Adhesive coacervate-based drug delivery system and method for prreparing same

Abstract

The present invention relates to a coating optimized drug delivery enhancement (COD2E; Dopa-fucoidan / PPL) system composed of dopamine-functionalized fucoidan and poly-lysine to facilitate secretome delivery, wherein the introduction of dopamine enhanced the adhesive strength by at least a factor of seven compared to existing coacervates (fucoidan / PLL), thereby imparting the ability to rapidly (5 minutes) and uniformly coat collagen sponges. The COD2E system was able to encapsulate fibroblast growth factor (FGF2) and extend the half-life of FGF2. In addition, biocompatibility, improved stability, antioxidant properties, and blood compatibility were demonstrated through in vitro and in vivo experiments, and thus the present invention may be useful as a regeneration accelerating agent due to having improved bioactivity and antioxidant effects.

Description

Adhesive coacervate-based drug delivery system and method for preparing the same

[0001] The present invention relates to an adhesive coacervate-based drug delivery system and a method for producing the same.

[0002] The human body's inherent regenerative capacity is significantly enhanced by the secretome, comprised of various molecules such as growth factors, cytokines, mRNA, and exosomes. The secretome is a key element in regenerative medicine, serving as a fundamental mediator in cell-to-cell interactions, and secretome-based therapies are emerging as a viable alternative to cell-based treatments. The importance of the secretome has been particularly highlighted by the successful use of mRNA vaccines during the COVID-19 pandemic, demonstrating their potential for disease prevention. However, clinical applications of the secretome face significant challenges due to rapid degradation at the injection site (shortened half-life due to proteolytic enzymes) and diffusion (diffusion into tissue fluids). This highlights the need to improve the persistence of the secretome in our bodies to enhance therapeutic efficacy.

[0003] Direct injection of secretome into the affected area is a method to promote tissue regeneration. However, due to its short biological half-life, frequent or high-dose administration may be necessary, which can lead to various side effects. Bone morphogenetic protein 2 (BMP-2) is known for its role in bone tissue regeneration, but high doses of BMP-2 can induce unwanted epitope formation, triggering an immune response that leads to abnormal bone growth. Interleukin 2 (IL-2) was the first cytokine approved by the U.S. Food and Drug Administration (FDA) for immunotherapy. However, due to its short half-life, high doses of IL-2 are typically administered, which can lead to severe systemic inflammation and respiratory problems. Furthermore, the enzymatic degradation and structural instability of proteins and mRNA emphasize the need for a precise delivery system. Such a system must protect the secretome from proteases and nucleases while enabling localized and sustained release.

[0004] The ultra-low surface tension of coacervates allows them to be coated on various surfaces, particularly collagen sponges used in various medical products. However, previous studies have shown that a 2-hour stabilization time is required for coating, which can be a limitation in clinical settings. To address this issue, this study introduced dopamine (DOPA) into the coacervate system. The catechol group of DOPA imparts adhesive properties and is widely used in tissue engineering. Specifically, DOPA conjugation to D-Fuc was performed prior to coacervation with PLL. The coacervation between D-Fuc and PLL was successfully achieved, and this system was termed the Coating and Optimized Drug Delivery Enhancement (COD2E) system. The introduction of DOPA did not affect the coacervation or protein encapsulation capabilities. COD2E significantly extended the biological half-life and enhanced the therapeutic efficacy of the encapsulated FGF2 by protecting it from various substances. COD2E provided rapid and uniform coating on a wide range of surfaces within a short set-up time (5 minutes). Furthermore, the DOPA functional group of COD2E confers adhesive strength as well as a rapid setting time. This study demonstrates that FGF2-encapsulated COD2E cysteine ​​can function as a secretome delivery coating aid for various medical devices.

[0005] [Prior Art Literature]

[0006] [Patent Document]

[0007] 1. Republic of Korea Publication Patent No. 10-2019-0070343.

[0008] The purpose of the present invention is to provide a coacervate (COD2E) based on a dopamine-substituted fucoidan anionic polymer (D-Fuc) and a poly-l-lysine cationic polymer (PLL) and a method for producing the same.

[0009] Another object of the present invention is to provide a collagen sponge comprising the coacervate (COD2E).

[0010] Another object of the present invention is to provide a drug delivery system comprising the coacervate (COD2E).

[0011] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating antioxidant-related diseases, which comprises the coacervate (COD2E).

[0012] Another object of the present invention is to provide a health functional food composition for preventing or improving antioxidant-related diseases, including the coacervate (COD2E).

[0013] Another object of the present invention is to provide an antioxidant cosmetic composition comprising the coacervate (COD2E).

[0014] Another object of the present invention is to provide an adhesive composition comprising the coacervate (COD2E).

[0015] To achieve the above object, the present invention provides a coacervate (COD2E) based on a dopamine-substituted fucoidan anionic polymer (D-Fuc) and a poly-l-lysine cationic polymer (PLL).

[0016] In addition, the present invention provides a collagen sponge comprising the coacervate (COD2E).

[0017] In addition, the present invention provides a drug delivery system comprising the coacervate (COD2E).

[0018] In addition, the present invention provides a pharmaceutical composition for preventing or treating antioxidant-related diseases comprising the coacervate (COD2E).

[0019] In addition, the present invention provides a health functional food composition for preventing or improving antioxidant-related diseases, including the coacervate (COD2E).

[0020] In addition, the present invention provides an antioxidant cosmetic composition comprising the coacervate (COD2E).

[0021] In addition, the present invention provides an adhesive composition comprising the coacervate (COD2E).

[0022] In addition, the present invention provides a method for producing a coacervate according to claim 1, comprising the steps of: i) dissolving fucoidan and dopamine in MES (2-morpholin-4-4ylethanesulfonic acid) and mixing them; ii) further adding EDC (n-(3-dimethylaminopropyl)-n-ethylcarbodiimide hydrochloride) to the mixture of step i), mixing them, and purifying them to synthesize a dopamine-substituted fucoidan anionic polymer; and iii) dissolving a poly-l-lysine cationic polymer in PBS (phosphate-buffered saline) and mixing them with the anionic polymer of step ii).

[0023] The present invention relates to an adhesive coacervate-based drug delivery system, wherein the introduction of dopamine increased adhesiveness, exhibited an adhesive force 7 times higher than that of conventional coacervates, exhibited excellent loading rate and release tendency as a drug delivery system, and showed a protective effect against external proteolytic enzymes, and exhibited biocompatibility, bioactivity, antioxidant activity, and blood compatibility through in vitro and in vivo experiments.

[0024] Figure 1 is a schematic diagram of the COD2E system. (a) illustrates the advantages of the COD2E system, and (b) illustrates the composition and coacervation process of the COD2E system. The electrical interaction between dopamine-conjugated fucoidan (D-Fuc) and poly-lysine (PLL) polyelectrolyte forms a complex coacervate or microdroplet (COD2E). Fibroblast growth factor (FGF2) can be encapsulated within the COD2E system during the coacervation process. (c) The COD2E system protects the encapsulated growth factor from proteolytic enzymes and reactive oxygen species, thereby enhancing the half-life of the growth factor.

[0025] Figure 2 shows data confirming D-Fuc.

[0026] Figures 3 and 4 show the physical properties of DOPA functionalized coacervate (hereinafter referred to as COD2E).

[0027] Figure 5 shows data confirming the protein encapsulation efficiency release profile and protective ability of COD2E.

[0028] Figure 6 shows data confirming the biocompatibility, bioactivity, and antioxidant capacity of COD2E coating in vitro.

[0029] Figure 7 shows the antioxidant function of the components that make up COD2E, and shows the structural stability and adhesiveness in a ROS environment.

[0030] Figure 8 shows the skin regeneration effect due to the antioxidant effect of COD2E in vivo.

[0031] Figure 9 shows the effect of FGF2 (Fibroblast growth factor 2) encapsulating COD2E coated collagen sponge on re-epithelialization, collagen deposition and dermal remodeling.

[0032] Figure 10 shows data confirming the enhancement of cell proliferation, angiogenesis, and collagen expression through FGF2 encapsulating COD2E coating.

[0033] Figure 11 shows the evaluation of COD2E coating on macrophage distribution.

[0034] Hereinafter, the present invention will be described in more detail.

[0035] In this study, we chose to incorporate dopamine (DOPA) into the coacervate system because the catechol group of DOPA is known to impart adhesive properties. DOPA binding to D-Fuc was performed prior to coacervation with PLL. Coacervation between D-Fuc and PLL was successfully achieved, and we named this system the Coating and Optimized Drug Delivery Enhancement (COD2E) system (Fig. 1a). DOPA modification did not affect coacervation or protein encapsulation ability (Fig. 1b). COD2E significantly extended the biological half-life and enhanced the therapeutic efficacy by protecting the encapsulated FGF2 from various substances (Fig. 1c). COD2E provided rapid and uniform coating on various surfaces within a short setup time (5 min). Furthermore, the DOPA functional group of COD2E not only provided rapid coating time but also enhanced adhesive properties. This study demonstrates that the FGF2-encapsulated COD2E system can function as a secretome delivery coating supplement for various medical devices and has potential applications in various fields.

