WO2025150711A1 - Adhesive coacervate-based drug delivery system and method for prreparing same - Google Patents

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 producing the same

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

The inherent regenerative capacity of the human body is greatly enhanced by the secretome, which consists of various molecules such as growth factors, cytokines, mRNA, and exosomes. The secretome is a key element of regenerative medicine, acting as a fundamental mediator in cell-to-cell interactions, and secretome-based therapies are emerging as a valid 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, which demonstrate their potential for disease prevention. However, the clinical application of the secretome faces significant challenges due to its 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.

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 required, which may result in various side effects. Bone morphogenetic protein 2 (BMP-2) is known for bone tissue regeneration, but high doses of BMP-2 may induce unwanted epitope formation, which may trigger an immune response with abnormal bone growth. Interleukin 2 (IL-2) is the first cytokine approved by the US Food and Drug Administration (FDA) for immunotherapy. However, due to its short half-life, high doses of IL-2 are usually administered, which may result in severe systemic inflammation and respiratory problems. In addition, enzymatic degradation and structural instability of proteins and mRNA emphasize the need for precise delivery systems. Such systems should enable local and sustained release while protecting the secretome from proteases and nucleases.

The ultra-low surface tension of coacervates allows them to be coated on various surfaces, especially collagen sponges used in various medical products. However, previous studies required a coating stabilization time of 2 hours, which can be a limiting factor in clinical settings. To address this issue, dopamine (DOPA) was introduced into the coacervate system in this study. The catechol group of DOPA is widely used in tissue engineering to impart adhesiveness. In particular, DOPA conjugation to D-Fuc was performed before coacervation with PLL. The coacervation between D-Fuc and PLL was successfully achieved, and was named Coating and Optimized Drug Delivery Enhancement (COD ₂ E) system. The introduction of DOPA did not affect the coacervation and protein encapsulation ability. COD ₂ E significantly extended the biological half-life and enhanced the therapeutic effect by protecting the encapsulated FGF2 from various substances. COD ₂ E provided rapid and uniform coating on a wide range of material surfaces within a short set-up time (5 min). Additionally, the DOPA functional group of COD ₂ E imparted adhesive strength as well as a fast setting time. In this study, we demonstrate that FGF2-encapsulated COD ₂ E cysteine can function as a secretome delivery coating aid for various medical devices.

[Prior art literature]

[Patent Document]

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

The purpose of the present invention is to provide a coacervate (COD ₂ E) 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.

Another object of the present invention is to provide a collagen sponge comprising the coacervate (COD ₂ E).

Another object of the present invention is to provide a drug delivery system comprising the coacervate (COD ₂ E).

Another object of the present invention is to provide a pharmaceutical composition for preventing or treating antioxidant-related diseases comprising the coacervate (COD ₂ E).

Another object of the present invention is to provide a health functional food composition for preventing or improving antioxidant-related diseases, which comprises the coacervate (COD ₂ E).

Another object of the present invention is to provide an antioxidant cosmetic composition comprising the coacervate (COD ₂ E).

Another object of the present invention is to provide an adhesive composition comprising the coacervate (COD ₂ E).

To achieve the above object, the present invention provides a coacervate (COD ₂ E) based on a dopamine-substituted fucoidan anionic polymer (D-Fuc) and a poly-l-lysine cationic polymer (PLL).

In addition, the present invention provides a collagen sponge comprising the coacervate (COD ₂ E).

In addition, the present invention provides a drug delivery system comprising the coacervate (COD ₂ E).

In addition, the present invention provides a pharmaceutical composition for preventing or treating antioxidant-related diseases comprising the coacervate (COD ₂ E).

In addition, the present invention provides a health functional food composition for preventing or improving antioxidant-related diseases, including the coacervate (COD ₂ E).

In addition, the present invention provides an antioxidant cosmetic composition comprising the coacervate (COD ₂ E).

In addition, the present invention provides an adhesive composition comprising the coacervate (COD ₂ E).

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).

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

Figure 1 is a schematic diagram of the COD ₂ E system. (a) shows the advantages of the COD ₂ E system, and (b) shows the composition and coacervation process of the COD ₂ E system. The electrical interaction between dopamine-conjugated fucoidan (D-Fuc) and poly-lysine (PLL) polyelectrolyte is shown to form complex coacervates or microdrops (COD ₂ E). Fibroblast growth factor (FGF2) can be encapsulated within the COD ₂ E system during the coacervation process. In (c), the COD ₂ E system protects the encapsulated growth factor from proteolytic enzymes and reactive oxygen species, thereby enhancing the half-life of the growth factor.

Figure 2 shows the data confirming D-Fuc.

Figures 3 and 4 show the physical properties of DOPA functionalized coacervate (hereinafter referred to as COD ₂ E).

Figure 5 shows data confirming the protein encapsulation efficiency release profile and protective ability of COD ₂ E.

Figure 6 shows data confirming the biocompatibility, bioactivity, and antioxidant capacity of the COD ₂ E coating in vitro .

