Phenolic derivatives modified pectin and uses thereof

Abstract

The present application aims to provide a hydrogel that exhibits equivalent or higher effects using a naturally occurring substance, pectin. Specifically, a hydrogel or patch is provided that is stable, has strong adhesion, is advantageous for drug delivery, and can be used for sustained treatment, has significantly low cytotoxicity, has low possibility of immune response induction, and thus can be applied to various body parts, especially the oral cavity. Also, a hydrogel patch is provided that adopts a pre-crosslinking freezing method, and thus exhibits excellent adhesion compared to a post-crosslinking freezing method.

Description

Technical Field

[0001] This invention relates to a gallol derivative-modified pectin and its uses. Background Technology

[0002] Globally, the market for functional medical materials technologies related to drug delivery, cell transplantation, and medical procedures such as hemostasis and suturing is growing rapidly. According to statistics, the market for modulated release drug delivery is projected to grow at an average annual rate of 13.8% by 2025, driven by increased research and development and the needs of elderly and pediatric patients. Furthermore, the global stem cell market is expected to grow rapidly from $62.8 billion in 2017 to $394.4 billion by 2025.

[0003] According to this forecast, demand for drug delivery and cell transplantation materials is expected to surge. Furthermore, the market size for hemostatic and suture materials is projected to grow to 4.5 trillion won by 2024, with further expansion anticipated.

[0004] In applications such as stable drug delivery into living organisms, cell transplantation, hemostasis at bleeding sites, and wound suturing, biomaterials with adhesive properties to the surfaces of various materials or biological tissues are required. As a result, various adhesive hydrogel technologies have been developed, some of which are also applied in clinical practice.

[0005] The oral cavity, typically referring to the area extending from the lips to the pharynx, is composed of a mucous membrane. This oral mucosa plays a crucial role in maintaining homeostasis, acting as the first line of defense against external infections, and serving as a pathway for the absorption of nutrients and medications. Mucosal damage can lead to bacterial infections and organ dysfunction caused by dryness of the submucosal tissue. Diseases occurring in the oral cavity include oral cancer, stomatitis, vitiligo, hand-foot-mouth disease, oral tuberculosis, oral candidiasis, and periodontitis. Recently, oral mucositis has become one of the most serious side effects of cancer treatment. Furthermore, infections associated with oral mucositis can trigger systemic sepsis in patients with weakened immune systems due to cancer treatment, potentially leading to death. These refractory oral ulcers cause pain and reduce nutrient intake, increasing the need for intravenous nutrition and thus lowering the patient's quality of life. In particular, it is a high-risk factor for sepsis in cancer patients with neutropenia, contributing to 10% of deaths among those with severe oral mucositis. Furthermore, cancer recurrence rates are increasing due to factors such as reduced doses of planned anticancer treatments and radiation therapy, or treatment interruptions. Moreover, if oral mucositis develops, additional treatments and surgeries are required, such as prescriptions for pain management with anesthetic analgesics, antibiotic prescriptions for infection prevention, and intravenous nutrition. Therefore, when oral ulcers occur during anticancer treatment, total medical costs will more than double. In the past six years, the number of patients with oral mucositis in South Korea has increased by more than 30%, and treatment costs have risen by approximately 70%.

[0006] As mentioned above, a wide variety of oral diseases, including stomatitis, can occur, and their incidence is on the rise. However, due to the flow of saliva in the mouth, medications cannot be maintained for a long time. Moreover, the convoluted nature of the oral cavity makes it difficult to apply medications such as patches. Therefore, in order to improve the treatment efficiency of a wide range of oral diseases, there is an urgent need to develop mucosal adhesive patches that can effectively adhere to the curved mucosal surfaces of the oral cavity and continuously release medication over a long period of time.

[0007] To address the aforementioned issues, Korean Patent No. 10-1942220 discloses a chitosan membrane containing catechol groups and a method for preparing the same. This invention is characterized by its ability to effectively adhere to the surface of the curved oral mucosa and to capture and continuously release various drugs over a long period.

[0008] In addition, in existing literature such as Korean Patent No. 10-2018-0127634, a cross-linking followed by freeze-drying method was used to prepare hydrogels, which resulted in a decrease in adhesive strength. Summary of the Invention

[0009] The problem that the invention aims to solve

[0010] This application provides a hydrogel that, compared to Korean Patent No. 10-1942220 and Korean Patent Publication No. 10-2018-0127634, utilizes the naturally occurring substance pectin to exhibit equivalent or superior effects. Specifically, it provides a hydrogel or patch that is stable, has strong adhesion, facilitates drug delivery, enables continuous treatment, exhibits significantly low cytotoxicity, and has a low likelihood of inducing immune responses, thus making it suitable for various body parts, especially the oral cavity. Furthermore, it provides a hydrogel patch that employs a pre-crosslinking freezing method, thereby exhibiting superior adhesion compared to post-crosslinking freezing methods.

[0011] However, the problems addressed in this application are not limited to those described above, but should be interpreted as encompassing all problems within the scope that can be understood by a person skilled in the art.

[0012] Solution for solving the problem

[0013] To address the aforementioned problems, according to a first aspect of this application, a pectin derivative modified with a galloyl group (Pec-PG) is provided.

[0014] According to a second aspect of this application, a hydrogel is provided which is formed by oxidizing the above-mentioned pectin derivative.

[0015] According to a third aspect of this application, a patch is provided that comprises the above-mentioned pectin derivative.

[0016] According to a fourth aspect of this application, a method for preparing pectin modified with a galloyl derivative is provided, comprising the step of reacting pectin, EDC, NHS, and a galloyl derivative in a predetermined ratio.

[0017] This is merely one example of a solution for solving a problem and should be interpreted as encompassing all solutions for solving a problem within the scope that can be understood by a person of ordinary skill.

[0018] Invention Effects

[0019] The pectin modified with gallol derivatives according to the present invention can regulate the swelling rate and the degree of decomposition. Strong binding occurs between mucosal tissue and gallol groups, which can regulate the delivery rate of the carried drug. Due to its low cytotoxicity, it exhibits excellent biocompatibility and has a low probability of inducing an intracellular immune response. As a stable hydrogel, it is suitable for various body parts, especially for oral areas that are difficult to adhere to. Attached Figure Description

[0020] Figure 1a A diagram illustrating an example of the process for preparing galloyl modified pectin. Figure 1b The graph shows the experimental results to confirm whether different concentrations of Pec-PG form hydrogels. Figure 1cA graph showing the experimental results used to confirm the appropriate concentration for the spray formulation.

[0021] Figure 2a and Figure 2b A graph showing the results of hydrogel formation kinetics analysis when crosslinking pectin-derived materials was performed according to Example 2.

[0022] Figure 3a and Figure 3b To illustrate the swelling ratio in the physical properties of the gallophenol-modified pectin hydrogel according to Example 3 ( Figure 3a ) and resolution ( Figure 3b The results of the analysis are shown in a graph.

[0023] Figure 4a and Figure 4b Figure (a) shows the results of confirming the internal structure of the hydrogel by scanning electron microscopy (SEM), hematoxylin and eosin (H&E) staining, and toluidine blue (TB) staining according to Example 4, and Figure (b) shows the results of confirming the pore size of the hydrogel by porosimeter analysis.