[0036] The present invention provides a coacervate (COD2E) based on a dopamine-substituted fucoidan anionic polymer (D-Fuc) and a poly-l-lysine cationic polymer (PLL).

[0037] The above coacervate may have a weight ratio of fucoidan anionic polymer and poly-l-lysine cationic polymer of 1 to 5:1.

[0038] The above coacervate can be encapsulated by carrying FGF2 (Fibroblast growth factor 2).

[0039] The above coacervate can be formed in the form of microdroplets.

[0040] In addition, the present invention provides a collagen sponge comprising the coacervate (COD2E).

[0041] In addition, the present invention provides a drug delivery system comprising the coacervate (COD2E).

[0042] The above drug delivery system may be in an encapsulated form.

[0043] In addition, the present invention provides a pharmaceutical composition for preventing or treating antioxidant-related diseases comprising the coacervate (COD2E).

[0044] The above antioxidant-related diseases may include, but are not limited to, wounds, skin aging, pigmentation, and skin cancer.

[0045] In another embodiment of the present invention, the pharmaceutical composition may further comprise one or more additives selected from the group consisting of suitable carriers, excipients, disintegrants, sweeteners, coating agents, bulking agents, lubricants, glidants, flavoring agents, antioxidants, buffers, bacteriostatic agents, diluents, dispersants, surfactants, binders and lubricants commonly used in the manufacture of pharmaceutical compositions.

[0046] Specifically, carriers, excipients, and diluents may include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like. These solid preparations may be prepared by mixing at least one excipient, for example, starch, calcium carbonate, sucrose or lactose, gelatin, and the like, into the composition. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Liquid preparations for oral administration include suspensions, solutions, emulsions, and syrups. In addition to commonly used simple diluents such as water and liquid paraffin, they may contain various excipients such as wetting agents, sweeteners, flavoring agents, and preservatives. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspending agents can be propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases can include witepsol, macrogol, tween 61, cacao butter, laurin butter, and glycerogelatin.

[0047] According to one embodiment of the present invention, the pharmaceutical composition can be administered to a subject in a conventional manner via intravenous, intraarterial, intraperitoneal, intramuscular, intraarterial, intraperitoneal, intrasternal, transdermal, intranasal, inhalational, topical, rectal, oral, intraocular, or intradermal routes.

[0048] The dosage of the active ingredient according to the present invention may vary depending on the condition and weight of the subject, the type and degree of the disease, the drug form, the route and period of administration, and may be appropriately selected by a person skilled in the art, and the daily dosage may be 0.01 mg / kg to 200 mg / kg, preferably 0.1 mg / kg to 200 mg / kg, and more preferably 0.1 mg / kg to 100 mg / kg. Administration may be once a day or divided into several times, and the scope of the present invention is not limited thereby.

[0049] In addition, the present invention provides a health functional food composition for preventing or improving antioxidant-related diseases, including the coacervate (COD2E).

[0050] The above health functional food may contain various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavoring agents and natural flavoring agents, coloring agents and thickening agents (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc.

[0051] In addition, it may contain fruit pulp for the production of natural fruit juice, synthetic fruit juice, and vegetable drinks. These ingredients may be used independently or in combination. Furthermore, the health functional food composition may be in the form of any one of meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, gum, ice cream, soup, beverage, tea, functional water, drink, alcohol, and vitamin complex.

[0052] In addition, the above health functional food may additionally contain food additives, and its suitability as a “food additive” is determined by the specifications and standards for the relevant item in accordance with the general provisions and general testing methods of the Food Additives Codex approved by the Ministry of Food and Drug Safety, unless otherwise provided.

[0053] Examples of items listed in the above "Food Additives Codex" include chemically synthesized products such as ketones, glycine, potassium citrate, nicotinic acid, and cinnamic acid; natural additives such as persimmon pigment, licorice extract, crystalline cellulose, kohlrabi pigment, and guar gum; and mixed preparations such as sodium L-glutamate preparations, alkaline agents added to noodles, preservative preparations, and tar color preparations.

[0054] At this time, the content of the effective ingredient added to the food during the process of manufacturing the health functional food can be appropriately increased or decreased as needed, and preferably, it can be added so that it is included in an amount of 1 to 90 parts by weight per 100 parts by weight of the food.

[0055] The above health functional food composition may be an inner beauty food.

[0056] According to a preferred embodiment of the present invention, the health functional food composition of the present invention has the advantage of having a more excellent antibacterial effect and biofilm formation inhibition effect against acne-causing bacteria when consumed in the form of inner beauty food. The inner beauty is referred to as an edible cosmetic or beauty food, and refers to a food that changes the skin constitution to a healthy one by allowing various skin-friendly ingredients to be absorbed into the body. Just as one selects cosmetics that are suitable for one's skin type, one can select and consume inner beauty food that is suitable for one's skin condition and lifestyle. More preferably, when a cosmetic containing the cosmetic composition is used in combination with the inner beauty food, the antibacterial effect and biofilm formation inhibition effect against acne-causing bacteria are significantly enhanced compared to using cosmetics alone, thereby having the advantage of seeing a more effective acne improvement effect.

[0057] In addition, the present invention provides an antioxidant cosmetic composition comprising the coacervate (COD2E).

[0058] The cosmetic composition of the present invention is not particularly limited in its formulation, and may be formulated as cosmetics such as, for example, a softening toner, an astringent toner, a nourishing toner, a nourishing cream, a massage cream, an essence, an eye cream, an eye essence, a cleansing cream, a cleansing foam, a cleansing water, a pack, a powder, a body lotion, a body cream, a body oil, and a body essence, and may be applied in a form that is applied to the skin or in a form that is absorbed into the skin using microneedles or the like.

[0059] In the cosmetic composition of the present invention, pharmaceutically or cosmetically acceptable carriers may vary depending on the formulation, but include hydrocarbons such as petrolatum, liquid paraffin, and gelling hydrocarbons (also known as plastibase); animal or vegetable oils such as medium-chain fatty acid triglycerides, lard, hard fat, and cacao fat; higher fatty acid alcohols and fatty acids and esters thereof such as cetanol, stearyl alcohol, stearic acid, and isopropyl palmitate; water-soluble bases such as macrogol (polyethylene glycol), 1,3-butylene glycol, glycerol, gelatin, sucrose, and sugar alcohols; emulsifiers such as glycerin fatty acid esters, polyoxyl stearate, and polyoxyethylene hydrogenated castor oil; adhesives such as acrylic acid esters and sodium alginate; propellants such as liquefied petroleum gas and carbon dioxide; Examples of preservatives include parahydroxybenzoic acid esters, and the external preparation of the present invention can be manufactured using these agents according to conventional methods. In addition to these agents, stabilizers, fragrances, colorants, pH adjusters, diluents, surfactants, preservatives, antioxidants, and the like can be blended as needed. The external preparation of the present invention can be applied to a local wound using conventional methods.

[0060] In addition, the present invention provides an adhesive composition comprising the coacervate (COD2E).

[0061] In addition, the present invention provides a method for producing a coacervate according to claim 1, comprising the steps of: i) dissolving fucoidan and dopamine in MES (2-morpholin-4-4ylethanesulfonic acid) and mixing them; ii) further adding EDC (n-(3-dimethylaminopropyl)-n-ethylcarbodiimide hydrochloride) to the mixture of step i), mixing them, and purifying them to synthesize a dopamine-substituted fucoidan anionic polymer; and iii) dissolving a poly-l-lysine cationic polymer in PBS (phosphate-buffered saline) and mixing them with the anionic polymer of step ii).

[0062] Hereinafter, to aid understanding of the present invention, examples and other embodiments will be described in detail. However, the following examples and other embodiments merely illustrate the content of the present invention and are not intended to limit the scope of the present invention. The examples and other embodiments of the present invention are provided to more fully explain the present invention to those of average skill in the art.