Figure 7 shows the antioxidant function of the components constituting COD ₂ E, and shows the structural stability and adhesiveness in a ROS environment.

Figure 8 shows the skin regeneration effect due to the antioxidant effect of COD ₂ E in vivo .

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

Figure 10 shows data confirming the enhancement of cell proliferation, angiogenesis, and collagen expression through FGF2 encapsulating COD ₂ E coating.

Figure 11 shows the evaluation of COD ₂ E coating on macrophage distribution.

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

In this study, we chose to introduce dopamine (DOPA) into the coacervate system because the catechol group of DOPA is known to impart adhesiveness. DOPA binding to D-Fuc was performed before coacervation with PLL. The coacervation between D-Fuc and PLL was successfully accomplished, which was named Coating and Optimized Drug Delivery Enhancement (COD ₂ E) system (Fig. 1a). DOPA modification did not affect the coacervation and protein encapsulation ability (Fig. 1b). COD ₂ E could significantly extend the biological half-life and enhance the therapeutic efficacy by protecting the encapsulated FGF2 from various substances (Fig. 1c). COD ₂ E provided rapid and uniform coating on various material surfaces within a short setup time (5 min). In addition, the DOPA functional group of COD ₂ E imparted adhesiveness as well as rapid coating time. In this study, we demonstrate that the COD ₂ E system encapsulating FGF2 can function as a secretome delivery coating supplement for various medical devices and may be utilized in various fields.

The present invention provides a coacervate (COD ₂ E) based on a dopamine-substituted fucoidan anionic polymer (D-Fuc) and a poly-l-lysine cationic polymer (PLL).

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

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

The above coacervate can be configured in the form of microdroplet formation.

In addition, the present invention provides a collagen sponge comprising the coacervate (COD ₂ E).

In addition, the present invention provides a drug delivery system comprising the coacervate (COD ₂ E).

The above drug delivery vehicle may be in an encapsulated form.

In addition, the present invention provides a pharmaceutical composition for preventing or treating antioxidant-related diseases comprising the coacervate (COD ₂ E).

The above antioxidant related diseases may be, but are not limited to, any of wounds, skin aging, pigmentation and skin cancer.

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, dispersing agents, surfactants, binders and lubricants commonly used in the manufacture of pharmaceutical compositions.

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, and solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and such solid preparations can be prepared by mixing at least one excipient, for example, starch, calcium carbonate, sucrose or lactose, gelatin, etc., into the composition. In addition to simple excipients, lubricants such as magnesium stearate and talc can also be used. Liquid preparations for oral administration include suspensions, solutions, emulsions, and syrups, and in addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, flavoring agents, and preservatives may be included. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspending agents can include 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.

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, inhalation, topical, rectal, oral, intraocular or intradermal routes.

The dosage of the effective 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.

In addition, the present invention provides a health functional food composition for preventing or improving antioxidant-related diseases, including the coacervate (COD ₂ E).

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 regulators, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc.

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

In addition, the above health functional food may additionally contain food additives, and its suitability as a “food additive” shall be determined by the standards and criteria for the relevant item in accordance with the general provisions and general test methods of the Food Additives Codex approved by the Ministry of Food and Drug Safety, unless otherwise specified.

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.

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 as to be included in an amount of 1 to 90 parts by weight per 100 parts by weight of the food.

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

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, which is an inner beauty food, and refers to a food that changes the skin constitution to a healthy one by absorbing various ingredients that are good for the skin into the body. Just as you select a cosmetic that suits your skin type, you can select and consume an inner beauty food that suits you by considering your skin condition and lifestyle. More preferably, when a cosmetic containing the cosmetic composition is used together with the inner beauty food, the antibacterial effect and biofilm formation inhibition effect against acne-causing bacteria are significantly increased compared to using only the cosmetic, so that a more effective acne improvement effect can be observed.

In addition, the present invention provides an antioxidant cosmetic composition comprising the coacervate (COD ₂ E).

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.

In the cosmetic composition of the present invention, pharmaceutically or cosmetically acceptable carriers may vary depending on their formulations, but include hydrocarbons such as petrolatum, liquid paraffin, and gelled 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; Preservatives such as paraoxybenzoic acid esters can be mentioned, and the external preparation of the present invention can be manufactured using these by a conventional method. In addition to these, stabilizers, fragrances, colorants, pH adjusters, diluents, surfactants, preservatives, antioxidants, etc. can also be blended as needed. The external preparation of the present invention can be applied to a local wound by a conventional method.

In addition, the present invention provides an adhesive composition comprising the coacervate (COD ₂ E).

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).

Hereinafter, in order to help understand the present invention, examples and the like will be given to explain in detail. However, the following examples and the like only illustrate the content of the present invention, and the scope of the present invention is not limited to the following examples and the like. The examples and the like of the present invention are provided to more completely explain the present invention to a person having average knowledge in the art.