[0024] Figure 5a and Figure 5b The figure shows the results of Fourier-transform infrared spectroscopy (FT-IR) analysis (a) and ultraviolet-visible spectrophotometry analysis (b) according to Example 5.

[0025] Figure 6a and Figure 6b The graph shows the results of measuring the elastic modulus of the hydrogel using a rheological analysis apparatus in frequency sweep mode according to Example 6.

[0026] Figures 7a to 7d Figures (a, b) show the results of analyzing the morphology of the reaction between Pec-PG and mucin located on the surface of the mucosa by atomic force microscopy (AFM) according to Example 7, figure (c) shows the results of UV-Vis spectrophotometric analysis, and figure (d) shows the results of analyzing the potential difference of the mixture of Pec-PG and mucin.

[0027] Figure 8A graph showing the results of comparing the release characteristics of the protein bovine serum albumin (BSA) according to Example 8.

[0028] Figure 9a and Figure 9b Figure (a) shows the cell viability confirmed by the live / dead staining method according to Example 9, and Figure (b) shows the proliferation rate of stem cells cultured in Pec-PG hydrogel confirmed by MTT analysis.

[0029] Figures 10a to 10c Figure (a) shows the results of comparing the TNF-α secretion levels between the Pec-PG hydrogel of this application and the control group according to Example 10, and the immune response was confirmed using mice by H&E staining (b) and TB staining (c).

[0030] Figure 11a and Figure 11b Figure (a) shows the results of comparing the weight of major organs of rats with that of a control group to evaluate the safety of the hydrogel of this application by oral administration experiment according to Example 11, and Figure (b) shows the results of histological analysis by H&E staining.

[0031] Figures 12a to 12c Figure (a) shows the results of confirming the tissue adhesion of the hydrogel according to the present application in pig tongue tissue according to Example 12, Figure (b) shows the results of confirming the tissue adhesion of the hydrogel according to the present application in live rat tongue tissue, and Figure (c) shows the results of applying a hydrogel mixed with fluorescent material to rat tongue tissue and confirming the tissue adhesion of the hydrogel according to the present application.

[0032] Figures 13a to 13d The figure shows the experimental results of confirming the rate of water evaporation and moisturizing function of Petit's culture dish (a), sponge (b), and live rat tongue tissue (c and d) according to Example 13.

[0033] Figures 14a to 14c The graph shows the results of analyzing the swelling rate (b) and disintegration rate (c) of the patch (a) according to this application, based on Example 14.

[0034] Figure 15a and Figure 15b Figure (a) shows the results of the analysis of the pore size of the patch by a porosimeter according to Example 15, and a figure comparing the results of the pore size between the hydrogel and the patch.

[0035] Figure 16a and Figure 16b A graph showing the results of measuring the elastic modulus of the hydrogel patch using a rheological analysis apparatus according to Example 16.

[0036] Figure 17a and Figure 17b A graph showing the results of comparing the tissue adhesion of hydrogel patches according to Example 17 by means of a tack test.

[0037] Figure 18 A graph showing the results of drug release confirmed by HPLC analysis according to Example 18.

[0038] Figures 19a to 19c A graph showing the results of analyzing the antioxidant function according to Example 19.

[0039] Figures 20a to 20g Figure (a) shows the results of confirming the gingival regeneration and inflammation treatment effect in rats according to Example 20; Figures (b, c) show the results of confirming the area of ​​the lesion and the effect of tissue regeneration; Figures (d to f) show the results of H&E staining and immunostaining of epithelial tissue markers (cytokeratin 5 (CK5); cytokeratin 13 (CK13)) and quantitative analysis based on the staining images; and Figure (g) shows the results of confirming the number of inflammatory cells by immunostaining for the leukocyte marker CD11b.

[0040] Figure 21a and Figure 21b To illustrate the results of atomic force microscopy (AFM) analysis of the reactivity and interactions of mucin and its derivatives according to Comparative Example 1, Figure 21c According to Figure 21a and Figure 21b The results of the actual adhesion test analysis were used to compare the mucosal adhesion of Pec-PG hydrogel patch, HA-PG hydrogel patch and Chi-CA hydrogel patch in porcine gingival mucosa.

[0041] Figure 22 A graph showing the results of comparing the preparation methods of the gallophenol-modified pectin hydrogel patch with those of the prior art, according to Comparative Example 2. Detailed Implementation

[0042] Hereinafter, embodiments of this application will be described in detail with reference to the accompanying drawings, so that those skilled in the art can readily implement this application. However, this application is not limited to the embodiments described herein and can be implemented in many different ways. Furthermore, for the purpose of clearly illustrating this application, parts unrelated to the description have been omitted in the drawings, and similar reference numerals have been assigned to similar parts throughout the specification.

[0043] Throughout this application specification, when it is mentioned that a component is "on" another component, it includes not only the case where one component is in contact with another component, but also the case where there are other components between the two components.

[0044] Throughout this application specification, when a part "comprises" a component, it means, unless specifically stated otherwise, that it also includes other components, rather than excluding other components.

[0045] The degree terms “about”, “actually”, etc., used throughout this application specification are used to indicate the inherent permissible errors in manufacturing and materials, meaning their numerical values ​​or close to their numerical values, and are intended to prevent unscrupulous infringers from improperly using the disclosed content of accurate or absolute numerical values ​​mentioned for ease of understanding. The degree terms “~(of) step” or “~ step” used throughout this application specification do not mean “for the ~ step”.

[0046] Throughout this application, the term "the combination thereof" included in the Markush form of expression means a mixture or combination of one or more of the structural elements described in the Markush form of expression, and means including one or more of the structural elements described in the above-mentioned group.

[0047] Throughout this application, the reference to "A and / or B" means "A, B or A and B".

[0048] Throughout this application specification, examples of certain substances are provided for illustrative purposes only and do not imply that the corresponding substances are limited to specific examples.

[0049] In the entirety of this application specification, "pectin" refers to a hydrogel surrounding a cellulose-hemicellulose network, which is a polysaccharide composed primarily of galacturonic acid, an oxide of galactitol. It acts as a hydrophilic filler to prevent network aggregation and collapse, and is responsible for determining the porosity of the polymers in the cell wall. Its main component consists of α-1,4 bonds in D-galacturonic acid units.

[0050] In the full text of this application specification, "gall group derivatives" refers to pyrogallol (or pyrogallol) expressed by C6H3(OH)3 or compounds having the structure of chemical formula 1.

[0051] [Chemical Formula 1]

[0052]

[0053] In the entirety of this application, "stomatitis" refers to inflammation occurring in the oral cavity, generally encompassing inflammatory diseases of the oral mucosa (tongue, gums, lips, and inner cheeks, etc.). Causes include bacterial, viral, and fungal infections, and severe inflammation can lead to blisters and ulcers. Examples include oral mucositis, oral cancer, stomatitis, vitiligo, hand-foot-mouth disease, oral tuberculosis, oral candidiasis, mucosal trauma, periodontitis, aphthous ulcers, Behcet's syndrome, lichen planus, and herpetic stomatitis, but it should be interpreted as encompassing all inflammations occurring in the oral cavity.