[0063] [Example 1] Materials

[0064] Fucoidan (purity ≥95%) derived from Fucus vesiculosus, poly-L-lysine hydrochloride (PLL; molecular weight = 15~30 kDa), dopamine hydrochloride (hereinafter referred to as DOPA), hydrochloric acid (hydrochloric acid 37%; HCl), EDC (n-(3-dimethylaminopropyl)-n-ethylcarbodiimide hydrochloride), D2O (deuterium oxide), NaOH (sodium hydroxide solution 30%), CH3COONa (sodium acetate), NaHCO3 (sodium bicarbonate), Na2CO3 (sodium carbonate), BSA-FITC, cOmplete™ (protease inhibitor cocktail tablets), HSCH2CH2OH (2-mercaptoethanol), methanol (CH3OH), phenylmethylsulfonyl Fluoride (phenylmethylsulfonyl fluoride) and dimethyl sulfoxide were purchased from Sigma-Aldrich (USA). Tris-HCl (1.5 M, pH 8.8), radioimmunoprecipitation assay buffer, Bradford reagent, SDS (sodium dodecyl sulfate), 10× TBST with Tween 20, 10× Tris glycine buffer (without SDS), 10× Tris glycine buffer (with SDS), 5× SDS-page loading buffer, TEMED (tetramethylethylenediamine), 10% ammonium persulfate solution, and isopropyl alcohol were purchased from Biosesang (Korea). DME (Dulbecco's phosphate-buffered saline, Dulbecco's modified Eagle's medium), PS (penicillin-streptomycin), FBS (fetal bovine serum), and LIVE / DEAD™ viability / cytotoxicity kit were purchased from Thermo Fisher Scientific (USA). P44 / 42 MAPK rabbit mAb (1:1000) and phospho-p44 / 42 MAPK rabbit mAb (1:2000) were purchased from Cell Signaling Technology (USA). Human / mouse / rat β-actin mAb (1:5000), rabbit lgG horseradish peroxidase (HRP) Ab (1:1000), mouse IgG HRP Ab (1:1000), and mouse / rat FGF basic / FGF2 Quantikine® ELISA kits were purchased from R&D Systems (USA). Collagen type I (10.9 mg mL). -1) and 0.25% trypsin-EDTA were purchased from Corning (USA). MES (2-(N-morpholino)ethanesulfonic acid) buffer (0.1 M, pH 6.0) was purchased from Tech and Innovation (Korea). Spectra / por2 dialysis membrane (MWCO = 12-14 kDa) was purchased from RepLigen (USA). FGF2 (16.3 kDa) was purchased from PeproTech (USA). Collagen type II (290 U mg -1 ) was purchased from Worthington (USA). CCK-8 was purchased from Dojindo (Japan). Transwell™ plates (6- and 24-well, SPLInsert™ Hanging) were purchased from SPL Life Sciences (Korea). Blotting membrane (PVDF) was purchased from GVS (Korea). Precision Plus Protein™ Dual Color Standards were purchased from Bio-Rad (USA). BSA was purchased from Bovogen (Australia). Chemiluminescent substrate and protein quantification kit (bicinchoninic acid) were purchased from Biomax (Korea).

[0065] [Example 2] Synthesis and identification of D-Fuc

[0066] Fucoidan (200 mg) and DOPA (248 mg) were dissolved in 30 mL of MES (0.1 M, pH 6.0). After complete dissolution, 410 mg of EDC was added to the solution. The mixture was stirred at room temperature for 2 h under an argon gas atmosphere, and the reaction product was purified for 3 days using a purification membrane (MWCO: 12-14 kDa). Acidic distilled water (DI water; 1 mL of 5 M HCl in 1 L of DI water) was used for D-Fuc purification. The final product was dried after freezing and stored in a refrigerator. To confirm whether DOPA was bound to fucoidan (D-Fuc), 1 A H NMR spectrometer (Bruker Avance III™ 400 MHz, Bruker, USA) was used. The concentration of D-Fuc in D2O solvent was 10 mg.mL -1 It has a resolution of 4 cm. -1 2,000-500 cm with a Fourier transform infrared spectrometer (Nicolet™ IS™ 10, Thermo Scientific, USA) in the -1 The scans were performed 32 times in the range. The degree of DOPA binding was measured by a UV-Vis spectrometer (Lambda 25, PerkinElmer, USA). The D-Fuc concentration was 1 mg.mL -1 DI water was used as the solvent. 0.02-0.1 mg.mL -1 The DOPA calibration curve was measured at 280 nm over the concentration range.

[0067] [Example 3] Measurement of the surface charge, i.e., zeta potential, of COD2E components

[0068] The surface charge of fucoidan (hereinafter referred to as Fuc), D-Fuc, DOPA, and PLL was analyzed using a zeta potential analyzer (Zetasizer Nano ZS, Malvern, UK). Each COD2E composition sample was dissolved in buffers of different pH values. The concentration of each sample was 0.1 mg. mL. -1This was the pH solution. For pH 3-6, 0.1 M sodium acetate was used, for pH 7-9, 0.1 M Tris-HCl was used, and for pH 10-11, 0.1 M sodium bicarbonate and sodium carbonate were used.

[0069] [Example 4] Preparation of D-Fuc / PLL complex coacervate (COD2E)

[0070] D-Fuc and PLL were dissolved in PBS (pH 7.4) and coacervates were formed at different weight ratios. To determine the maximum turbidity of the coacervate, D-Fuc and PLL were added at 6.25 mg mL each. -1 The solution was dissolved in a concentration and mixed in different ratios. The optical density was measured at a wavelength of 600 nm using a microreader (VersaMax™, Molecular Devices, USA). Turbidity measurements were performed immediately after coacervate formation. The morphological changes of COD2E over time were photographed using an optical microscope (DMIL LED, Leica, Germany). For D-Fuc, the pH was adjusted because it forms coacervate by itself. Turbidity and optical microscopy images were analyzed at pH 7, 8, and 8.5 using D-Fuc. The pH of all D-Fuc solutions was adjusted to 8.5-9 before use.

[0071] [Example 5] Adhesion test

[0072] 12.5, 25, and 50 mg.mL in PBS -1 D-Fuc and PLL were dissolved in concentrations of 10 × 10 × 1 mm, and coacervates were prepared with a mixing ratio of Fuc:PLL of 70:30 (weight ratio) and a mixing ratio of D-Fuc:PLL of 80:20 (weight ratio). Each sample was centrifuged at 12,000 rpm and 4°C, and sedimented for 10 minutes. The supernatant thus obtained was filtered into a 10 × 10 × 1 mm 3It was applied to pig skin (Apures, Korea). For the minimum coating using COD2E drop coating, D-Fuc and PLL were prepared in the same concentration and mixing order as before. After COD2E was formed, it was immediately applied to the collagen sponge. After the coating was maintained for 5 minutes to stabilize, it was coated on pig skin. ASTM F2258 test (25 × 25 mm) 2 ) The tensile strength of the adhesive test was measured by attaching the tool to the pig skin. The measurement conditions were 1-kN load cell, speed 100 mm min -1 The adhesive strength was calculated using the following mathematical formula:

[0073] [Mathematical Formula 1]

[0074] Adhesive strength = F / A

[0075] Here, F and A represent the force at the point where the maximum force is divided by the contact area.

[0076] Before evaluation, oil and foreign substances were removed from the surface of the pig skin, washed with pH 12-14 DI water for 15 minutes, and then washed five times with DI water for 5 minutes each. Remaining water was removed from the surface, and dried at room temperature for 3 to 4 hours.

[0077] [Example 6] Preparation of COD2E-coated collagen sponge

[0078] COD2E-coated collagen sponges (Teruplug, Olympus Terumo Biomaterials, Tokyo, Japan) were evaluated using a confocal laser scanning microscope (Leica TCS STED CW, Leica Camera AG). Collagen sponges with a diameter of 8 mm and a thickness of 1.5 mm were used. BSA (5 μg) was encapsulated in the COD2E-coated collagen sponges. Protein yield tests were performed, and the sponges were stored at room temperature for 1, 3, or 5 min to stabilize the coating. The coated collagen sponges were washed twice with 100 μL of PBS to collect uncoated BSA. Fluorescence values ​​were measured at λex = 480 nm and λem = 530 nm using a microanalyzer. The coating yields of free BSA-FITC and loaded COD2E were calculated using the following mathematical equation (2):

[0079] [Equation 2]

[0080] Protein coating yield = (W_total - W_wash) / W_total × 100

[0081] Here, W_total and W_wash represent the weight of the total BSA-FITC and the washed solution, respectively.

[0082] The morphology of COD2E-coated collagen sponges was observed in top, bottom, and 3D images using a confocal laser scanning microscope, and measured at λex=480 nm and λem=530 nm.

[0083] [Example 7] Protein encapsulation evaluation

[0084] When the D-Fuc:PLL weight ratio was 80:20 (weight ratio), maximum coacervate formation was achieved. The loading efficiency was evaluated to compare it with the case where the D-Fuc:PLL weight ratio was 50:50 (weight ratio), taking into account the loaded protein.

[0085] The encapsulation efficiency was evaluated by mixing the protein solution and PLL solution and then mixing with the D-Fuc solution (Method 1) or mixing the protein solution and D-Fuc solution and then mixing with the PLL solution (Method 2). The total concentrations of D-Fuc and PLL were 12.5 mg.mL each. -1 was fixed at , and the concentration of model protein (BSA-FITC) was 25 μg.mL -1 After encapsulation, the COD2E mixture was centrifuged at 12,000 rpm for 10 minutes at room temperature. Each supernatant was collected, and the coacervate phase of COD2E was present at the bottom of the test tube. The amount of unloaded BSA-FITC was analyzed by fluorescence intensity at λex = 480 nm and λem = 530 nm using a microanalyzer. The protein encapsulation yield was quantified using the equation given below.

