared after applying Pec-PG hydrogel and an existing artificial saliva product to a sponge that had absorbed water (see Figure 13b).

[0131] The results showed that moisture evaporated most quickly in the group treated with Control 1 product, while the groups treated with Control 2 product, 2% (w / v) Pec-PG, and 4% (w / v) Pec-PG hydrogel evaporated at similar rates.

[0132] Therefore, it was confirmed that Pec-PG hydrogel has the same or superior moisturizing ability as existing artificial saliva products.

[0133] As can be seen from the above examples, considering that the tissue adhesion of existing artificial saliva products is very low, Pec-PG hydrogel, which has both excellent tissue adhesion and excellent moisturizing properties, is expected to have great potential for development into a new artificial saliva product for the treatment of xerostomia that overcomes the limitations of existing products.

[0134] To compare moisturizing ability in actual oral tissue, commercially available artificial saliva products Control 1 (product name: Xerova; Kolmar) and Control 2 (product name: Biotene; GSK) and Pec-PG hydrogel were sprayed onto the tongue tissue of living rats, and in the case of Pec-PG, crosslinking was induced through natural oxidation to attach, and the moisture content of the rat tongue tissue was compared using a moisture measuring device (see Figure 13c). For the accuracy of the analysis, the rat tongue was dried in the same way, and then treated with 2% (w / v) Pectin, 2% (w / v) Pec-PG hydrogel, and existing artificial saliva, and the moisture content was measured.

[0135] The analysis confirmed that the tongue tissue coated with Pec-PG hydrogel maintained moisture the best (see Figure 13d).

[0136] Therefore, when applied to actual oral tissue, it was confirmed that Pec-PG hydrogel has superior moisturizing ability and excellent functionality as an artificial saliva compared to existing artificial saliva products.

[0137] Example 14. Analysis of the physical properties of gallol-modified pectin hydrogel patches

[0138] The Pec-PG solution was poured into a mold (e.g., a disc with a diameter of 8 mm) having a desired pattern and size, and then frozen at -80°C and lyophilized to prepare a Pec-PG patch (see FIG. 14a; Before). 4.5 mg / ml NaIO4 oxidant was sprayed onto the prepared Pec-PG patch to induce crosslinking (see FIG. 14a; After).

[0139] The Pec-PG patches prepared at two concentrations (2% (w / v) and 4% (w / v)) were sprayed with 4.5 mg / ml NaIO4 oxidant to induce crosslinking, and the swelling ratio of the Pec-PG hydrogel patches was then measured.

[0140] When maintained under physiological conditions (37°C in PBS buffer) for 7 days, the 2% (w / v) Pec-PG hydrogel patch exhibited a higher degree of swelling than the 4% (w / v) Pec-PG hydrogel patch (see Figure 14b).

[0141] To compare the degradation profile of the Pec-PG hydrogel patches, they were treated with pectin-degrading enzyme. When treated with the same concentration of pectin-degrading enzyme (2 U / sample), the 4% (w / v) Pec-PG hydrogel patch degraded more slowly due to increased cross-linking between gallol groups (see Figure 14c).

[0142] In other words, it was confirmed that the physical properties such as the swelling degree and degradation behavior of the Pec-PG hydrogel patch can be controlled by adjusting the concentration of Pec-PG.

[0143] Example 15. Analysis of the porous structure of gallol-modified pectin hydrogel patches

[0144] The size of the pores in the 2% (w / v) Pec-PG hydrogel patch was determined by porosimeter analysis, and it was found that most of the pores had a size of 10 μm (see FIG. 15a).

[0145] Comparing with the pore size of the Pec-PG hydrogel, it was confirmed that the Pec-PG hydrogel patch had a smaller porous structure (see FIG. 15b).

[0146] In other words, the freeze-dried patch-form Pec-PG hydrogel has a denser internal porous structure than Pec-PG hydrogel crosslinked in a solution state, and therefore is expected to have improved mechanical properties and adhesiveness due to an increased adhesive surface area.

[0147] Example 16. Analysis of mechanical properties of gallol-modified pectin hydrogel patch

[0148] The elastic modulus of Pec-PG hydrogel patches with two different concentrations (2% (w / v) and 4% (w / v)) was measured using a rheological analyzer in frequency sweep mode (see Figure 16a). It was confirmed that the storage modulus (G') was always higher than the loss modulus (G'') for the 2% (w / v) and 4% (w / v) Pec-PG hydrogel patches, confirming the formation of a stable polymer network structure inside the hydrogel patch.