[0054] In the full text of this application specification, a "patch" is a membrane that can be attached to the surface of the oral mucosa. It also contains therapeutic drugs and can release drugs for a long time. Therefore, it can also be used to treat diseases. Even without additional drug components, it can be attached to the surface of a wound in the tissue to inhibit further infection of the wound, etc., and thus be used for treatment.

[0055] In the full text of this application, "adhesion" mainly refers to the bonding between mucosal tissue and phenolic derivatives. Possible bonds include covalent bonds, hydrogen bonds, hydrophobic interactions, π-π interactions, and other physical non-covalent bonds.

[0056] The first aspect of this application provides a pectin derivative modified with a gallol group (Pec-PG). Due to the outstanding tissue adhesion ability of the gallol group, the Pec-PG derivative is expected to be used in various pharmaceutical and bioengineering fields, especially as an effective artificial saliva or oral disease drug carrier with important adhesion to mucous membranes. Furthermore, due to the natural oxidation ability of the gallol group, the solution of Pec-PG can be sprayed or applied to the oral cavity via a spraying method, thereby applying it to hard-to-reach oral disease sites.

[0057] The gallophenol derivative can be selected from the group consisting of 5-hydroxydopamine, tannic acid, gallic acid, epigallocatechin, epicatechin gallate, epigallocatechingallate, 2,3,4-trihydroxybenzaldehyde, 2,3,4-trihydroxybenzoic acid, 3,4,5-trihydroxybenzaldehyde, 3,4,5-trihydroxybenzamide, 5-tert-Butylpyrogallol, and 5-methylpyrogallol.

[0058] The second aspect of this application provides a hydrogel formed by oxidizing the pectin derivative of this application. The hydrogel of this application possesses all the features of the first aspect, and in particular, through the prominent reactivity of the oxidized gallol group with various nucleophilic functional groups, it can sustainably release the carried drug, thus making it suitable for various diseases and sites depending on the hydrogel dosage form.

[0059] The oxidation of hydrogels can be achieved by adding oxidants, enzymes, or pH adjusters. The oxidants can be periodate salt or hydrogen peroxide; the enzymes can be selected from the group consisting of peroxidase, horseradish peroxidase, and tyrosinase; and the pH adjusters can be 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.

[0060] Furthermore, the oxidation of hydrogels can also be caused by natural oxidation. Therefore, Pec-PG in solution can be sprayed or applied to the oral cavity via a spraying method, thereby applying it to hard-to-reach areas of oral disease.

[0061] The hydrogel of this application is applicable to sites including the oral mucosa, nasal mucosa, ocular mucosa, gastric mucosa, intestinal mucosa, bronchial mucosa, heart, lungs, or urinary system, and can thus be used to treat mucosal tissue diseases. In particular, it is effective in treating mucosal tissue diseases including oral mucositis, oral cancer, stomatitis, vitiligo, hand-foot-mouth disease, oral tuberculosis, oral candidiasis, mucosal trauma, periodontitis, oral ulcers, Behcet's syndrome, lichen planus, or herpetic stomatitis.

[0062] Furthermore, the hydrogel of this application can also be used to regulate the delivery of drugs within a biological organism. The drug can be encapsulated or loaded within the hydrogel, and the drug may comprise one or more of the following groups: DNA, mRNA, siRNA, miRNA, antisense oligonucleotides, antihistamines, growth factors, corticosteroid-based steroids, immune cell activators, anticancer agents, therapeutic antibodies, antibiotics, antibacterial agents, antiviral agents, anti-inflammatory agents, protein drugs, growth factors, cytokines, peptide drugs, and anesthetics.

[0063] The aforementioned anticancer agents can be selected from anthracyclines such as doxorubicin, daunomycin, epirubicin, and idarubicin; taxanes such as paclitaxel, docetaxel, cabazitaxel, and testataxel; alkaloids such as curcumin, camptothecin, berberine, evodiamine, matrine, piperine, sanguisorbine, tetrandrine, thalidomide, and roserine; vinca alkaloids such as vinca alkaloids, vincristine, vinblastine, and vinorelbine; platinum-based drugs such as cisplatin, carboplatin, and oxaliplatin; antimetabolites such as 5-fluorouracil, capecitabine, methotrexate, and gemcitabine; and topoisomerase inhibitors such as irinotecan, topotecan, etoposide, teniposide, and acridine. One or more of the following groups: inhibitors, bleomycin, actinomycin, mitomycin, mitoxantrone, etc.; alkylating agents such as cyclophosphamide, nitrogen mustard, chlorambucil, melphalan, and nitrosourea; azacitidine, azathioprine, cytarabine, and deoxyfluorouridine.

[0064] A third aspect of this application provides a patch comprising a pectin derivative according to this application. The patch according to this application can be manufactured by freeze-thinning a Pec-PG solution followed by freeze-drying, allowing for drug delivery by pre-loading the desired drug into the hydrogel or patch. In particular, prior art, such as Korean Patent Publication No. 10-2018-0127634, primarily employs a post-crosslinking freeze-drying method to manufacture patches, but this application uses a pre-crosslinking freeze-drying method. Due to this difference in method, the patch of this application exhibits significantly superior adhesion compared to existing patches.

[0065] Furthermore, the pre-loaded drug-containing Pec-PG hydrogel patch offers great user convenience and is therefore easily applicable to living organisms.

[0066] The fourth aspect of this application provides a method for preparing pectin modified with gallophenol derivatives.

[0067] The above preparation method may include the step of reacting pectin, EDC, NHS and galloyl derivatives in a predetermined ratio, which may be pectin:EDC:NHS:PG = 1:2:2:2, but is not limited thereto.

[0068] All common elements in the first through fourth aspects of this application shall apply.

[0069] The embodiments and examples of this application are described in detail below with reference to the accompanying drawings. However, this application is not limited to these embodiments, examples, and drawings.

[0070] Example 1. Synthesis of gallophenol-modified pectin derivatives, preparation of hydrogels, and confirmation of suitable concentration.

[0071] Pec-PG hydrogels were prepared by modifying pectin (Pec), which is commonly used as a food additive or food supplement, with the addition of gallophenol (PG).

[0072] Pec-PG was synthesized using 5-hydroxydopamine and a chemical reaction of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) / N-hydroxysuccinimide (NHS) at a molar ratio of pectin:EDC:NHS:PG = 1:2:2:2 (see reference). Figure 1a ).

[0073] Regarding Pec-PG concentrations, at concentrations below 1% (w / v), no hydrogel was formed when treated with 4.5 mg / ml NaIO4 oxidant. At a 1% concentration, a weak hydrogel with a structure that easily collapsed under slight shaking was formed. Stable hydrogels formed starting from a 2% (w / v) concentration of Pec-PG. Therefore, in the embodiments of this application, Pec-PG concentrations of 2% (w / v) or higher were used (see reference). Figure 1b However, this does not imply a limitation on the Pec-PG concentration in this application.

[0074] In addition, to determine the suitable concentration for spray formulations, experiments were conducted involving the spraying of Pec-PG solutions of various concentrations through a spray nozzle. Higher Pec-PG concentrations required greater spraying force (see reference). Figure 1c (Left side).