[Example 1] Materials

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), D ₂ O (deuterium oxide), NaOH (sodium hydroxide solution 30 %), CH ₃ COONa (sodium acetate), NaHCO ₃ (sodiumbicarbonate), Na ₂ CO ₃ (sodium carbonate), BSA-FITC, cOmplete™ (protease inhibitor cocktail tablets), HSCH ₂ CH ₂ OH (2-mercaptoethanol), methanol (CH ₃ OH), phenylmethylsulfonyl fluoride, and dimethyl sulfoxide were purchased from Sigma-Aldrich (USA). Tris-HCl (1.5 M, pH 8.8), radioimmunoprecipitation assay buffer, Bradford reagent, sodium dodecyl sulfate (SDS), 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 Bioworld (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). 2-(N-morpholino)ethanesulfonic acid (MES) 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).

[Example 2] Synthesis and confirmation of D-Fuc

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. After that, the mixture was stirred at room temperature for 2 h under an argon gas atmosphere, and the reaction product was purified using a purification membrane (MWCO: 12-14 kDa) for 3 days. 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 freeze-dried, dried, and stored in a refrigerator. ¹ H NMR spectrometer (Bruker Avance III™ 400 MHz, Bruker, USA) was used to confirm whether DOPA was bound to fucoidan (D-Fuc). The concentration of D-Fuc in D ₂ O solvent is 10 mg.mL ^-1 . The range of 2,000-500 cm ^-1 was scanned 32 times with a Fourier transform infrared spectrometer (Nicolet™ IS™ 10, Thermo Scientific, USA) at a resolution of 4 cm ^-1 . The degree of DOPA binding was measured by a UV-Vis spectrometer (Lambda 25, PerkinElmer, USA). DI water with a D-Fuc concentration of 1 mg mL ^-1 was used as the solvent. A DOPA calibration curve was measured at 280 nm in the concentration range of 0.02-0.1 mg mL ^-1 .

[Example 3] Measurement of surface charge, i.e. zeta potential, of COD ₂ E component

The surface charges of fucoidan (hereinafter referred to as Fuc), D-Fuc, DOPA, and PLL were analyzed using a zeta potential analyzer (Zetasizer Nano ZS, Malvern, UK). Each COD ₂ E composition sample was dissolved in buffers with different pH values. The concentration of each sample was 0.1 mg. mL ^-1 . 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.

[Example 4] Preparation of D-Fuc/PLL complex coacervate (COD ₂ E)

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 dissolved at a concentration of 6.25 mg.mL ^-1 , respectively, and mixed at 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 the formation of the coacervate. The morphological changes of COD ₂ E over time were photographed using an optical microscope (DMIL LED, Leica, Germany). In the case of D-Fuc, since it forms coacervate by itself, the pH was adjusted. 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.

[Example 5] Adhesion test

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

[Mathematical Formula 1]

Adhesive strength = F/A

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

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

[Example 6] Preparation of COD ₂ E coated collagen sponge

COD ₂ E-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 COD ₂ E-coated collagen sponges. Protein yield tests were performed and stored at room temperature for 1, 3, or 5 min for coating stabilization. The coated collagen sponges were washed twice with 100 μL 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 COD ₂ E were calculated by the following mathematical equation 2:

[Mathematical formula 2]

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

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

The morphology of COD ₂ E-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.

[Example 7] Protein encapsulation evaluation

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) considering the loaded protein.

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

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

[Mathematical Formula 3]

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

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.

Also, the encapsulation efficiency of D-Fuc:PLL 50:50 (by weight) ratio and various concentrations (25, 50, 100, and 200 μg.mL ^-1 ) of model protein (BSA-FITC) was measured using Method 2 above. 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, the amount of unloaded FGF2 was evaluated using FGF2 ELISA kit at various concentrations (25, 50, 100, and 200 μg.mL ^-1 ) of target protein (FGF2). The protein encapsulation yield was quantified using Equation 3 above.

To analyze the drug encapsulation ratio according to the concentration of the components as drug carriers, the final D-Fuc and PLL concentrations were evaluated using Method 2 with a D-Fuc:PLL 80:20 (wt%) ratio of 12.5, ²⁵ and 50 mg.mL -1 , respectively. The model protein (BSA) concentration was 25 μg.mL ^-1 . 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. The protein encapsulation yield was quantified using the above mathematical equation 3.

[Example 8] Protein release profile

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

[Mathematical Formula 4]

Protein release yield = W_supernatant / W_total × 100

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

[Example 9] Protection experiment

The ability of COD ₂ E to be protected by proteolytic enzymes was evaluated using collagen hydrogels and collagenases. Fifty μL each of BSA-FITC-loaded COD ₂ E and free BSA-FITC were added to 50 μL of collagen free-hydrogel solution, and the final concentrations were adjusted to 25 μg.mL ^-1 BSA-FITC and 3 mg.mL ^-1 collagen (collagen type I, 10.9 mg.mL ^-1 ). The solution was neutralized by adding 1 N NaOH and 10× Dulbecco's phosphate buffer. Free BSA-FITC and BSA-FITC-loaded COD ₂ E were gelated after reaction for 1 h at 37 °C. 100 U.mL ^-1 collagenase II (290 U.mL ^-1 ) was added to 100 μL of collagen gel sample and reacted for 16 h. Each sample was observed with fluorescence and bright-field images (Leica TCS STED CW).