[0149] The average elastic modulus of the 2% (w / v) Pec-PG hydrogel patch was approximately 10 kPa, whereas the 4% (w / v) Pec-PG hydrogel patch had an average elastic modulus of approximately 40 kPa, confirming that the elastic modulus increased in proportion to the Pec-PG concentration (see Figure 16b).

[0150] In other words, it was confirmed that the mechanical properties of the Pec-PG hydrogel patch could be adjusted by adjusting the concentration of Pec-PG.

[0151] When comparing the Pec-PG hydrogel molded into a patch with a hydrogel crosslinked with a Pec-PG solution of the same concentration, it was confirmed that the elastic modulus of the Pec-PG hydrogel patch was significantly increased (the elastic moduli of the 2% (w / v) and 4% (w / v) Pec-PG hydrogels were 1.7 kPa and 4.9 kPa, respectively, and the elastic moduli of the 2% (w / v) and 4% (w / v) Pec-PG patches were 10 kPa and 40 kPa, respectively). It is expected that the Pec-PG hydrogel patch will be able to be maintained stably even in an internal environment such as oral tissue, which has high fluidity and is subject to large physical deformation.

[0152] Example 17. Measurement of tissue adhesive strength of gallol group-modified pectin hydrogel patch

[0153] The tissue adhesive strength of 2% (w / v) and 4% (w / v) Pec-PG hydrogel patches was compared through tack test analysis. The adhesive strength to porcine gum tissue was evaluated.

[0154] After the pig gum tissue was attached to the plate and probe of a rheological analyzer, a Pec-PG hydrogel patch that was cross-linked through natural oxidation was applied between the tissues to adhere them to each other, and the force at which the tissue was detached was measured while pulling the tissue (see Figure 17a).

[0155] It was confirmed that both the 2% (w / v) and 4% (w / v) Pec-PG hydrogel patches had excellent tissue adhesion strength at the level of about 10 to 12 kPa (see FIG. 17b).

[0156] It is expected that the excellent tissue adhesion of the Pec-PG hydrogel patch will allow the hydrogel patch to adhere and remain in place for a longer period of time in an oral environment that is subject to a lot of movement and stimuli, such as food intake and chewing movements, thereby enabling the drug delivery effect in the oral cavity to be sustained for a longer period of time compared to existing materials.

[0157] Example 18. Analysis of drug release profile of gallol-modified pectin hydrogel patch

[0158] To verify the sustained-release drug delivery function of the Pec-PG hydrogel patch, the release profile of TA (triamcinolone acetonide), a drug used to treat stomatitis, was analyzed.

[0159] The release profile of the TA drug was observed while treating with pectinase (0.1 U / sample) under physiological conditions (37° C. in PBS buffer) (see FIG. 18).

[0160] The drug release profile was confirmed through HPLC analysis, and the sustained release behavior of the TA drug was confirmed from the Pec-PG hydrogel patch.

[0161] Therefore, it is expected that the drug-loaded Pec-PG hydrogel patch can be used to effectively deliver drugs to stomatitis and induce a sustained therapeutic effect.

[0162] Example 19. Analysis of antioxidant function of gallol-modified pectin hydrogel and patch molding

[0163] Experiments were conducted to determine whether the Pec-PG hydrogel and patch had the ability to scavenge reactive oxygen species (ROS) and various harmful radicals through the oxidized derivatives of the modified gallol group.

[0164] To evaluate the antioxidant function of the Pec-PG hydrogel and patch, their scavenging effects on ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) radical and hydroxyl radical were analyzed.

[0165] For the antioxidant test, we used ABTS solution, which turns blue-green and transparent when it reacts with samples with high antioxidant function (see Figure 19a; upper diagram). As a negative control, we used ABTS buffer solution, and ascorbic acid, known as vitamin C with high antioxidant function, as a positive control.

[0166] In addition, H2O2 and Fe 2+ The ions react to form Fe 3+ Using a Fenton reaction reagent that produces ions and hydroxyl radicals that induce DNA damage and cell death, it was confirmed whether the reaction was prevented in the presence of Pec-PG hydrogel and patch (see Figure 19a; lower panel). The negative control group was buffer only, and the positive control group was ascorbic acid.

[0167] The 4% (w / v) Pec-PG hydrogel and patch were placed in a solution made using ABTS solution and Fenton reaction, and after 30 minutes of incubation, the hydrogel and patch were removed and the color of the remaining solution was measured by UV-vis spectroscopy to confirm whether the ABTS radical and hydroxyl radical had been eliminated (see Figures 19b and 19c). As a result, it was confirmed that both the Pec-PG hydrogel and patch had the antioxidant function of only ascorbic acid, the positive control.