[0075] The higher the concentration of Pec-PG, the more varied the spray volume per application. In particular, with 4% Pec-PG, the amount sprayed decreases significantly after more than three applications. This indicates that higher Pec-PG concentrations result in lower spray usability (see reference). Figure 1c (Right side).

[0076] Therefore, it has been confirmed that a 2% concentration of Pec-PG is suitable for preparation as a spray formulation. This does not mean that other concentrations of Pec-PG cannot be used, but rather that a 2% concentration is suitable only for water mist formulations.

[0077] Example 2. Analysis of hydrogel formation kinetics during pectin-derived crosslinking.

[0078] The hydrogel formation kinetics of pectin-derived crosslinking were analyzed. After modifying gallol groups (PG) on citrus peel and apple pectin via EDC / NHS reaction, crosslinking induced the formation of hydrogels from the synthesized pectin derivatives, and the gelation kinetics were analyzed.

[0079] The process of hydrogel formation was analyzed when 4.5 mg / ml NaIO4 oxidant was mixed with 2% (w / v) citrus Pec-PG pre-gel solution and 2% (w / v) apple Pec-PG pre-gel solution and cross-linking was induced.

[0080] In a 2% (w / v) citrus Pec-PG pregel solution, the loss modulus (G″) was higher than the storage modulus (G′), but when an oxidant was mixed in for crosslinking, the G′ value was confirmed to be higher than the G″ value. This means that citrus peel-derived Pec-PG can stably form hydrogels (see reference). Figure 2a ).

[0081] In a 2% (w / v) apple Pec-PG pregel solution, the loss modulus (G″) remained higher than the storage modulus (G′), but no change was observed when an oxidant was mixed in for crosslinking. This suggests that apple-derived Pec-PG is difficult to form hydrogels (see [reference]). Figure 2b ).

[0082] In other words, it was confirmed that due to variations in pectin structure depending on its source, even with modification of the gallol group, hydrogel formation is sometimes impossible. Furthermore, it was confirmed that the degree of esterification of pectin and the amount of galacturonic acid with modifiable functional groups (COOH groups) can affect the synthesis of Pec-PG derivatives. In fact, citrus peel-derived pectin contains over 70% galacturonic acid, thus possessing sufficient functional groups for gallol group modification, posing no problem for Pec-PG derivative synthesis and hydrogel formation. Conversely, apple-derived pectin has a relatively low proportion of galacturonic acid and is more esterified, making it difficult to synthesize Pec-PG derivatives and form hydrogels.

[0083] Example 3. Analysis of the physical properties of pectin hydrogels modified with gallol groups.

[0084] After 4.5 mg / ml NaIO4 oxidant was mixed with Pec-PG pregel solutions at two commonly used concentrations (2% (w / v) and 4% (w / v)) to induce crosslinking and prepare hydrogels, the swelling ratio of each hydrogel was measured.

[0085] When maintained under physiological conditions (PBS buffer at 37°C) for 7 days, up to day 4, the 2% (w / v) hydrogel showed a higher swelling rate than the 4% (w / v) hydrogel (see reference). Figure 3a This means that the lower the concentration, the lower the degree of polymer cross-linking, and therefore the higher the expansion rate.

[0086] To compare the decomposition of Pec-PG hydrogels, pectinase treatment was performed. When treated with the same concentration of pectinase (1 U / sample), the 4% (w / v) hydrogel showed a higher proportion of cross-linked gallophenol groups. Therefore, it was confirmed that hydrogel decomposition occurred later under high concentration conditions (see reference). Figure 3b ).

[0087] That is, it was confirmed that physical properties such as the swelling rate and decomposition behavior of Pec-PG hydrogels can be adjusted by regulating the Pec-PG concentration.

[0088] Example 4. Analysis of the internal structure of gallol-modified pectin hydrogel

[0089] Hydrogels were prepared by mixing 4.5 mg / ml NaIO4 oxidant into a 2% (w / v) Pec-PG pregel solution.

[0090] The internal structure of the hydrogel was confirmed by scanning electron microscopy (SEM), hematoxylin and eosin (H&E) staining, and toluidine blue (TB) staining, revealing a dense, porous structure at the micrometer level (see reference). Figure 4a ).

[0091] Analysis using a porosimeter confirmed the pore size within the hydrogel, revealing numerous pores approximately 100 μm in size (see reference). Figure 4b ).

[0092] The hydrogel according to this application has the above-mentioned porous structure, thus providing an environment conducive to cell attachment and proliferation, and can effectively carry and release drugs.

[0093] Example 5. Analysis of the chemical properties of pectin hydrogels modified with gallol groups.

[0094] The chemical structures of Pec-PG (1 mg / ml) before oxidation (Pec-PG) and after oxidation (Pec-PG+NaIO4) were analyzed by Fourier-transform infrared spectroscopy (FT-IR) (see reference). Figure 5a ).

[0095] Observed all ~3435cm -1 Peak.

[0096] Observed all CH stretching groups at ~2925 cm -1peak.

[0097] Observed all ~1743 cm groups representing ester carbonyl stretching groups -1 peak.

[0098] Observed all stretching groups representing carboxylate ions at ~1620 cm -1 Peak and ~1440cm -1 peak.

[0099] All ~1100cm segments representing the galacturonan backbone were observed. -1 Peak and ~1020cm -1 peak.

[0100] 784 cm represents the formation of biphenol and biphenyl ether. -1 The peak was observed only in oxidized Pec-PG.

[0101] Thus, cross-linking between oxidized gallophenol groups (PGs) was confirmed only in Pec-PG treated with oxidants.

[0102] The chemical reactions that occurred after oxidation of Pec-PG solution with the addition of oxidant (NaIO4) were analyzed by UV-vis spectrophotometry (see reference). Figure 5b ).

[0103] The addition of the oxidant increased the peaks at 280 nm and 350 nm. This indicates that the gallophenol group (PG) forms semi-quinone and phenoxyl radicals during oxidation, confirming the cross-linking process.

[0104] Example 6. Analysis of the mechanical properties of gallol-modified pectin hydrogels

[0105] Hydrogels were prepared by mixing 4.5 mg / ml NaIO4 oxidant with two different concentrations (2% (w / v) and 4% (w / v)) of Pec-PG pregel solutions.

[0106] The elastic modulus of the hydrogel was determined using a rheological analysis apparatus in frequency sweep mode (refer to...). Figure 6a ).

[0107] In both 2% (w / v) and 4% (w / v) Pec-PG hydrogels, the measured storage modulus (G′) values ​​were consistently higher than the loss modulus (G″) values, thus confirming the formation of a stable polymer network structure within the hydrogel.

[0108] The average elastic modulus of 2% (w / v) Pec-PG hydrogel is approximately 1.7 kPa, compared to approximately 4.9 kPa for 4% (w / v) Pec-PG hydrogel. This confirms that the elastic modulus increases proportionally to the Pec-PG concentration (see reference). Figure 6b ).

[0109] That is, it was confirmed that the mechanical properties of Pec-PG hydrogels can be adjusted by regulating the Pec-PG concentration.