[Example 10] Biocompatibility test

In vitro toxicity tests were performed based on ISO 10993-5 to evaluate biocompatibility. In vitro cytotoxicity tests were performed using the NIH-3T3 cell line (Korean Cell Line Bank, Korea) that was a fibroblast cell line. NIH- _3T3 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. Initially, cells were seeded at a density of 1 × ¹⁰³ cells/well in 96-well plates (SPL Life Sciences) and cultured for 1 day at 37°C in a humidified atmosphere containing 5% _CO2 . The cultured medium was then replaced with fresh DMEM supplemented with COD ₂ E extract. Each sample was cultured for 1, 3, or 5 days at 37°C in a humidified atmosphere containing 5% _CO2 . Cell viability was quantitatively measured by measuring the absorbance at 450 nm after treatment with 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 Ethidium homodimer-1 (Ethd-1) and observed using a confocal laser scanning microscope (Leica TCS STED CW) for 30 min at 37°C.

[Example 11] Proliferation experiment

To determine the effect of COD ₂ E on the FGF2 release rate, the proliferation of NIH-3T3 cells was evaluated. 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 . Cells were seeded at a density of 0.5 × ¹⁰⁴ cells/well in a 24-well plate (SPLInsert™ Hanging) and cultured for 1 day at 37°C in a humidified atmosphere of 5% _CO2 . The medium was then 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 COD ₂ E (D-Fuc and PLL, final concentration = 12.5 mg mL ^-1 ) was loaded with various FGF2 concentrations (0, 25, 50, 100, 200 μg mL ^-1 ). The mixing ratio and method described above 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.

[Example 12] Evaluation of livability

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. Cells were seeded at densities of 2 × ¹⁰⁵ , 1 × ¹⁰⁵ , and 2.5 × ¹⁰⁴ cells/well in 6-well plates (SPLInsert™ Hanging) and cultured for 1, 3, and 5 days, respectively. After culturing for 1 day at 37°C and 5% _CO2 , the medium was replaced with fresh DMEM. Each sample was placed in a 6-well Transwell™. Collagen sponges in pure state, free (saturated with 200 μg.mL ^-1 FGF2), and COD ₂ E-coated [final concentration of D-Fuc and PLL (12.5 mg mL ^-1) and various concentrations of FGF2 (0, 25, 50, 100, 200 μg mL ^-1 )] were loaded with the mixing ratios and methods mentioned above. After culturing for 1, 3, and 5 days at 37°C under 5% CO ₂ , NIH-3T3 cells were harvested and proteins were extracted. Cells were lysed using RIPA buffer and protease inhibitor cocktail. The concentration of the extracted proteins was adjusted to 10 μg.mL ^-1 with SDS loading buffer, 2-mercaptoethonal, and PBS. Concentrated protein solutions were qualitatively evaluated by Western blot analysis (Mini-PROTEAN® Tetra Cell 4-gel Hand Casting system and PowerPac™ HC Power supply; Bio-Rad) to confirm ERK activation. Proteins were separated on 10% acrylamide gels using SDS-PAGE (stacking; 80 V, 30 min; running; 100 V, 90 min). BSA (2 w/v%) was used for blocking. Proteins were transferred to membranes 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). After the membrane was washed five times in TBST buffer, fluorescent chemiluminescence was detected with an Amersham™ lmager 680 (GE, USA).

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 the Western blot analysis. NIH-3T3 cells were harvested after culturing for 1, 3, and 5 days at 37°C in a 5% CO ₂ atmosphere, 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 adjusted to 0.1 μg.mL ^-1 using a protein quantification kit (bicinchoninic acid). 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:

[Mathematical Formula 5]

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

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

[Example 13] Antioxidant test

The antioxidant properties were evaluated using a DPPH assay kit according to the manufacturer's instructions. COD ₂ E and Coa, and their respective components D-Fuc, Fuc, and PLL were prepared in liquid form. The DPPH reagent was generally used as a dark purple solid. To prepare the working solution of the reagent, 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 the assay buffer and DPPH working solution were added to the Eppendorf tube containing 100 μL of each sample. The reaction was maintained at room temperature in the dark for 30 min, and a color change from purple to yellow indicated the antioxidant function.

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

[Example 14] Waterproofing effect

The waterproofing effect and sealability of COD ₂ E were investigated by evaluating its liquid-liquid phase separation properties. Untreated (blank), Free (PBS-saturated), and COD ₂ E-coated (6.25, 12.5, and 25 mg.mL ^-1 concentrations) collagen sponges were tested using a 6-well plate. The samples were treated as described above. The surface of the treated collagen sponges and blood were allowed to interact for 10 or 60 s, respectively. After the interaction, each sample and blood were thoroughly immersed in DI water until they were completely wetted. Accelerated coagulation was transparent, and the color of DI water changed to red when coagulation was inhibited. Therefore, the color of the surrounding DI water was used to evaluate the waterproofing effect.