[0086] The encapsulation ratio for the target protein FGF2 was evaluated in the same manner. The above-described mixing ratio [D-Fuc:PLL 80:20 (wt%)] and Method 2 were used for FGF2 encapsulation. After FGF2 was loaded with COD2E, the mixture was centrifuged at 12,000 rpm at room temperature for 10 minutes. The amount of FGF2 unloaded in the supernatant was analyzed using an FGF2 ELISA kit according to the manufacturer's guidelines. The protein encapsulation yield was quantified using the following mathematical formula (3).

[0087] [Equation 3]

[0088] Protein encapsulation yield = (W_total - W_supernatant) / W_total ×100

[0089] Here, W_total and W_supernatant represent the weight of the total model protein (BSA) or target protein (FGF2) and the weight of the supernatant solution, respectively.

[0090] Additionally, D-Fuc:PLL 50:50 (weight ratio) ratio, various concentrations (25, 50, 100 and 200 μg.mL) of model protein (BSA-FITC) were prepared using Method 2 above. -1 ) were measured for encapsulation efficiency. Each sample was centrifuged and the detection process was performed. The supernatant was analyzed by fluorescence intensity at λex=480 nm and λem=530 nm using a microanalyzer. In addition, various concentrations (25, 50, 100, and 200 μg.mL) of the target protein (FGF2) were measured. -1 ) was used to evaluate the amount of unloaded FGF2 using the FGF2 ELISA kit. The protein encapsulation yield was quantified using the above mathematical formula 3.

[0091] To analyze the drug encapsulation ratio according to the concentration of the components as drug carriers, 12.5, 25 and 50 mg.mL, respectively. -1 In the case of D-Fuc:PLL 80:20 (wt%) ratio, the final D-Fuc and PLL concentrations were evaluated using Method 2. The model protein (BSA) concentration was 25 μg.mL -1 Each sample was centrifuged and subjected to a detection process. The supernatant was analyzed using a microanalyzer for fluorescence intensities at λex=480 nm and λem=530 nm. The protein encapsulation yield was quantified using the above mathematical equation (3).

[0092] [Example 8] Protein release profile

[0093] The FGF2 protein release profile of COD2E-coated collagen sponges manufactured using Method 2 was analyzed by ELISA. 1.25 and 2.5 μg of FGF2 were loaded onto COD2E. COD2E was 12.5 mg mL -1It consisted of D-Fuc and PLL. The mixture was coated on a collagen sponge and stabilized for 5 minutes. After the coating was stabilized, the release was performed by immersing the mixture in 200 μL of PBS at 37°C. To measure the amount of FGF2 released over 1, 3, 5, 10, 20, 30, and 60 days, centrifugation was performed at 12,000 rpm, 37°C for 1 minute, and the supernatant was measured using an ELISA kit. After collecting the supernatant, 200 μL of PBS was freshly added and continuously incubated at 37°C. The release profile of the target protein (FGF2) was determined using the following mathematical formula 4:

[0094] [Equation 4]

[0095] Protein release yield = W_supernatant / W_total × 100

[0096] Here, W_total and W_supernatant represent the weight of total target protein (FGF2) and the weight of supernatant solution, respectively.

[0097] [Example 9] Protection experiment

[0098] The ability of COD2E to be protected by proteolytic enzymes was evaluated using collagen hydrogels and collagenase. 50 μL of COD2E loaded with BSA-FITC and free BSA-FITC were each added to 50 μL of collagen free hydrogel solution, resulting in a final concentration of 25 μg mL -1 BSA-FITC and 3 mg.mL -1 Collagen (Collagen type I, 10.9 mg.mL -1 ) was adjusted. The solution was neutralized by adding 1 N NaOH and 10× Dulbecco's phosphate buffer. Free BSA-FITC and COD2E loaded with BSA-FITC were gelled after reacting at 37°C for 1 hour. 100 U.mL -1 Collagenase II (290 U.mL) -1) was added to 100 μL of collagen gel sample and reacted for 16 hours. Each sample was observed by fluorescence and bright field images (Leica TCS STED CW).

[0099] [Example 10] Biocompatibility test

[0100] Biocompatibility was evaluated by performing an in vitro toxicity test based on ISO 10993-5. In vitro cytotoxicity tests were performed using a living fibroblast NIH-3T3 cell line (Korea Cell Line Bank, Korea). NIH-3T3 cells were cultured in DMEM supplemented with 10% (v / v) FBS and 1% (v / v) PS at 37°C under humidified conditions containing 5% CO2. Initially, 1 × 10 cells were seeded in 96-well plates (SPL Life Sciences). 3 Cells were seeded at a density of 10 cells / well and cultured for 1 day at 37°C in a 5% CO2 atmosphere. The culture medium was then replaced with fresh DMEM supplemented with COD2E extract. Each sample was cultured for 1, 3, or 5 days at 37°C in a 5% CO2 atmosphere. Cell viability was quantitatively measured by measuring the absorbance at 450 nm after treatment with the CCK-8 reagent. Cell viability was determined using the LIVE / DEAD™ staining kit. At each time point, cells were treated with Calcein-AM (Calcein acetoxymethyl) and Ethd-1 (Ethidium homodimer-1) and observed using a confocal laser scanning microscope (Leica TCS STED CW) for 30 minutes at 37°C.

[0101] [Example 11] Proliferation experiment

[0102] To determine the effect of COD2E on the FGF2 release rate, the proliferation of NIH-3T3 cells was assessed. Cells were cultured in DMEM supplemented with 10% (v / v) FBS and 1% (v / v) PS at 37°C in a humidified atmosphere containing 5% CO2. 0.5 × 10 4 Cells were seeded at a density of 10 cells / well and cultured for 1 day at 37°C in a 5% CO2 atmosphere. Afterwards, the medium was replaced with fresh DMEM. Each sample was then placed in a 24-well Transwell™, and a net collagen sponge was used as a control, and COD2E (D-Fuc and PLL, final concentration = 12.5 mg mL -1 ) at various FGF2 concentrations (0, 25, 50, 100, 200 μg mL -1 ) were loaded. The above-mentioned mixing ratio and method were used. After culturing at 37°C for 1, 3, and 5 days, cell proliferation was quantitatively measured by measuring the absorbance at 450 nm after treatment with CCK-8 reagent. A microplate reader was used.

[0103] [Example 12] Evaluation of Viability

[0104] NIH-3T3 cells were cultured in DMEM supplemented with 10% (v / v) FBS and 1% (v / v) PS at 37°C and 5% CO2 in a humidified atmosphere. 2 × 10 5 , 1×10 5 and 2.5×10 4 Cells were seeded at a density of 10 cells / well and cultured for 1, 3, and 5 days, respectively. After culturing for 1 day at 37°C in 5% CO2 conditions, the medium was replaced with fresh DMEM. Each sample was placed in a 6-well Transwell™. Pure state, Free (200 μg.mL -1 (FGF2-soaked) and COD2E-coated [D-Fuc and PLL final concentration 12.5 mg mL-1 and various FGF2 concentrations (0, 25, 50, 100, 200 μg mL -1 )] Collagen sponges were loaded with the mixing ratio and method mentioned above. After culturing for 1, 3, and 5 days at 37°C under 5% CO2, NIH-3T3 cells were collected and proteins were extracted. Cells were lysed using RIPA buffer and a protease inhibitor cocktail. The extracted proteins were diluted with SDS loading buffer, 2-mercaptoethanol, and PBS to a concentration of 10 μg.mL. -1 was adjusted. The concentration-adjusted protein solution was qualitatively evaluated by Western blot analysis (Mini-PROTEAN® Tetra Cell 4-gel Hand Casting system and PowerPac™ HC Power supply; Bio-Rad) to confirm the activation of ERK. The proteins were separated on a 10% acrylamide gel using SDS-PAGE (stacking; 80 V, 30 min, running; 100 V, 90 min). BSA (2 w / v%) was used for blocking. The proteins were transferred to the membrane and treated with p44 / 42 MAPK rabbit mAb (1:1000) and phospho-p44 / 42 MAPK rabbit mAb (1:2000) and human / mouse / rat beta-actin mAb (1:5000) for 1 h, and washed five times in TBST buffer. Antigen-antibody reactions were performed on each sample using rabbit IgG HRP Ab (1:1000) and mouse IgG HRP Ab (1:1000). The membrane was washed five times in TBST buffer, and luminescence chemiluminescence was detected using an Amersham™ lmager 680 (GE, USA).