[0110] Example 7. Analysis of mucosal surface adhesion of pectin modified with gallol groups

[0111] The morphology of the reaction between Pec-PG and mucin located on the surface of the mucosa was analyzed by atomic force microscopy (AFM).

[0112] Small aggregates were observed forming on the surface when mucin was present alone or mixed with pectin (2 mg / ml pectin). When mucin was mixed with 2 mg / ml Pec-PG, significant large aggregate stratification was confirmed on the surface (see reference). Figure 7a and Figure 7b It is likely that adhesion is achieved through a strong binding reaction and interaction between the gallophenol group of Pec-PG and the nucleophilic functional groups of mucins present in mucosal tissues.

[0113] Mucin present on the surface of the mucosal layer was mixed with Pec-PG solution, and the chemical reaction between mucin and gallophenol (PG) was analyzed by UV-vis spectrophotometry. When 0.5 mg / ml Pec-PG and 0.5 mg / ml mucin were mixed, the peaks at ~280 nm and ~350 nm increased. This confirmed the occurrence of a strong covalent bond between the gallophenol group and the nucleophilic functional group of mucin present in the mucosal tissue (see reference). Figure 7c ).

[0114] Zeta potential analysis was performed to analyze the reaction between Pec-PG and mucin located on the mucosal surface at different time points. Mucin and 2% (w / v) Pec-PG were mixed, and it was confirmed that the zeta potential of the reactants gradually became negatively charged over time due to the strong binding reaction and interaction between the gallophenol groups of Pec-PG and the nucleophilic functional groups of mucin. This implies that a strong covalent bond is formed between the gallophenol groups of Pec-PG and the nucleophilic functional groups of mucin (see reference). Figure 7d ).

[0115] Therefore, the Pec-PG according to this application can be stably adhered to mucosal tissue.

[0116] Example 8. Analysis of drug release characteristics of gallophenol-modified pectin hydrogel

[0117] To verify the sustained-release drug delivery function of Pec-PG hydrogel, the following two groups were compared under physiological conditions (37°C PBS buffer) to determine the methodological characteristics of bovine serum albumin (BSA) (see reference). Figure 8 ).

[0118] 1) BSA supported on 2% (w / v) Pec-PG hydrogel (Pec-PG 2%)

[0119] 2) BSA supported on 4% (w / v) Pec-PG hydrogel (Pec-PG 4%)

[0120] The results of the BCA assay confirmed the drug release characteristics. Due to the strong binding between the gallophenol group and various nucleophilic functional groups of the protein, sustained-release drug release behavior of Pec-PG hydrogel was observed.

[0121] The 2% Pec-PG hydrogel at lower concentrations decomposes slightly faster, thus releasing BSA more quickly. For the 4% Pec-PG hydrogel, the BSA release time is longer compared to the 2% hydrogel.

[0122] Therefore, it is expected that drug-carrying Pec-PG hydrogels can effectively deliver drugs to sites where drug delivery is difficult, such as sites with oral diseases, and the concentration can be adjusted as needed to induce continuous treatment in various tissues.

[0123] Example 9. Evaluation of the cytotoxicity of gallophenol-modified pectin hydrogels

[0124] Human adipose-derived cells (hADSCs) were encapsulated in a Pec-PG pregel solution, and after adding 4.5 mg / ml NaIO4 oxidant to induce cross-linking, the stem cells were cultured in three dimensions within the Pec-PG hydrogel.

[0125] Cell viability was confirmed using a live / dead staining method during the 7-day culture period. A high cell viability of over 94% was observed during the culture period, thus confirming that the Pec-PG hydrogel is non-cytotoxic and possesses excellent biocompatibility (see reference). Figure 9a ).

[0126] The proliferation rate of stem cells cultured in Pec-PG hydrogel was confirmed using MTT assay. Normal stem cell proliferation was confirmed during the 7-day culture period (see reference). Figure 9b ).

[0127] Therefore, Pec-PG hydrogels do not exhibit cytotoxicity and thus have excellent biocompatibility.

[0128] Example 10. Evaluation of the immunoreactivity of gallol-modified pectin hydrogel

[0129] By co-culturing macrophages and Pec-PG hydrogels, the levels of pro-inflammatory cytokines tumor necrosis factor-α (TNF-α) secreted by macrophages were measured using enzyme-linked immunosorbent assay (ELISA), and the degree of immune response induction was confirmed.

[0130] Macrophages were cultured at the bottom of well plates, and after adding trans-wells, 2% (w / v) Pec-PG hydrogel was placed on the trans-wells for co-culture, where the hydrogel was only affected by components eluted from the hydrogel without direct contact with the cells. Lipopolysaccharide (LPS) was used as a control group.

[0131] As a result, when co-cultured with Pec-PG hydrogel, the TNF-α secretion level of macrophages showed a similar level to that of the no-treatment group, and showed a significantly different difference from the control group (see reference). Figure 10a ).

[0132] As another experiment, mice were subcutaneously injected with a 2% (w / v) Pec-PG pregel solution that had not been treated with an oxidant, and cross-linking was induced by natural oxidation caused by the oxidative environment present in the body. The injected hydrogel tissues were recovered on days 3, 7 and 14 for histological analysis.

[0133] As a result, H&E staining confirmed that the tissue injected with the hydrogel was not significantly different from normal tissue and did not induce a severe immune response. It was also confirmed that surrounding cells flowed into the interior of the hydrogel (see reference). Figure 10b ).

[0134] Similar to normal tissue, TB staining confirmed that almost no mast cells were observed in the tissue injected with the hydrogel, thus confirming that no immune response to the external substance occurred (see reference). Figure 10c ).

[0135] Therefore, the likelihood of Pec-PG hydrogel inducing an immune response in tissues is very low.

[0136] Example 11. Evaluation of the safety of taking gallophenol-modified pectin hydrogel

[0137] Due to the nature of products suitable for oral use, to evaluate the safety of oral Pec-PG hydrogel, 2% (w / v) Pec-PG pregel solution or PBS buffer was orally administered to rats for 28 days. Afterwards, the rats' body weight and feed intake were periodically measured and compared during the feeding period (see reference). Figure 11a ).

[0138] As a result, no significant differences in body weight or the weight of feed consumed were observed between the group receiving PBS buffer and the group receiving Pec-PG solution.

[0139] In addition, after administration of Pec-PG hydrogel, major organs (heart, liver, spleen, lung, and kidney) of rats were harvested and weighed on day 28, and histological analysis was performed by H&E staining (see reference). Figure 11b ).

[0140] As a result, no significant difference in organ weight was observed between the PBS group and the Pec-PG hydrogel group (upper part), and histological analysis also showed no tissue damage or immune response (lower part).

[0141] Therefore, it is hoped that Pec-PG hydrogels can be used in humans without any safety issues.

[0142] Example 12. Determination of tissue adhesion of gallol-modified pectin hydrogel

[0143] In pig tongue tissue, the tissue adhesion of 2% (w / v) and 4% (w / v) Pec-PG hydrogels and commercial artificial saliva products was compared using a tack test (reference). Control 1 (product name: Xerova; Kolmar) and Control 2 (product name: Biotene; GSK) were compared. Figure 12a ).