[Example 15] Static model

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. Then, porcine skin was attached to the cap, and a 2-mm hole was created in the porcine skin using a biopsy punch. All sponges were perfectly fixed to the porcine skin to utilize the liquid-liquid phase separation property of coacervate. Collagen sponges were treated with PBS and COD ₂ E. COD ₂ E was prepared in a volume ratio of 8:2 (D-Fuc:PLL). The samples were centrifuged at 12,000 rpm and 4 °C. The supernatant of COD ₂ E polymer was removed, and the remaining portion, the lower layer, was coated with the 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.

[Example 16] Blood compatibility test

Blood compatibility was assessed 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 stirring up and down and stored at 4°C before use. The collected blood was centrifuged at 2000 rpm for 5 min, and the supernatant was removed. Sample volumes of 900 μL were prepared with different concentrations of COD ₂ E (6.25, 12.5, and 25 mg.mL ^-1 ). PBS and Triton™ X-100 (0.2 v/v%) were used as controls. 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 hour. After incubation, centrifugation was performed at 3000 rpm for 5 minutes. 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 formula 6:

[Mathematical Formula 6]

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

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

[Example 17] Animal experiment

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 cared for in compliance with the guidelines of Laboratory Animals, 8th edition, NRC (2010).

Thirty-two male Sprague-Dawley rats, 6–8 weeks old, weighing 220–250 g, were divided into four study groups: Control, Free, COD ₂ E0-coated, and COD ₂ E200-coated. Four full-thickness wounds, each 10 mm in diameter, were made on the backs of the rats. Silicone rings were used to ensure that wound healing occurred primarily by granulation and re-epithelialization rather than skin contraction. Each group was treated with 25 μL of collagen sponges (8 mm in diameter, 5 mm in thickness; Olympus Terumo Biomaterials) dispersed in PBS. These coated sponges were usually 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 more in-depth analysis, and three rats were euthanized after 3 weeks.

[Example 18] Histological evaluation

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

For immunohistochemical procedures, sections of paraffin-embedded samples were mounted on slides. After deparaffinization, the slides were treated with the following specific 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). The staining process was performed with hematoxylin as a counterstain. Semiquantitative analysis of immunohistochemistry 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, with a resolution of 640×480 or 1280×960 pixels, respectively.

[Example 19] Quantitative reverse transcription PCR

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'-CTTGGGACTGATGCTGGTGACA-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.

[Example 20] Statistical analysis

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.

[Experimental Example 1] Characteristic Analysis of D-Fuc

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

Since DOPA becomes insoluble in water upon oxidation, its functionalization with fucoidan was investigated at various pH levels of deionized (DI) water. As a result, DOPA was confirmed to be successfully bound to fucoidan, as shown in Figs. 2a to 2c. The DOPA-specific peaks (ranging from 6.9 to 7.6) and the fucoidan-specific peaks were identified using ¹ H nuclear magnetic resonance spectroscopy (Fig. 2a). Subsequent Fourier transform infrared spectroscopy confirmed the presence of DOPA by showing peaks at 1550 cm ^-1 (amide II band, N-H bending) and 1650 cm ^-1 (amide I band, C=O stretching) (Fig. 2b). UV-Vis spectroscopy also confirmed the presence of DOPA with a peak at 280 nm (Fig. 2c). The DOPA content in D-Fuc, determined by comparing the concentration measured by UV-Vis spectroscopy, was found to be in the range of 9 to 15 wt%.

Since complex coacervation occurs between oppositely charged polyelectrolytes through electrostatic interactions, anionic and cationic polyelectrolytes should maintain surface charges during coacervation, and coacervation should occur and persist at pH 7.4 because biomedical applications should be performed under physiological conditions. Therefore, the surface charges of D-Fuc and PLL were investigated in the range of pH 3–11. According to 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 was possible under physiological conditions of pH 7.4.

[Experimental Example 2] Functional Verification of COD ₂ E

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

A D-Fuc:PLL ratio of 80:20 (by weight) exhibited the highest turbidity, indicating optimal COD ₂ E formation. In addition, the spontaneous formation of opaque coacervates was confirmed upon mixing (Fig. 3b), and aggregation of fine droplets was observed over time. This aggregation occurs when oppositely charged polymer electrolytes experience electrostatic equilibrium in which they displace water molecules due to their increased surface hydrophobicity, and the subsequent hydrophobic interactions can promote the formation of bulk coacervates. On the other hand, when the surface charges of the polymer electrolytes are similar, a repulsive force is formed between the polymer electrolytes, causing the coacervate to dissociate, which can be reversibly dissociated and reformed by pH adjustment. The reversibility of COD ₂ E was evaluated by changing the pH of the solution, as shown in Fig. 4, after coacervation between D-Fuc and PLL, the pH was 7-8, and the introduction of NaOH to raise the pH (pH 12) induced the spontaneous dissociation of COD ₂ E. When HCl was added to lower the pH to 8-9 to re-establish the coacervate, COD ₂ E was effectively revived. This confirmed that the properties of the drug carrier itself can be controlled by external factors such as pH, and this phenomenon also showed the possibility of programmed drug release from COD ₂ E by controlling pH.