[0105] The effect of FGF2 was quantitatively evaluated using the phospho-ERK (Thr202 / Tyr204; Thr185 / Tyr187) / total ERK1 / 2 assay whole cell lysate kit (MULTI-SPOT assay system, Meso Scale Discovery®, USA). The MSD assay was performed according to the manufacturer's instructions. Cell culture was performed in the same manner as Western blot analysis. NIH-3T3 cells were harvested after culturing for 1, 3, and 5 days at 37°C in 5% CO2, and proteins were extracted. Proteins were extracted by adding phenylmethylsulfonyl fluoride and dimethyl sulfoxide to the lysis buffer provided for the MSD assay. The concentration of the protein extract was 0.1 μg.mL using a protein quantification kit (bicinchoninic acid). -1 The levels of p-ERK and ERK were measured using MESO SECTOR S 600MM (Meso Scale Discovery®), and the p-ERK / total ERK ratio was calculated using the following equation:

[0106] [Equation 5]

[0107] % Phosphorylated Protein = (Phosphorylation Signal) / (Total Signal) × 100

[0108] Here, phosphorylated and total signals refer to p-ERK and total ERK, respectively.

[0109] [Example 13] Antioxidant test

[0110] Antioxidant properties were assessed using a DPPH assay kit according to the manufacturer's instructions. COD2E, Coa, and their respective components, D-Fuc, Fuc, and PLL, were prepared in liquid form. The DPPH reagent was typically a dark purple solid. To prepare a working solution, ethanol (100%) was added to the DPPH reagent and mixed until completely dissolved. Additional ethanol was then added to make the final volume 10 mL, and the working solution was mixed. 400 μL of assay buffer and DPPH working solution were added to Eppendorf tubes containing 100 μL of each sample. The reaction was incubated in the dark at room temperature for 30 minutes, and a color change from purple to yellow indicated antioxidant activity.

[0111] DCFDA assay (Abcam) was performed according to the manufacturer's instructions, with COD2E prepared at 12.5, 25, and 50 mg / ml, and Coa prepared at 12.5 mg / ml, and 2Х10 in 96-well plates. 4 Cells were seeded per well and allowed to attach overnight. The wells were then divided into positive control (1 mM ROS treatment), negative control (untreated), and experimental groups (COD2E and Coa treatment). Each group was incubated with 10 μM DCFDA for 30 min at 37°C in the dark, then washed with PBS to remove any residual DCFDA. Finally, fluorescence intensity was measured using a microplate reader (VersaMax, Molecular Devices, USA) with excitation at 485 nm and emission at 530 nm. Cell imaging was performed using a confocal microscope (Leica TCS STED CW).

[0112] [Example 14] Waterproofing effect

[0113] The waterproofing effect and sealing potential of COD2E were investigated by evaluating its liquid-liquid phase separation properties. 6-well plates were used to test untreated (blank), free (PBS-wetted), and COD2E-coated (6.25, 12.5, and 25 mg mL -1 (Concentration) collagen sponges were tested. The samples were treated as described above. The surface of the treated collagen sponges was allowed to interact with blood for 10 or 60 seconds, respectively. After the interaction, each sample and blood were thoroughly immersed in deionized water until completely wetted. Accelerated coagulation was transparent, and when coagulation was inhibited, the color of the deionized water changed to red. Therefore, the color of the surrounding deionized water was used to evaluate the waterproofing effect.

[0114] [Example 15] Static model

[0115] The model was used to compare the leakage resistance of control (untreated), free (loaded with PBS), and D-Fuc-coated collagen sponges. Collagen sponges with a diameter of 8 mm and a height of 5 mm were used. An 8-mm hole was created in each cap of a 15-mL conical tube using a biopsy punch. Porcine skin was then attached to the cap, and a 2-mm hole was created in the porcine skin using a biopsy punch. All sponges were completely fixed to the porcine skin to take advantage of the liquid-liquid phase separation properties of coacervates. The collagen sponges were treated with PBS and COD2E. COD2E was prepared in a volume ratio of 8:2 (D-Fuc:PLL). The samples were centrifuged at 12,000 rpm at 4°C. The supernatant of COD2E polymer was removed, and the remaining portion, the lower layer, was coated with paste. The tubes were inverted using DI water (10 mL of DI water added to each 15 mL conical tube) and the time required for a leak to occur and the mass of DI water were measured.

[0116] [Example 16] Blood compatibility test

[0117] Blood compatibility was evaluated using rat blood samples. Blood was prepared by separating erythrocytes from whole blood, and sodium citrate (3.2 w / v%) was prepared by dissolving 320 mg of sodium citrate in 10 mL of DI water. The pH was adjusted to 7.4–7.6 using HCl. Blood (9 mL) was mixed with the prepared sodium citrate solution (1 mL) by gentle and slow mixing up and down, and stored at 4°C before use. The collected blood was centrifuged at 2000 rpm for 5 min, and the extracted supernatant was removed, and then the blood was treated with different concentrations of COD2E (6.25, 12.5, 25 mg mL -1 ) was prepared with a sample volume of 900 μL. A control group was prepared using PBS and Triton™ X-100 (0.2 v / v%). The sample and red blood cell solution were mixed in a ratio of 9:1, and the mixture was incubated at 37°C for 1 h. After incubation, it was centrifuged at 3000 rpm for 5 min. The absorbance of the supernatant was measured at a wavelength of 415 nm using a microplate reader. The platelet lysis rate was calculated using the following mathematical equation:

[0118] [Equation 6]

[0119] Platelet lysis rate (%) = (ODt-ODn) / (ODp-ODn)*100

[0120] Here, ODt represents the sample group, and ODp and ODn represent the positive (PBS) and negative (Triton™ X-100) controls.

[0121] [Example 17] Animal experiment

[0122] All animal experiments were approved by the Institutional Animal Care and Use Committee of Seoul National University Hospital (Approval No. 22-0070-S1A0). Animals were cared for in a facility accredited by AAALAC International (AAALAC International, #001169) and were treated in compliance with the guidelines of the American Association for Laboratory Animal Care and Use (AAALAC), 8th edition, NRC (2010).

[0123] Thirty-two male Sprague-Dawley rats, aged 6–8 weeks, weighing 220–250 g, were divided into four study groups: control, free, COD2E0-coated, and COD2E200-coated. Four full-thickness wounds, each 10 mm in diameter, were created on the backs of the rats. A silicone ring was used to ensure that wound healing occurred primarily through granulation and reepithelialization rather than skin contraction. Each group received 25 μL of collagen sponges (8 mm in diameter, 5 mm in thickness; Olympus Terumo Biomaterials) dispersed in phosphate-buffered saline (PBS) as a coating. These coated sponges were typically applied directly to the wounds immediately after surgery. Wound healing was monitored weekly, and photographs were taken to assess the rate of wound closure. Wound area was measured using ImageJ software (National Institutes of Health). Five rats were euthanized after 2 weeks for further analysis, and three rats were euthanized after 3 weeks.

[0124] [Example 18] Histological evaluation

[0125] Skin samples were first fixed in 4% paraformaldehyde, then dehydrated and embedded in paraffin wax. The paraffin-embedded tissue was sectioned at 4 μm thickness. The sectioned tissue was then deparaffinized and stained with hematoxylin-eosin and Masson's trichrome.

[0126] For immunohistochemical procedures, sections from paraffin-embedded samples were mounted on slides. After deparaffinization, the slides were treated with the following primary antibodies: PCNA (ab29, Abcam, Boston, MA, USA); CD31 (GTX130274, GeneTex, Irvine, CA, USA); α-SMA (GTX100034, GeneTex); CD68 (ab125212, Abcam), inducible nitric oxide synthase (ab15323, Abcam), and arginase (D4E3M, Cell Signaling Technology). The sections were then treated with secondary antibodies (Invitrogen, USA). Staining was performed with hematoxylin counterstaining. Semiquantitative analysis of the immunohistochemical results was performed using ImageJ software. Two areas from each section were analyzed from five subjects in each group. All images were digitized to ensure 400× and 20× original magnifications, respectively, with a resolution of 640×480 or 1280×960 pixels.