[0144] After attaching pig tongue tissue to the plate and probe of a rheological analysis device, Pec-PG hydrogel cross-linked by natural oxidation or an existing artificial saliva product is applied between the tissues to bind them together. The force of tissue debonding is then measured by pulling the tissues.

[0145] The adhesion strength was measured at approximately 0.2–0.5 kPa in control groups 1 and 2, which confirmed that the tissue adhesion strength was very low. In contrast, the Pec-PG hydrogel was confirmed to have excellent tissue adhesion strength at approximately 6–6.5 kPa.

[0146] In live rat tongue tissue, the adhesion of 2% (w / v) and 4% (w / v) Pec-PG hydrogels and commercial artificial saliva products was compared between control group 1 (product name: Xerova; Kolmar) and control group 2 (product name: Biotene; GSK).

[0147] Liquid Pec-PG solution was sprayed into rat tongue tissue using a sprayer. Cross-linking and adhesion were induced by in vivo natural oxidation without additional oxidant treatment. The tissue was recovered after 30 minutes for histological analysis (see reference). Figure 12b ).

[0148] In the cases of control group 1 and control group 2 products, it was confirmed that there was no residue in the tongue tissue, 2% (w / v) Pec-PG hydrogel was applied thinly with residue (arrow), and 4% (w / v) Pec-PG hydrogel was applied thicker with residue (arrow).

[0149] The hydrogel containing the mixed fluorescent substances was applied to rat tongue tissue, washed with PBS buffer, and the adhesion was confirmed under conditions simulating the oral cavity environment with saliva (see reference). Figure 12c ).

[0150] In control groups 1 and 2, it was confirmed that the fluorescence signal disappeared with increasing PBS washing repetitions. In 2% (w / v) and 4% (w / v) Pec-PG hydrogels, it was confirmed that the fluorescence signal was well maintained and observed even after 3 PBS washings.

[0151] Therefore, thanks to the outstanding adhesive strength of Pec-PG hydrogel, it can adhere to and maintain the hydrogel for a longer period of time in mucosal tissues, especially in the oral environment with more movement and stimulation such as eating and chewing. Compared with existing materials, it is expected to maintain oral moisture and achieve long-term continuous drug delivery.

[0152] Example 13. Analysis of the moisturizing power of gallophenol-modified pectin hydrogel

[0153] To evaluate the moisturizing effect of Pec-PG hydrogel, the water evaporation rate was compared between Pec-PG hydrogel and commercial artificial saliva products: Control Group 1 (product name: Xerova; Kolmar) and Control Group 2 (product name: Biotene; GSK). Figure 13a Pec-PG hydrogel and existing artificial saliva products were applied to Petit's culture dishes, and their weights were measured at different times. The amount of water evaporation was also observed.

[0154] It was confirmed that during the 100-minute observation period, the moisture in control product 1 evaporated rapidly, resulting in a rapid decrease in weight. The moisture content of 2% (w / v) Pec-PG, 4% (w / v) Pec-PG, and control product 2 decreased at a similar rate.

[0155] In addition, to analyze the moisturizing and retaining capacity of Pec-PG hydrogel, Pec-PG hydrogel and existing artificial saliva products were applied to water-absorbing sponges, and the amount of water evaporation from the sponges was compared (refer to...). Figure 13b ).

[0156] The results confirmed that the fastest water evaporation occurred in the group treated with product control 1, while water evaporated at a similar rate in the groups treated with product control 2, 2% (w / v) Pec-PG, and 4% (w / v) Pec-PG hydrogel.

[0157] Therefore, it has been confirmed that Pec-PG hydrogel has equivalent or better moisturizing power compared to existing artificial saliva products.

[0158] As confirmed in the foregoing embodiments, considering the very low tissue adhesion of existing artificial saliva products, it is hoped that Pec-PG hydrogel, which has both outstanding tissue adhesion and excellent moisturizing properties, can be developed into a new artificial saliva product for the treatment of xerostomia that exceeds the limitations of existing products.

[0159] To compare the moisturizing power in actual oral tissue, commercial artificial saliva products Control Group 1 (Xerova; Kolmar), Control Group 2 (Biotene; GSK), and Pec-PG hydrogel were sprayed onto the tongue tissue of live rats via a spray method. For Pec-PG, after natural oxidation-induced cross-linking and adhesion, the hydration rate of the rat tongue tissue was compared using a corneometer (see reference). Figure 13c To ensure analytical accuracy, rat tongues were dried and then treated with 2% (w / v) pectin, 2% (w / v) Pec-PG hydrogel, and existing artificial saliva before hydration rates were determined.

[0160] The analysis results confirmed that the tongue tissue coated with Pec-PG hydrogel maintained the best moisture content (refer to...). Figure 13d ).

[0161] Therefore, when applied to actual oral tissues, Pec-PG hydrogel exhibits superior moisturizing power and excellent functionality as an artificial saliva compared to existing artificial saliva products.

[0162] Example 14. Analysis of the physical properties of pectin hydrogel patches modified with gallol groups.

[0163] Pec-PG solution was poured into a mold of the desired shape and size (example: an 8 mm diameter petri dish), and then freeze-dried at -80°C to manufacture the Pec-PG patch (see reference). Figure 14a Before (2010) – Spray 4.5 mg / ml NaIO4 oxidant onto the manufactured Pec-PG patch to induce cross-linking (see [reference]). Figure 14a ;After)

[0164] After spraying 4.5 mg / ml NaIO4 oxidant onto Pec-PG patches manufactured under two concentrations (2% (w / v) and 4% (w / v)) and inducing cross-linking, the swelling ratio of the Pec-PG hydrogel patches was measured.

[0165] After 7 days of maintenance under physiological conditions (PBS buffer at 37°C), the 2% (w / v) Pec-PG hydrogel patch showed a higher swelling rate compared to the 4% (w / v) Pec-PG hydrogel patch (see reference). Figure 14b ).

[0166] To compare the decomposition characteristics of Pec-PG hydrogel patches, pectinase treatment was performed. When treated with the same concentration of pectinase (2U / sample), the 4% (w / v) Pec-PG hydrogel patch decomposed more slowly due to the further increased cross-linking between gallol groups (see reference). Figure 14c ).

[0167] That is, it was confirmed that the physical properties of Pec-PG hydrogel patches, such as swelling rate and decomposition behavior, can be adjusted by regulating the Pec-PG concentration.

[0168] Example 15. Analysis of the porous structure of pectin hydrogel patch modified with gallol group.

[0169] Analysis using a porosimeter confirmed the pore size of the 2% (w / v) Pec-PG hydrogel patch, confirming that most pores were 10 μm in size (refer to...). Figure 15a ).

[0170] When compared with the pore size of Pec-PG hydrogel, it was confirmed that the Pec-PG hydrogel patch has a smaller porous structure (see reference). Figure 15b ).

[0171] That is, the freeze-dried patch-like Pec-PG hydrogel has a denser internal porous structure in solution compared to cross-linked Pec-PG hydrogel, thus increasing the adhesive surface area and expecting to have further improved mechanical and adhesive properties.

[0172] Example 16. Analysis of the mechanical properties of pectin hydrogel patches modified with gallol groups.