COD ₂ E with various polyelectrolyte concentrations (12.5, 25, and 50 mg mL-1) was compared with fucoidan/PLL coacervate (Coa without DOPA bonds). As shown in Fig. 3c, the bonding strength was significantly enhanced at all concentrations of COD ₂ E. Specifically, Coa exhibited bonding 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, COD ₂ E exhibited bonding 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 adhesion of COD ₂ E was at least 7.7 times higher than that of Coa alone. This improvement was attributed to DOPA, which provides strong adhesion.

The adhesion strength of COD ₂ E was further evaluated by cyclic loading tests involving several attachment-detachment cycles. A polyelectrolyte concentration of 12.5 mg.mL ^-1 was applied to the porcine skin substrate. As shown in Fig. 3d, the average adhesion strength was 183 kPa and increased with subsequent cycles. This could be due to the evaporation of water molecules between COD ₂ E and porcine skin, which led to the agglomeration of COD ₂ E microcapsules into a bulk structure, enabling stronger adhesion. Furthermore, the covalent bond formation between the organic surface (porcine skin) and COD ₂ E could be caused by the reaction with the abundant sulfate and amide side chains following DOPA oxidation.

To investigate the coating ability of COD ₂ E, COD ₂ E solution was applied to collagen sponge to evaluate the surface morphology and coating yield. Bovine serum albumin (BSA)-fluorescein isothiocyanate (FITC) conjugate was encapsulated in COD ₂ E. After dropping COD ₂ E solution onto the collagen sponge, the collagen sponge was visualized using fluorescence imaging 1, 3, and 5 min later. As shown in Fig. 3e, the top and bottom surfaces of the sponge were uniformly coated. In particular, the coating yield of COD ₂ E was higher than that of Coa regardless of the polyelectrolyte concentration and required less coating or curing time (Fig. 3f). Specifically, the previous Coa coating required 2 h of curing time, whereas the COD ₂ E coating required only 5 min. All COD ₂ E coating yields exceeded 83%, while the highest coating yield of Coa was 77.57 ± 4.95%. The enhanced coating ability could possibly be attributed to the adhesiveness of DOPA. The rapid and homogeneous coating ability of COD ₂ E confirmed its potential as an effective bioactive coating for delivery of various proteins and drugs.

[Experimental Example 3] COD ₂ E, a drug delivery coating system

To investigate the encapsulation ability of COD ₂ E, BSA-FITC conjugate was used as a model protein. The protein mixing order and mixing ratio were compared as shown in Fig. 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 maintained at 25 μg.mL ^-1 . The encapsulation yield of the model protein exceeded 80% regardless of various parameters. Although there was no significant difference among the mixing orders, the encapsulation yield (93.76 ± 1.19%) was higher when the mixing ratio was 50:50. In addition, Method 2 with a mixing ratio of 50:50 (weight ratio) was used to evaluate the effect of BSA-FITC and polyelectrolyte concentrations on the encapsulation efficiency. As shown in Fig. 5b, the highest encapsulation rate of 94.72 ± 1.17% was observed at the lowest BSA-FITC concentration (25 μg.mL ^-1 ). Also, similar encapsulation rates were 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 the loss of COD ₂ E. Increasing COD ₂ E concentration resulted in adhesion to the Eppendorf tube and pipette tips. Therefore, a concentration of 12.5 mg.mL ^-1 was used in the subsequent experiments.

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

To evaluate the release profile of FGF2 from COD ₂ E-coated collagen sponges, FGF2 concentrations of 100 and 200 μg.mL were encapsulated in COD ₂ E and a coating stabilization period of 5 min 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 incubation, which was consistent with previous studies involving Coa encapsulating BMP-2 and IL-2. The cumulative release rates at concentrations of 100 and 200 μg.mL ^-1 were 8.98 ± 0.03% and 8.22 ± 0.18%, respectively.

Proteolytic enzymes are secreted to degrade the damaged extracellular matrix at the wound site. Proteases directly interact with growth factors to reduce their biological half-life. To evaluate the protective effect of COD ₂ E against proteolytic enzymes, collagenase treatment tests were performed based on our previous studies. Briefly, BSA-FITC and BSA-FITC-encapsulated COD ₂ E were embedded in collagen hydrogels and then 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 COD ₂ E-embedded hydrogels (Fig. 5j). Reflecting the results obtained from Coa, COD ₂ E was confirmed to protect the encapsulated protein from proteolytic enzymes.