[0168] Therefore, it is expected that the Pec-PG hydrogel and patch, in addition to functioning as a sustained-release drug delivery vehicle, can also contribute to reducing tissue damage through its excellent antioxidant function.

[0169] Example 20. Evaluation of the therapeutic efficacy of gallol-modified pectin hydrogel patch for diabetic stomatitis in rats

[0170] After fasting for 24 hours, rats were injected intraperitoneally with streptozotocin (65mg / kg) to induce diabetes. After 48 hours, fasting blood glucose was confirmed to be 200mg / dL or higher.

[0171] A stomatitis model was created by treating the gums of diabetic rats with 80% acetic acid to induce tissue damage of about 4 mm in diameter. A Pec-PG hydrogel patch loaded with triamcinolone acetonide (TA), a stomatitis drug, was applied to the stomatitis-induced site on the day of tissue damage induction and on the fourth day using natural oxidation without an oxidizing agent, and the effects of gum regeneration and inflammation treatment were confirmed (see Figure 20a).

[0172] The following six groups were used in the experiment:

[0173] 1) No treatment

[0174] 2) TA only

[0175] 3) Pec-PG patch only (Patch only)

[0176] 4) Applying a commonly used drug with TA 25μm (Control: Product name Aftach; Donghwa Pharmaceuticals)

[0177] 5) Pec-PG patch with TA 12.5μm (Patch+TA(1x))

[0178] 6) Pec-PG patch with TA 25μm (Patch+TA(2x))

[0179] When a Pec-PG patch loaded with the TA drug was attached and the inflamed area was visually observed on the 4th and 7th days, it was confirmed that the area of ​​the damaged area was reduced the most and the tissue regeneration effect was also most enhanced when the Pec-PG patch loaded with TA (Patch+TA(1x), Patch+TA(2x)) was applied (see Figures 20b and 20c).

[0180] To evaluate the degree of tissue regeneration at the site of stomatitis, H&E staining and epithelial tissue marker (cytokeratin 5; CK5, cytokeratin 13; CK13) immunostaining and quantitative analysis of the stained images were performed on the 7th day after treatment. This confirmed that epithelial tissue regeneration was significantly promoted in the groups that delivered TA using the Pec-PG patch (Patch+TA(1x), Patch+TA(2x)) (see Figures 20d-f).

[0181] Immunostaining for CD11b, a leukocyte marker, revealed that numerous inflammatory cells were observed at the site of tissue damage in the no treatment group, the group treated with only the Pec-PG patch or TA drug, and the group that received the existing product, whereas a significantly smaller number of inflammatory cells was observed in the groups that received the TA drug via the Pec-PG patch (Patch+TA(1x), Patch+TA(2x)) (see Figures 20d and 20g).

[0182] In conclusion, it was confirmed that the Pec-PG patch can be well fixed at the inflamed site, protect the damaged tissue from external stimuli, and promote the regeneration of epithelial tissue through its antioxidant effect and effective drug delivery function.

[0183] Comparative Example 1. Comparison of mucoadhesion between gallol-modified pectin hydrogel and the prior art

[0184] An analysis was carried out to verify that the Pec-PG hydrogel developed in the present invention has superior mucoadhesion to the HA-PG and Chi-CA hydrogels (Korean Patent Registration Publication No. 10-1942220) developed in the prior art. Like Pec-PG, HA-PG and Chi-CA are materials that are applied in the past by inducing crosslinking through natural oxidation.

[0185] The reactivity and interaction of mucin protein with each derivative was analyzed by atomic force microscopy (AFM) (see Figure 21a). It was confirmed that when mucin protein solution was mixed with Pec-PG solution, larger aggregates were formed on the surface than when mixed with HA-PG or Chi-CA solution (see Figure 21b).

[0186] Based on these results, the mucoadhesion strength of the Pec-PG hydrogel patch, HA-PG hydrogel patch, and Chi-CA hydrogel patch on pig gingival mucosa tissue was compared through an actual tack test analysis (see Figure 21c). It was confirmed that the Pec-PG hydrogel patch had a mucoadhesion strength of approximately 8.3 kPa, while the HA-PG hydrogel patch and Chi-CA patch had mucoadhesion strengths of approximately 2.3 kPa and 3.5 kPa, respectively. Therefore, it was confirmed that the Pec-PG hydrogel patch had the best mucoadhesion strength.

[0187] Comparative Example 2. Comparison of the manufacturing method of the gallol group-modified pectin hydrogel patch with that of the prior art

[0188] According to the patch manufacturing technology of the prior art (Korean Patent Publication No. 10-2018-0127634, etc.), when manufacturing the Pec-PG patch, Pec-PG was crosslinked with an oxidizing agent of 4.5 mg / ml NaIO4, and then freeze-dried to produce the patch (freeze-dried patch after crosslinking).