[0173] The elastic modulus of two different concentrations (2% (w / v) and 4% (w / v)) of Pec-PG hydrogel patches was determined using a rheological analysis instrument in frequency sweep mode (refer to...). Figure 16a In 2% (w / v) and 4% (w / v) Pec-PG hydrogel patches, it was confirmed that the storage modulus (G′) value was always higher than the loss modulus (G″) value, thus confirming the formation of a stable polymer network structure inside the hydrogel patch.

[0174] The average elastic modulus of the 2% (w / v) Pec-PG hydrogel patch was confirmed to be approximately 10 kPa, compared to approximately 40 kPa for the 4% (w / v) Pec-PG hydrogel patch. This confirms that the elastic modulus increases proportionally with the Pec-PG concentration (see reference). Figure 16b ).

[0175] That is, it was confirmed that the mechanical properties of Pec-PG hydrogel patches can be adjusted by regulating the Pec-PG concentration.

[0176] When comparing the Pec-PG hydrogel patch formulation with the hydrogel crosslinked in the same concentration of Pec-PG solution, it was confirmed that the elastic modulus of the Pec-PG hydrogel patch was significantly increased (the elastic moduli of 2% (w / v) and 4% (w / v) Pec-PG hydrogel were 1.7 kPa and 4.9 kPa, respectively, and the elastic moduli of 2% (w / v) and 4% (w / v) Pec-PG patches were 10 kPa and 40 kPa, respectively). Thus, the Pec-PG hydrogel patch can be stably and well maintained in the in vivo environment of oral tissues, which have high fluidity and large physical deformation.

[0177] Example 17. Determination of tissue adhesion of gallol-modified pectin hydrogel patch

[0178] The tissue adhesion of 2% (w / v) and 4% (w / v) Pec-PG hydrogel patches was compared using a tack test. The adhesion in porcine gingival tissue was evaluated.

[0179] After attaching porcine gingival tissue to the plate and probe of a rheological analysis device, a Pec-PG hydrogel patch, cross-linked by natural oxidation, was applied between the tissues to allow them to adhere together. The force required to de-adhere the tissues was then measured by pulling on them. (Reference) Figure 17a ).

[0180] Both 2% (w / v) and 4% (w / v) Pec-PG hydrogel patches were confirmed to have excellent tissue adhesion at a level of approximately 10–12 kPa (refer to...). Figure 17b ).

[0181] Due to the outstanding tissue adhesion of Pec-PG hydrogel patches, they can adhere and maintain for a longer period of time in the oral environment, which is characterized by high movement and stimulation, such as eating and chewing. Therefore, compared with existing materials, they are expected to provide a longer-lasting intraoral drug delivery effect.

[0182] Example 18. Analysis of drug release characteristics of pectin hydrogel patch modified with gallol group.

[0183] To verify the sustained-release drug delivery function of the Pec-PG hydrogel patch, the release characteristics of triamcinolone acetonide (TA), a drug used to treat oral inflammation, were analyzed.

[0184] Pectinase (0.1 U / sample) was treated under physiological conditions (PBS buffer at 37°C), and the drug release characteristics of TA were observed (refer to...). Figure 18 ).

[0185] The results of HPLC analysis confirmed the drug release characteristics, and sustained-release behavior of TA drug was observed in Pec-PG hydrogel patch.

[0186] Therefore, drug-carrying Pec-PG hydrogel patches can be used to achieve effective drug delivery for stomatitis and induce sustained therapeutic effects.

[0187] Example 19. Analysis of the antioxidant function of pectin hydrogels and patch formulations modified with gallol groups.

[0188] Experiments were conducted on Pec-PG hydrogels and patches to determine their ability to remove reactive oxygen species (ROS) and various harmful free radicals caused by the oxidation of modified gallophenol groups.

[0189] To verify the antioxidant function of Pec-PG hydrogel and patch, the scavenging effect of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical and hydroxyl radical was analyzed.

[0190] ABTS solution was used in the antioxidant test. When ABTS solution reacts with a sample exhibiting high antioxidant activity, it becomes transparent within a blue-green color (see reference). Figure 19a (Upper part). ABTS solution was used as a buffer solution as a negative control group, and ascorbic acid, a well-known antioxidant of vitamin C, was used as a positive control group.

[0191] Furthermore, utilizing H2O2 and Fe 2+ Fe is generated by ionic reaction. 3+ The Fenton reaction reagent, which involves ions and reactive oxygen species (hydroxyl radicals) that induce DNA damage and apoptosis, was used to confirm whether the corresponding reaction was prevented in the presence of Pec-PG hydrogel and patch (see reference). Figure 19a (Lower part). The negative control group used only buffer solution, while the positive control group used ascorbic acid.

[0192] 4% (w / v) Pec-PG hydrogel and patch were added to ABTS solution and a solution prepared using the Fenton reaction. After incubation for 30 minutes, the hydrogel and patch were removed, and the color of the remaining solution was determined by UV-visspectroscopy to confirm whether ABTS radicals and hydroxyl radicals were eliminated (see reference). Figure 19b and Figure 19c The results confirmed whether both the Pec-PG hydrogel and the patch possessed antioxidant functions equivalent to those of the ascorbic acid in the positive control group.

[0193] Therefore, in addition to their function as sustained-release drug delivery systems, Pec-PG hydrogels and patches are expected to reduce tissue damage through their outstanding antioxidant properties.

[0194] Example 20. Evaluation of the therapeutic efficacy of gallol-modified pectin hydrogel patch in diabetic stomatitis in rats.

[0195] After fasting for 24 hours, rats were injected intraperitoneally with streptozotocin (65 mg / kg) to induce diabetes. Forty-eight hours later, fasting blood glucose levels were confirmed to be above 200 mg / dL.

[0196] A stomatitis model was established in diabetic rats by inducing tissue damage of approximately 4 mm in diameter through gingival treatment with 80% acetic acid. Pec-PG hydrogel patches carrying the stomatitis drug triamcinolone acetonide (TA) were applied to the stomatitis-inducing sites on the day of tissue damage induction and on day 4 using a natural oxidation method without the use of oxidants, thereby confirming the efficacy of gingival regeneration and inflammation treatment (see reference). Figure 20a ).

[0197] The experiment used the following 6 groups.

[0198] 1) No treatment was given (No treatment)

[0199] 2) Applicable only to TA drugs (TA only)

[0200] 3) Only applicable to Pec-PG patches (Patch only)

[0201] 4) Applicable to commercially available pharmaceutical products carrying 25 μg of TA (Control group; Product name: Aftach; Dong-Wha Corporation)

[0202] 5) Pec-PG patch carrying 12.5 μg of TA (Patch + TA (1x))

[0203] 6) Pec-PG patch carrying 25μg of TA (Patch + TA (2x))

[0204] When Pec-PG patches carrying TA were applied and the inflamed areas were observed visually on days 4 and 7, a significant reduction in the area of ​​the damaged site and a maximum enhancement in tissue regeneration were confirmed when using TA-carrying Pec-PG patches (Patch + TA (1x) and Patch + TA (2x)). (Ref) Figure 20b and Figure 20c ).