[0127] [Example 19] Quantitative reverse transcription PCR

[0128] RNA was extracted from mouse skin tissue samples using TRIzol™ reagent (Invitrogen). The RNA was reverse transcribed into cDNA using ReverTra Ace™ qPCR RT Master Mix (Toyobo, Osaka, Japan). PCR was performed on an Applied Biosystems QuantStudio™ 5 system using SYBR™ Green PCR Master Mix (Toyobo).The primer sequences used in PCR were as follows: COL1A1, 5'-AGGGAACAACTGATGGTGCTACTG-3' (Forward) and 5'-GGACTGCTGTGCCAAAATAAGAGA-3' (Reverse); COL3A1, 5'-AGGGAACAACTGATGGTGCTACTG-3' (Forward) and 5'-GGACTGCTGTGCCAAAATAAGAGA-3' (Reverse); MMP-2, 5'-TACAGGATCATTGGCTACACACC-3' (Forward) and 5'-GGTCACATCGCTCCAGACT-3' (Reverse); TIMP-2, 5'-TCTCGACATCGAGGACCCAT-3' (Forward) and 5'-TGGACCAGTCGAAACCCTTG-3' (Reverse); tumor necrosis factor-α, 5'-CCGCTCGTTGCCAATAGTGATG-3' (Forward) and 5'-CATGCCGTTGGCCAGGAGGG-3' (Reverse); IL-1β, 5'-GCACTACAGGCTCCGAGATGAA-3' (Forward) and 5'-GTCGTTGCTTGGTTCTCCTTGT-3' (Reverse); IL-6, 5'-CTTGGGACTGATGCTGGGTGACA-3' (Forward) and 5'-GCCTCCGACTTGTGAAGTGGTA-3' (Reverse); IL-10, 5'-CTTACTGACTGGCATGAGGATCA-3' (Forward) and 5'-GCAGCTCTAGGAGCATGTGG-3' (Reverse); IL-4, 5'-TGCACCGAGATGTTTGTACC-3' (Forward) and 5'-GGATGCTTTTTAGGCTTTCC-3' (Reverse); TATA-binding protein 1 (TBP-1) (reference gene), 5'-AAGGGAGAATCATGGACCAG-3' (Forward) and 5'-CCGTAAGGCATCATTGGACT-3' (Reverse).PCR results were calculated using the comparative CT method and quantified relative to the internal reference gene TBP-1.

[0129] [Example 20] Statistical Analysis

[0130] Comparisons between groups were performed using one-way ANOVA followed by Tukey's post hoc analysis. All results were analyzed and graphically presented using Prism version 8 (GraphPad Software, La Jolla, CA, USA) and SPSS version 22 (IBM, Armonk, NY, USA). A difference was considered statistically significant at p<0.05. * indicates p<0.05, ** indicates p<0.01, *** indicates p<0.001, and **** indicates p<0.0001.

[0131] [Experimental Example 1] Characteristic Analysis of D-Fuc

[0132] Inspired by the underwater adhesive properties of DOPA, a novel and simple composite coacervate was prepared using D-Fuc and PLL to enhance adhesion according to the above examples.

[0133] Since DOPA becomes insoluble in water upon oxidation, its functionalization with fucoidan was investigated at various pH levels in deionized (DI) water. As a result, it was confirmed that DOPA was successfully bound to fucoidan according to Fig. 2 a to c. The DOPA-specific peak (range 6.9 to 7.6) and the fucoidan-specific peak 1 H was confirmed using nuclear magnetic resonance spectroscopy (Fig. 2a). Subsequent Fourier transform infrared spectroscopy revealed a peak at 1550 cm -1 (amide II band, NH bend) and 1650 cm -1The presence of DOPA was confirmed by the peak at (amide I band, C=O stretching) (Fig. 2b). UV-Vis spectrometry also confirmed the presence of DOPA with a peak at 280 nm (Fig. 2c). The DOPA content in D-Fuc, determined by comparing it to the concentration measured by UV-Vis spectrometry, was found to be in the range of 9 to 15 wt%.

[0134] Complex coacervation occurs between oppositely charged polyelectrolytes through electrostatic interactions, so anionic and cationic polyelectrolytes must maintain surface charges during coacervation. Since biomedical applications must be performed under physiological conditions, coacervation must occur and persist at pH 7.4. Therefore, the surface charges of D-Fuc and PLL were investigated over the pH range of 3 to 11. As shown in Fig. 2d, D-Fuc and PLL exhibited opposite surface charges from pH 3 to 9. Although DOPA has a negative surface charge at pH 7-9, DOPA conjugation did not significantly affect the surface charge of D-Fuc. It was confirmed that D-Fuc / PLL coacervation is possible under physiological conditions at pH 7.4.

[0135] [Experimental Example 2] Functional Verification of COD2E

[0136] Turbidity was measured according to various D-Fuc and PLL mixing ratios, and the results are shown in a of Fig. 3a.

[0137] A D-Fuc:PLL ratio of 80:20 (weight ratio) exhibited the highest turbidity, indicating optimal COD2E formation. Furthermore, spontaneous formation of opaque coacervates was observed upon mixing (Fig. 3b), with flocculation of fine droplets observed over time. This flocculation occurs when oppositely charged polymer electrolytes experience electrostatic equilibrium, displacing water molecules due to increased surface hydrophobicity; subsequent hydrophobic interactions can promote the formation of bulk coacervates. Conversely, when the surface charges of the polymer electrolytes are similar, repulsion occurs between the polymer electrolytes, dissociating the coacervate. This allows for reversible dissociation and reformation by pH adjustment. The reversibility of COD2E was evaluated by varying the pH of the solution. As shown in Figure 4, the pH after coacervation between D-Fuc and PLL was 7-8, and the introduction of NaOH to raise the pH (pH 12) induced the spontaneous dissociation of COD2E. Addition of HCl to lower the pH to 8-9 to re-establish the coacervate effectively revived COD2E. This confirmed that the properties of the drug carrier itself can be modulated by external factors such as pH, and this phenomenon also demonstrated the possibility of programmed drug release from COD2E by controlling pH.

[0138] COD2E with various polyelectrolyte concentrations (12.5, 25, and 50 mg mL-1) was compared with fucoidan / PLL coacervate (Coa, a coacervate without DOPA bonds). As shown in Fig. 3c, the bond strength was significantly improved at all concentrations of COD2E. Specifically, Coa exhibited bond strengths of 27.71 ± 14.36, 36.75 ± 13.88, and 34.21 ± 32.4 kPa at polyelectrolyte concentrations of 12.5, 25, and 50 mg / mL, respectively. In contrast, COD2E exhibited bond strengths of 213.83 ± 28.45, 330.2 ± 51.15, and 332.38 ± 60.62 kPa at polyelectrolyte concentrations of 12.5, 25, and 50 mg / mL, respectively. In particular, the adhesive strength of COD2E was at least 7.7 times higher than that of Coa alone. This improvement was attributed to DOPA, which provides strong adhesive strength.

[0139] The bond strength of COD2E was further evaluated by cyclic loading tests involving multiple attachment-detachment cycles. 12.5 mg.mL -1 A concentration of polyelectrolyte was applied to the porcine skin substrate. As shown in Fig. 3d, the average bond strength was 183 kPa and increased with subsequent cycles. This may have occurred due to the evaporation of water molecules between COD2E and the porcine skin, which caused the COD2E microcapsules to aggregate into a bulk structure, enabling stronger adhesion. Furthermore, the covalent bond formation between the organic surface (porcine skin) and COD2E may occur due to the reaction with the abundant sulfate and amide side chains following DOPA oxidation.

[0140] To investigate the coating ability of COD2E, a COD2E solution was applied to collagen sponges to evaluate the surface morphology and coating yield. A bovine serum albumin (BSA)-fluorescein isothiocyanate (FITC) conjugate was encapsulated in COD2E. After dropping the COD2E solution onto the collagen sponges, the sponges were visualized using fluorescence imaging 1, 3, and 5 minutes later. As shown in Figure 3e, the top and bottom surfaces of the sponges were uniformly coated. Notably, the coating yield of COD2E was higher than that of Coa, regardless of the polyelectrolyte concentration, and shorter coating or curing times were required (Figure 3f). Specifically, while the previous Coa coating required 2 hours of curing time, the COD2E coating required only 5 minutes. All COD2E coating yields exceeded 83%, while the highest coating yield of Coa was 77.57 ± 4.95%. The enhanced coating ability may be attributed to the adhesive strength of DOPA. The rapid and homogeneous coating ability of COD2E confirmed its potential as an effective bioactive coating for delivery of various proteins and drugs.

[0141] [Experimental Example 3] COD2E, a drug delivery coating system

[0142] To investigate the encapsulation ability of COD2E, BSA-FITC conjugate was used as a model protein. The protein mixing order and mixing ratio were compared, as shown in Figure 5a. The mixing order was varied by introducing the model protein into PLL (Method 1) or D-Fuc (Method 2). Two different D-Fuc:PLL mixing ratios of 80:20 and 50:50 (weight ratio) were compared. The final BSA-FITC concentration was 25 μg.mL. -1was maintained. The encapsulation yield of the model protein exceeded 80% regardless of various parameters. Although there was no significant difference between the mixing orders, the encapsulation yield (93.76 ± 1.19%) was found to be higher when the mixing ratio was 50:50. In addition, Method 2 with a mixing ratio of 50:50 (by weight) was used to evaluate the effect of BSA-FITC and polyelectrolyte concentrations on the encapsulation efficiency. As shown in Fig. 5b, the lowest BSA-FITC concentration (25 μg.mL -1 ) was observed. Similar encapsulation rates were also observed with increased BSA-FITC concentrations (Fig. 5c). Similar trends were observed for various polyelectrolyte concentrations: 94.72 ± 1.17%, 82.88 ± 1.51%, and 82.08 ± 5.57% for 12.5, 25, and 50 mg / mL, respectively (Fig. 5d). A decrease in encapsulation efficiency was observed with increasing polyelectrolyte concentration, which was attributed to COD2E loss. Increasing COD2E concentration resulted in adhesion to Eppendorf tubes and pipette tips. Therefore, 12.5 mg.mL was used for subsequent experiments. -1 Concentration was used.