[Experimental Example 4] Evaluation of biocompatibility and bioactivity of in vitro COD ₂ E coating

The intrinsic toxicity of COD ₂ E as a drug carrier was evaluated. The biocompatibility of COD ₂ E was determined using the indirect contact method. NIH-3T3 fibroblasts were cultured with COD ₂ E extract for 5 days, and then both Cell Counting Kit-8 (CCK-8) and LIVE/DEAD™ assays were performed. The CCK-8 assay confirmed that the presence of COD ₂ E did not adversely affect cell viability (Fig. 6a). In addition, the proliferation and morphological elongation of fibroblasts were markedly increased in cells exposed to COD ₂ E compared to the control (Fig. 6b). The bioactivity was analyzed to confirm the intrinsic function of FGF2 throughout the encapsulation and release steps. To evaluate the effect of COD ₂ E coating on cell proliferation, the control (pure) and COD ₂ E-coated collagen sponges with 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 COD ₂ E-coated group decreased until the 3rd day of culture, which indicated that the catechol group inhibited cell proliferation at the early stage by chelating the ions present in the cell culture medium in COD ₂ E for proliferation. However, the proliferation rates of all samples increased after 5 days of culture. It was confirmed that this trend was probably due to the sudden release of FGF2 from COD ₂ E (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 inhibited cell proliferation beyond the ED50.

To further validate the bioactivity of COD ₂ E coating, the activation of extracellular signal-regulated kinase (ERK) was compared between control (pure), free (FGF2-soaked) and COD ₂ E-coated (various concentrations of FGF2; 0, 25, 50, 100, and 200 μg.mL ^-1 ) collagen sponges. Upon binding to FGF receptors, FGF2 triggers downstream signaling pathways, particularly the ERK pathway, specifically, the binding activates dimerization of cytoplasmic tyrosine kinases by promoting phosphorylation of tyrosine residues. These phosphorylated residues serve as anchor points for downstream signaling molecules such as Ras-Raf-MEK-MAPK and ERK sequences. The ERK activation observed in the control and free groups could be attributed to the absence of cytotoxic agents 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 among the different groups. However, after 3 days, the groups without FGF2 (control and COD ₂ E0 coating) showed a decreased p-ERK/total ERK ratio compared to the groups treated with FGF2. By day 5, COD ₂ E50 and COD ₂ E100-coated samples showed the most prominent ERK ratio, while the ERK activities of the other groups were similar to or lower than those of the control group. In particular, the ERK activation of COD ₂ E200-coated cells was lower than that of the control cells. This could be due to the excessive release of FGF2 beyond the known ED50 concentration. Overall, the in vitro bioactivity tests confirmed that COD ₂ E coating induced higher cell proliferation.

In addition, since the catechol group derived from DOPA is known to have antioxidant properties, the antioxidant properties of COD ₂ E were evaluated. This is important for promoting wound healing, as wounds induce various immune and inflammatory responses, resulting in high concentrations of reactive oxygen species (ROS). Appropriate ROS levels can promote wound healing, but excessive ROS levels can aggravate chronic wounds. As shown in Fig. 6f, both D-Fuc and COD ₂ E 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 captured the hydrogen atom of DOPA. In addition, as shown in Fig. 6g and h, the quantification of ROS levels was performed using the 2'7'-dichlorofluorescein diacetate (DCFDA) assay, which confirmed that the COD ₂ E system had antioxidant properties. Confocal laser scanning microscopy images show intracellular ROS levels, with higher green signals corresponding to increased ROS levels. Therefore, COD ₂ E may promote enhanced healing and tissue repair by modulating ROS levels at the wound site.

To evaluate the leak-proofing and blood compatibility of COD ₂ E, the liquid-liquid phase separation phenomenon was evaluated. Phase separation occurs when oppositely charged polyelectrolyte dispersions are mixed, which leads to an instantaneous net charge neutralization, which is characteristic of coacervation. Figure 6(i) shows the surface of commercial collagen sponges coated with various COD ₂ E concentrations and covered with blood. Collagen sponges known for their blood coagulation properties were used as controls. Blood coagulation was observed in the control group, which did not leak into the surrounding DI water. However, when COD ₂ E coating was applied to the sponge, blood was repelled by the phase separation properties of COD ₂ E, preventing coagulation and spreading into the DI water. Interestingly, significant blood repulsion occurred as the COD ₂ E concentration increased. This highlighted the waterproofing ability of COD ₂ E, which can act as a sealant to prevent blood leakage. This further confirmed the leak test results of COD ₂ E-treated collagen sponges shown in Figure 6(j). To eliminate bias due to adhesiveness of the material, the adhesive was used consistently in all test groups. The untreated control group showed liquid leakage within 5 minutes, while the COD ₂ E treated control group prevented leakage for up to 16 hours. Considering that standard blood clotting is usually completed within 3–5 minutes, this observation emphasized the waterproofing efficacy of the COD ₂ E treated collagen sponge. This suggested the potential of a novel clotting system using physical liquid-liquid phase separation to improve commercial dressings. The blood compatibility of COD ₂ E was shown in Fig. 6k. Rat blood was used as the test medium, and Triton™ X-100 and PBS were designated as negative and positive controls, respectively. COD ₂ E was used at concentrations of 6.25, 12.5, and 25 mg/mL for the experimental purposes. COD ₂ E at various concentrations did not induce any adverse effects in blood, as evidenced by values similar to those of the PBS control group. These results demonstrated that COD ₂ E is compatible with blood.