[0205] To evaluate the degree of tissue regeneration in the lesion site inducing stomatitis, H&E staining and immunostaining for epithelial tissue markers (cytokeratin 5 (CK5); cytokeratin 13 (CK13)) and quantitative analysis based on staining images were performed on day 7 post-treatment. Thus, Pec-PG patches confirmed significantly enhanced epithelial tissue regeneration in the TA delivery groups (Patch + TA (1x), Patch + TA (2x)) (see reference). Figures 20d to 20f ).

[0206] Immunostaining for the leukocyte marker CD11b revealed a higher number of inflammatory cells at the site of tissue injury in the no-treatment group, the Pec-PG patch group, the TA-only group, and the group using existing products. However, significantly fewer inflammatory cells were observed in the TA-delivered TA-patch group (Patch + TA (1x), Patch + TA (2x)) (see reference). Figure 20d and Figure 20g ).

[0207] Ultimately, it was confirmed that the Pec-PG patch adhered well to the site of inflammation, protected the damaged area from external irritants, and promoted epithelial tissue regeneration through its antioxidant effects and effective drug delivery.

[0208] Comparative Example 1. Comparison of mucosal adhesion between gallophenol-modified pectin hydrogels and existing technologies.

[0209] Analysis was conducted to verify that the Pec-PG hydrogel developed in this invention exhibits superior mucosal adhesion compared to HA-PG and Chi-CA hydrogels developed in existing literature (Korean Patent No. 10-1942220). Similar to Pec-PG, HA-PG and Chi-CA are materials suitable for cross-linking via natural oxidation induced in existing studies.

[0210] The reactivity and interactions of mucin and its derivatives were analyzed using atomic force microscopy (AFM) (see reference). Figure 21a When mucin solution and Pec-PG solution were mixed, larger aggregates were confirmed to form compared to when mixed with HA-PG solution or Chi-CA solution (see reference). Figure 21b ).

[0211] Based on these results, the mucosal adhesion of Pec-PG hydrogel patches, HA-PG hydrogel patches, and Chi-CA hydrogel patches in porcine gingival mucosa was analyzed and compared using a tack test (refer to...). Figure 21c The study confirmed that the HA-PG hydrogel patch and Chi-CA patch had mucosal adhesion forces of approximately 2.3 kPa and 3.5 kPa, respectively, compared to approximately 8.3 kPa for the Pec-PG hydrogel patch. Therefore, the Pec-PG hydrogel patch demonstrated the most significant mucosal adhesion force.

[0212] Comparative Example 2. Comparison of the manufacturing methods of the gallophenol-modified pectin hydrogel patch and the prior art.

[0213] According to existing literature (Korean Patent Publication No. 10-2018-0127634, etc.), when manufacturing Pec-PG patches, Pec-PG is cross-linked with an oxidant of 4.5 mg / ml NaIO4 and then freeze-dried to produce patches (cross-linked and freeze-dried patches).

[0214] According to this application, the Pec-PG patch is manufactured by introducing a Pec-PG solution into a mold and directly freeze-drying it without cross-linking (freeze-dried patch before cross-linking).

[0215] After cross-linking was induced using an oxidant of 4.5 mg / ml NaIO4, the tissue adhesion of each Pec-PG patch was analyzed and compared using a tack test after the patch was attached to a plate and probe in a rheometer. The results confirmed that the adhesion of the freeze-dried patch before cross-linking (the patch manufactured by the method of this patent) was significantly superior to that of the freeze-dried patch after cross-linking (the patch manufactured by conventional methods in existing literature) (see reference). Figure 22 ).

Claims

1. The use of a pectin derivative modified with a galloyl group in the preparation of a patch for treating oral mucosal tissue diseases, wherein, The gallophenol derivative is 5-hydroxydopamine. The pectin used as a source of the pectin derivative is citrus peel-derived pectin. The preparation method of the pectin derivative modified with the gallol group includes: The step of reacting pectin, EDC, NHS, and galloyl derivatives in a predetermined ratio. The patch is manufactured by freeze-drying the pectin derivative before crosslinking.

2. The use of the pectin derivative modified with galloyl group according to claim 1 in the preparation of patches for treating oral mucosal tissue diseases, wherein, The pectin derivative is oxidized to obtain a hydrogel.

3. The use of the pectin derivative modified with galloyl group according to claim 2 in the preparation of patches for treating oral mucosal tissue diseases, wherein, The oxidation is caused by the addition of oxidizing agents, enzymes, or pH adjusters.

4. The use of the pectin derivative modified with galloyl group according to claim 3 in the preparation of patches for treating oral mucosal tissue diseases, wherein, The oxidant is periodate or hydrogen peroxide.

5. The use of the pectin derivative modified with galloyl group according to claim 3 in the preparation of a patch for treating oral mucosal tissue diseases, wherein, The enzyme is selected from the group consisting of peroxidase and tyrosinase.

6. The use of the pectin derivative modified with galloyl group according to claim 5 in the preparation of a patch for treating oral mucosal tissue diseases, wherein, The peroxidase is horseradish peroxidase.

7. The use of the pectin derivative modified with galloyl group according to claim 3 in the preparation of a patch for treating oral mucosal tissue diseases, wherein, The pH adjuster is selected from the group consisting of sodium hydroxide, lithium hydroxide, potassium hydroxide, rubidium hydroxide, cesium hydroxide, calcium hydroxide, and strontium hydroxide.

8. The use of the pectin derivative modified with galloyl group according to claim 2 in the preparation of a patch for treating oral mucosal tissue diseases, wherein, The oxidation is caused by natural oxidation.

9. The use of the pectin derivative modified with galloyl group according to claim 2 in the preparation of a patch for treating oral mucosal tissue diseases, wherein, The hydrogel is used to regulate and deliver the drug.

10. The use of the pectin derivative modified with galloyl group according to claim 9 in the preparation of a patch for treating oral mucosal tissue diseases, wherein, The drug comprises one or more of the following groups: DNA, mRNA, siRNA, miRNA, antisense oligonucleotides, antihistamines, proteins, corticosteroid-based steroids, immune cell activators, antibiotics, taxanes, alkaloids, platinum-based drugs, topoisomerase inhibitors, antimetabolites, alkylating agents, antibacterial agents, antiviral agents, anti-inflammatory agents, growth factors, cytokines, peptide drugs, and anesthetics.

11. The use of the pectin derivative modified with galloyl group according to claim 10 in the preparation of a patch for treating oral mucosal tissue diseases, wherein, The drug comprises one or more of the following groups: doxorubicin, donomycin, epirubicin, anthracyclines, paclitaxel, docetaxel, carbamate, curcumin, camptothecin, berberine, evodiamine, matrine, piperine, sanguisorbine, tetrandrine, thalidomide, vincristine, vinblastine, cisplatin, carboplatin, 5-fluorouracil, capecitabine, methotrexate, irinotecan, topotecan, etoposide, teniposide, bleomycin, actinomycin, mitomycin, antitumor antibiotics, cyclophosphamide, nitrogen mustard, chlorambucil, melphalan, azacytidine, azathioprine, cytarabine, deoxyfluorouridine, and therapeutic antibodies.

12. The use of the pectin derivative modified with galloyl group according to claim 1 in the preparation of a patch for treating oral mucosal tissue diseases, wherein, The ratio is pectin:EDC:NHS:PG=1:2:2:2.

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