[0143] After establishing the encapsulation ability with a model protein, FGF2 was encapsulated into COD2E. FGF2 was selected to be integrated into the wound healing process due to its pivotal role in promoting cell proliferation, angiogenesis, and tissue repair. FGF2 is an important factor that stimulates fibroblasts, endothelial cells, and keratinocytes, accelerating wound closure and supporting regenerative mechanisms. Unlike BSA-FITC encapsulation, there was no significant difference in encapsulation yields across various mixing orders and mixing ratios (Fig. 5e). The encapsulation yield was >93% for all methods. Furthermore, to evaluate the effect of FGF2 concentration on loading yield, Method 2 was used to encapsulate 25, 50, 100, and 200 μg / mL FGF2 into COD2E at a mixing ratio of 80:20 (weight ratio). As shown in Fig. 5f, there was no difference in encapsulation yields.

[0144] To evaluate the release profile of FGF2 from COD2E-coated collagen sponges, FGF2 concentrations of 100 and 200 μg.mL were encapsulated in COD2E and a 5-min coating stabilization period was allowed. The coated sponges were immersed in PBS at 37°C and cultured for 60 days. As shown in Fig. 5g, after an initial burst release, a sustained release profile of FGF2 was observed after 60 days of culture, which was consistent with previous studies involving CoA containing BMP-2 and IL-2 encapsulated at 100 and 200 μg.mL. -1 The cumulative release rates of concentration were 8.98 ± 0.03% and 8.22 ± 0.18%, respectively.

[0145] Proteolytic enzymes are secreted to degrade damaged extracellular matrix at the wound site. Proteases directly interact with growth factors, reducing their biological half-life. To evaluate the protective effect of COD2E against proteolytic enzymes, we performed a collagenase treatment test based on previous studies. Briefly, BSA-FITC and BSA-FITC-encapsulated COD2E were embedded in collagen hydrogels and incubated with collagenase II for 16 h (Fig. 5h). After collagenase treatment, BSA-FITC in the lysate was visualized using fluorescence spectroscopy and brightfield microscopy. Collagen hydrogels loaded with unprotected BSA-FITC produced virtually undetectable BSA-FITC (Fig. 5i) due to collagenase-mediated proteolysis, whereas the presence of BSA-FITC was confirmed in the COD2E-embedded hydrogels (Fig. 5j). Reflecting the results obtained from Coa, COD2E was confirmed to protect encapsulated proteins from proteolytic enzymes.

[0146] [Experimental Example 4] In vitro evaluation of biocompatibility and bioactivity of COD2E coating

[0147] The intrinsic toxicity of COD2E as a drug carrier was evaluated. The biocompatibility of COD2E was determined using the indirect contact method. NIH-3T3 fibroblasts were cultured with COD2E extract for 5 days, and then both the Cell Counting Kit-8 (CCK-8) and LIVE / DEAD™ assays were performed. The CCK-8 assay confirmed that the presence of COD2E did not negatively affect cell viability (Fig. 6a). Furthermore, fibroblast proliferation and morphological elongation were significantly increased in cells exposed to COD2E compared to the control group (Fig. 6b). To confirm the intrinsic function of FGF2 throughout the encapsulation and release steps, bioactivity was analyzed. To evaluate the effect of COD2E coating on cell proliferation, the control (pure) and COD2E-coated collagen sponges containing various FGF2 concentrations (0, 25, 50, 100, and 200 μg / mL) were cultured in Transwell™ plates containing NIH-3T3 cells (Fig. 6c). As shown in Fig. 6d, the proliferation rate of the COD2E-coated group decreased until the third day of culture, indicating that the catechol group inhibited cell proliferation in the early stage by chelating ions present in the cell culture medium within COD2E for proliferation. However, after 5 days of culture, the proliferation rates of all samples increased. We confirmed that this trend was probably due to the sudden release of FGF2 from COD2E (Fig. 5g). The ED50 (effective dose to achieve 50% of the desired result) of FGF2 is known to be less than 0.2 ng / mL in terms of biological activity. The initial burst release of FGF2 during the first 3 days probably exceeded the ED50 and inhibited cell proliferation.

[0148] To further verify the bioactivity of COD2E coating, activation of extracellular signal-regulated kinase (ERK) was measured in control (pure), free (FGF2-soaked) and COD2E-coated (various concentrations of FGF2; 0, 25, 50, 100, and 200 μg.mL -1 ) were compared between collagen sponges. Upon binding to FGF receptors, FGF2 triggers downstream signaling pathways, particularly the ERK pathway. Specifically, the binding activates the dimerization of cytoplasmic tyrosine kinases by promoting the phosphorylation of tyrosine residues. These phosphorylated residues serve as anchor points for downstream signaling molecules such as Ras-Raf-MEK-MAPK and ERK sequences. We confirmed that the ERK activation observed in the control and free groups could be attributed to the absence of cytotoxic substances and the effect of fetal bovine serum (FBS) in the culture medium, which promotes cell proliferation. Quantification of the p-ERK / total ERK ratio was performed using the Meso Scale Discovery™ MULTI-SPOT assay (Fig. 6e). After 1 day of culture, there was no substantial difference between the different groups. However, after 3 days, the groups without FGF2 (control and COD2E0-coated) showed a decreased p-ERK / total ERK ratio compared to the groups treated with FGF2. By day 5, COD2E50 and COD2E100-coated samples exhibited the most pronounced ERK ratios, while ERK activity in the other groups was similar to or lower than that of the control group. Notably, ERK activation in COD2E200-coated cells was lower than that in the control cells. This may have been due to excessive FGF2 release, exceeding the known ED50 concentration. Overall, in vitro bioactivity tests confirmed that COD2E coating induced higher cell proliferation.

[0149] Furthermore, the antioxidant properties of COD2E were evaluated, as catechol groups derived from DOPA are known to have antioxidant properties. This is important for promoting wound healing, as wounds trigger various immune and inflammatory responses, resulting in high concentrations of reactive oxygen species (ROS). While moderate ROS levels can promote wound healing, excessive ROS levels can worsen chronic wounds. As shown in Figure 6f, both D-Fuc and COD2E effectively converted the purple hue of DPPH (1,1-diphenyl-2-picryl hydrazyl) solution to yellow. This color change indicates that the nitrogen atom of DPPH captures the hydrogen atom of DOPA. Furthermore, as shown in Figures 6g and h, quantification of ROS levels was performed using the 2'7'-dichlorofluorescein diacetate (DCFDA) assay, confirming the antioxidant properties of the COD2E system. Confocal laser scanning microscopy images show intracellular ROS levels, with higher green signals corresponding to increased ROS levels. Therefore, COD2E may promote enhanced healing and tissue repair by regulating ROS levels at the wound site.

[0150] To evaluate the leak-proofing and blood compatibility of COD2E, the liquid-liquid phase separation phenomenon was evaluated. Phase separation occurs when oppositely charged polyelectrolyte dispersions are mixed, leading to the instantaneous net charge neutralization characteristic of coacervation. Figure 6(i) shows the surfaces of commercial collagen sponges coated with various COD2E concentrations and covered with blood. Collagen sponges known for their blood-clotting properties were used as controls. Blood clotting was observed in the control group, which did not leak into the surrounding DI water. However, when COD2E coating was applied to the sponges, the blood repelled due to the phase separation properties of COD2E, preventing clotting and allowing it to diffuse into the DI water. Interestingly, increasing the COD2E concentration resulted in significant blood repulsion. This highlighted the waterproofing ability of COD2E, which can act as a sealant to prevent blood leakage. This further corroborated the leak test results of the COD2E-treated collagen sponges shown in Figure 6(j). To eliminate bias due to the adhesive properties of the material, the adhesive was used consistently across all test groups. The untreated control group showed liquid leakage within 5 minutes, whereas the COD2E-treated control group prevented leakage for up to 16 hours. Considering that standard blood clotting typically occurs within 3 to 5 minutes, this observation emphasized the waterproofing efficacy of the COD2E-treated collagen sponge. This suggests the potential of a novel coagulation system that utilizes physical liquid-liquid phase separation to improve commercial dressings. The blood compatibility of COD2E is shown in Figure 6k. Rat blood was used as the test medium, and Triton™ X-100 and PBS were designated as negative and positive controls, respectively. COD2E was used at concentrations of 6.25, 12.5, and 25 mg / mL for the experiment. The various concentrations of COD2E did not induce any adverse effects in the blood, as evidenced by values ​​similar to those of the PBS control group. These results demonstrate the blood compatibility of COD2E.