[0189] The Pec-PG patch according to the present invention was fabricated by pouring the Pec-PG solution into a mold and immediately freeze-drying it without cross-linking (freeze-dried patch before cross-linking).

[0190] After inducing crosslinking using 4.5mg / ml NaIO4 as an oxidizing agent, the patch was attached to a plate and a probe using a rheometer device, and the tissue adhesive strength of each Pec-PG patch was compared through a tack test analysis. As a result, it was confirmed that the lyophilized patch before crosslinking (a patch manufactured using our patented manufacturing method) had significantly better adhesive strength than the lyophilized patch after crosslinking (a patch manufactured using the general manufacturing method in existing prior art) (see Figure 22).

Claims

1. A hydrogel comprising a pyrogallol group-modified pectin derivative in which pectin derived from Citrus peel is modified with 5-hydroxydopamine.

2. The hydrogel described in claim 1, wherein the pyrogallol group modified pectin derivative is oxidized.

3. The hydrogel of claim 2 , wherein the oxidation is by addition of an oxidizing agent, an enzyme, or a pH adjusting agent.

4. The hydrogel of claim 3 , wherein the oxidizing agent is a periodate salt or hydrogen peroxide.

5. The hydrogel according to claim 3, wherein the enzyme is any one selected from the group consisting of peroxidase, horseradish peroxidase, and tyrosinase.

6. 4. The hydrogel according to claim 3, wherein the pH adjuster is any one selected from the group consisting of sodium hydroxide, lithium hydroxide, potassium hydroxide, rubidium hydroxide, cesium hydroxide, magnesium hydroxide, calcium hydroxide, strontium hydroxide, and barium hydroxide.

7. The hydrogel of claim 2 , wherein the oxidation is due to natural oxidation.

8. The hydrogel according to claim 1, wherein the application site of the hydrogel includes oral mucosa, nasal mucosa, ocular mucosa, gastric mucosa, intestinal mucosa, bronchial mucosa, heart, lungs, or urinary tract.

9. The hydrogel of claim 1 , wherein the hydrogel is used in the treatment of a mucosal tissue disorder.

10. The hydrogel of claim 9, wherein the mucosal tissue disease comprises oral mucositis, oral cancer, stomatitis, leukoplakia, hand, foot and mouth disease, oral tuberculosis, oral candidiasis, mucosal wounds, periodontitis, aphthous ulcers, Behcet's syndrome, lichen planus or herpetic stomatitis.

11. The hydrogel of claim 1 , wherein the hydrogel is used for controlled drug delivery.

12. The drugs include DNA, mRNA, siRNA, miRNA, antisense oligonucleotides, antihistamines, proteins (growth factors), corticosteroid-based steroids, immune cell activators, doxorubicin, daunomycin, epirubicin, anthracyclines, paclitaxel, docetaxel, cabazitaxel, taxanes, cocumin, campothecin, berberine, evodiamine, matrine, piperine, sanguinarine, tetrandrine, thalicarpine, alkaloids, vinblastine, vincristine, vindesine, vinca alkaloids, and the like. alkaloids), cisplatin, caboplatin, platinum, 5-fluorouracil, capecitabine, methotraxate, metabolic antagonists, irinotecan, topotecan, etoposide, teniposide, etoposide, topoisomerase inhibitors, bleomycin, actinomycin, mitomycin, antitumor antibiotics, cyclophosphamide, mechlorethamine, chlorambucil, melphalan, alkylating agents 12. The hydrogel of claim 11, comprising at least one of the following: therapeutic antibodies, antibiotics, antibacterial agents, antiviral agents, anti-inflammatory agents, protein drugs, growth factors, cytokines, peptide drugs, and anesthetic agents.

13. A patch comprising a pyrogallol group-modified pectin derivative in which pectin derived from Citrus peel is modified with 5-hydroxydopamine.

14. A method for producing a patch according to claim 13, The patch is produced by freeze-drying the pyrogallol group-modified pectin derivative before cross-linking the same.

15. A method for producing a pyrogallol group-modified pectin derivative in which pectin derived from Citrus peel is modified with 5-hydroxydopamine, comprising the steps of: A method for producing a pyrogallol group-modified pectin derivative, comprising the step of reacting pectin, EDC, NHS and 5-hydroxydopamine in a predetermined ratio.

16. The method for producing a pyrogallol group-modified pectin derivative according to claim 15, wherein the ratio is pectin:EDC:NHS:5-hydroxydopamine=1:2:2:2.

Images