[0151] [Experimental Example 5] Evaluation of the Effect of COD2E-Coated Collagen Sponge on In Vivo Wound Healing

[0152] To further investigate the underlying mechanisms of the COD2E system, additional animal studies were conducted comparing the wound healing effects of the control, coacervate (Coa), and COD2E. Animals were sacrificed on postoperative day 7 (POD 7), earlier than the primary in vivo analysis performed on day 14, to analyze ROS levels during the inflammatory phase of wound healing. Histological analysis revealed that wound healing was enhanced in the COD2E group (Fig. 7). Specifically, angiogenesis was observed throughout the full-thickness collagen sponge (CS), and early epithelialization was observed over the implanted CS (Fig. 7a). Furthermore, the expression of 8-hydroxy-2'-deoxygunosine (8-OHdG), a direct marker of oxidative DNA damage, and superoxide dismutase 2 (SOD2), an indirect marker of oxidative stress, was significantly reduced in the COD2E group compared to the control and / or Coa groups (Fig. 7b). The decrease in SOD2 levels reflects reduced oxidative stress in the COD2E group, suggesting that the system effectively scavenged ROS, reducing the need for endogenous antioxidant responses such as SOD2. SOD2 is a key antioxidant enzyme that catalyzes the conversion of superoxide radicals to oxygen and hydrogen peroxide, thereby protecting cells from oxidative damage. Therefore, the lower levels of 8-OHdG and SOD2 suggest that COD2E can directly alleviate oxidative stress and create a more favorable healing environment, which is consistent with our in vitro results (Figure 8).

[0153] The wound healing potential of collagen sponges enhanced with COD2E coating loaded with FGF2 was evaluated using a rat excisional wound splint model. Four groups were evaluated: control group (collagen sponge treated with 25 μL PBS), uncoated group (collagen sponge soaked in 5 μg FGF2 in 25 μL PBS), COD2E0 coating (collagen sponge with 25 μL COD2E coating), and COD2E200 coating (collagen sponge coated with 25 μL COD2E containing 5 μg FGF2) (Fig. 9a).

[0154] Figure 9b shows representative images of wounds across groups at various grafting time points (postoperative days 0, 7, and 14), among which the COD2E200-coated group exhibited an accelerated rate of wound closure. There were significant differences in the wound healing rate over the 14-day postoperative period. Specifically, the COD2E200-coated group showed a significantly reduced residual wound area of ​​7.4±3.9%, which was significantly less than the control group (36.5±8.0%), the glass group (20.5±9.2%), and the COD2E0-coated (17.1±5.4%) collagen sponge group (all p<0.05, Figure 9c). These observed differences prompted a more detailed analysis on postoperative day 14, where distinct differences between groups were macroscopically evident.

[0155] Histological analysis 14 days after surgery revealed rapid re-epithelialization of the wound margins followed by degradation of the collagen sponge. Notably, both the COD2E0 and COD2E200-coated groups exhibited significant cellular infiltration and angiogenesis within the adhesive coacervate-coated collagen sponge (Fig. 9d). Masson's trichrome staining revealed a significant increase in collagen deposition in the COD2E200-coated group compared to the other groups (all p <0.01, Fig. 9e and f). At 14 days after surgery, wounds treated with the COD2E200-coated collagen sponge exhibited nearly complete re-epithelialization over the largely degraded collagen sponge, highlighted by well-organized collagen fibers (Fig. 9d and e). Epithelialization was consistent across all groups until 3 weeks after surgery, but the COD2E200-coated group exhibited significantly enhanced tissue regeneration, particularly in the restoration of skin structures such as hair follicles (Fig. 9g). In the COD2E200 coating group, the combination of sustained FGF release and coacervation significantly promoted wound closure and tissue regeneration.

[0156] On the 14th day after surgery, immunohistochemical evaluation of proliferating cell nuclear antigen (PCNA) showed a significant increase in cell proliferation in the COD2E200-coated group compared to the other groups (all p<0.01, Fig. 9 a and b). This could be attributed to the enhanced mitogenic and chemoattractive properties of FGF promoted by the COD2E-coated collagen sponge, which plays a pivotal role in wound healing. The expression levels of cluster of differentiation (CD) 31 and alpha-smooth muscle actin (α-SMA), which indicate angiogenesis and vascular maturation, respectively, were significantly higher in the COD2E200-coated group (Fig. 9 a, c, and d). In addition, a significant recruitment of α-SMA-positive smooth muscle cells within the vessels was observed in the COD2E200-coated group, highlighting enhanced arteriogenesis (remodeling of existing arteries into larger conduits) compared to the other groups (all p<0.01).

[0157] Additionally, the COD2E200-coated group showed increased expression of fibronectin, an important scaffold for fibroblast migration and cell interaction during wound healing (Fig. 10a and e). Fibroblasts play a central role in wound healing by secreting extracellular matrix components such as fibronectin and collagen, and RT-PCR analysis confirmed a superior collagen type I / III ratio in the COD2E200-coated group compared to the other groups (all p<0.00001, Fig. 10f). The shift to a higher collagen type I / III ratio signified a transition from primarily immature collagen (type III) to a more mature wound state characterized by increased collagen type I. In addition, the MMP-2 / tissue inhibitor of TIMP-2 mRNA expression ratio, which is often associated with non-healing wounds, was decreased in the COD2E200-coated group (Fig. 10g). In summary, the ability of COD2E200 to ensure controlled release of FGF promoted an optimal collagen expression profile encompassing fibronectin and mature collagen type I.

[0158] During wound healing, the COD2E200-coated group exhibited an increased macrophage transition from the inflammatory M1 to the reparative M2 phenotype, with a marked increase in arginase-1 expression (Fig. 11a to d). Furthermore, analysis of the relative proportion of M2 macrophages among CD68-positive cells further supported this transition, highlighting the beneficial effects of COD2E on macrophage polarization (Fig. 11e). RT-PCR results confirmed a bias toward anti-inflammatory cytokines (IL-4 and IL-10) in the COD2E200-coated group (Fig. 11f and g). While the fucoidan in COD2E has been reported to suppress inflammatory responses, its combination with growth factors appeared to enhance tissue regeneration. These results confirm the potential of the FGF-encapsulating coacervate coating to promote M2 macrophage proliferation and advance the tissue remodeling phase.

[0159] The foregoing description of the present invention is for illustrative purposes only. Those skilled in the art will readily appreciate that modifications to other specific embodiments can be made without altering the technical spirit or essential characteristics of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive.

[0160] The scope of the present invention is indicated by the claims set forth below, and all changes or modifications derived from the meaning and scope of the claims and their equivalent concepts should be interpreted as being included in the scope of the present invention.

Claims

1. Coacervate (COD2E) based on dopamine-substituted fucoidan anionic polymer (D-Fuc) and poly-l-lysine cationic polymer (PLL).

2. In claim 1, the coacervate is a coacervate (COD2E) characterized in that the weight ratio of the fucoidan anionic polymer and the poly-l-lysine cationic polymer is 1 to 5:

1.

3. In claim 1, the coacervate is a coacervate (COD2E) characterized in that it encapsulates FGF2 (Fibroblast growth factor 2).

4. A coacervate (COD2E) according to claim 1, characterized in that the coacervate is formed in the form of microdroplet formation.

5. A collagen sponge comprising the coacervate (COD2E) of claim 1.

6. A drug delivery system comprising the coacervate (COD2E) of claim 1.

7. A pharmaceutical composition for preventing or treating antioxidant-related diseases, comprising the coacervate (COD2E) of claim 1.

8. A pharmaceutical composition according to claim 7, characterized in that the antioxidant-related disease is any one of wounds, skin aging, pigmentation, and skin cancer.

9. A health functional food composition for preventing or improving antioxidant-related diseases, comprising the coacervate (COD2E) of claim 1.

10. A health functional food composition according to claim 9, characterized in that the health functional food composition is an inner beauty food.

11. An antioxidant cosmetic composition comprising the coacervate (COD2E) of claim 1.

12. An adhesive composition comprising the coacervate (COD2E) of claim 1. 13.i) A step of dissolving and mixing fucoidan and dopamine in MES (2-morpholin-4-4ylethanesulfonic acid); ii) a step of further adding EDC (n-(3-dimethylaminopropyl)-n-ethylcarbodiimide hydrochloride) to the mixture of step i), mixing, and purifying to synthesize a dopamine-substituted fucoidan anionic polymer; and iii) A method for producing a coacervate according to claim 1, comprising the step of dissolving a poly-l-lysine cationic polymer in PBS (phosphate-buffered saline) and mixing the anionic polymer of step ii).

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