[Experimental Example 5] Evaluation of the Effect of COD ₂ E Coated Collagen Sponge on Wound Healing in Vivo

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

The wound healing potential of collagen sponges reinforced with COD ₂ E coating loaded with FGF2 was evaluated using a rat excision wound splint model. Four groups were evaluated: control (collagen sponge treated with 25 μL PBS), untreated group (collagen sponge soaked with 5 μg FGF2 in 25 μL PBS), COD ₂ E0 coating (collagen sponge with 25 μL COD ₂ E coating) and COD ₂ E200 coating (collagen sponge coated with 25 μL COD ₂ E containing 5 μg FGF2) (Fig. 9a).

Figure 9b shows representative images of wounds across groups at different grafting time points (post-surgery days 0, 7, and 14), among which the COD ₂ E200-coated group exhibited an accelerated wound closure rate. There was a significant difference in the wound healing rate over the 14-day post-surgery period. Specifically, the COD ₂ E200-coated group showed a significantly reduced residual wound area by 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 COD ₂ E0-coated (17.1±5.4%) collagen sponge groups (all p<0.05, Figure 9c). These observed differences prompted a more detailed analysis to be performed on post-surgery day 14, where distinct differences between the groups were macroscopically evident.

Histological analysis revealed rapid re-epithelialization of the wound margins followed by degradation of the collagen sponge at 14 days postoperatively. In particular, both COD ₂ E0 and COD ₂ E200-coated groups showed significant cellular infiltration and angiogenesis within the adhesive coacervate-coated collagen sponge (Fig. 9d). Masson's trichrome staining showed significantly increased collagen deposition in the COD ₂ E200-coated group compared with the other groups (all p<0.01, Fig. 9e and f). At 14 days postoperatively, wounds treated with the COD ₂ E200-coated collagen sponge showed almost complete epithelization over the largely degraded collagen sponge highlighted by well-organized collagen fibers (Fig. 9d and e). Epithelialization was consistent in all groups up to 3 weeks postoperatively, but the COD ₂ E200-coated group showed markedly enhanced tissue regeneration, particularly with respect to restoration of skin structures such as hair follicles (Fig. 9g). In the COD ₂ E200 coating group, the combination of sustained FGF release and coacervation significantly promoted wound closure and tissue regeneration.

On the 14th day after surgery, immunohistochemical evaluation of proliferating cell nuclear antigen (PCNA) showed that cell proliferation was significantly increased in the COD ₂ E200-coated group compared with 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 COD ₂ E-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 represent angiogenesis and vascular maturation, respectively, were prominent in the COD ₂ E200-coated group (Fig. 9 a, c and d). In addition, significant recruitment of α-SMA-positive smooth muscle cells within the vessels was observed in the COD ₂ E200-coated group, which highlighted enhanced arteriogenesis (remodeling of existing arteries into larger conduits) compared with the other groups (all p<0.01).

In addition, the COD ₂ E200 coating 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 COD ₂ E200 coating group compared with 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 COD ₂ E200 coating group (Fig. 10g). In summary, the ability of COD ₂ E200 to ensure controlled release of FGF promoted an optimal collagen expression profile encompassing fibronectin and mature collagen type I.

During wound healing, the COD ₂ E200-coated group showed an increased macrophage transition from the inflammatory M1 to the reparative M2 phenotype with a marked increase in the expression of arginase 1 (Fig. 11a to d). In addition, analysis of the relative proportion of M2 macrophages among CD68 positive cells further supported this transition, highlighting the beneficial effect of COD ₂ E on macrophage polarization (Fig. 11e). RT-PCR results confirmed the anti-inflammatory cytokine (IL-4 and IL-10) bias in the COD ₂ E200-coated group (Fig. 11f and g). While the fucoidan in COD ₂ E has been reported to suppress inflammatory responses, its combination with growth factors appeared to enhance tissue regeneration. These results confirmed the potential of the FGF-encapsulating coacervate coating in promoting M2 macrophage proliferation and advancing the tissue remodeling phase.

The above description of the present invention is for illustrative purposes only, and those skilled in the art will understand that the present invention can be easily modified into other specific forms without changing the technical idea or essential characteristics of the present invention. Therefore, it should be understood that the embodiments described above are illustrative in all respects and not restrictive.

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 (13)

Coacervates based on dopamine-substituted fucoidan anionic polymer (D-Fuc) and poly-l-lysine cationic polymer (PLL) (COD ₂ E). In claim 1, the coacervate is a coacervate (COD ₂ E) characterized in that the weight ratio of the fucoidan anionic polymer and the poly-l-lysine cationic polymer is 1 to 5:1. In claim 1, the coacervate is characterized in that it is encapsulated by carrying FGF2 (Fibroblast growth factor ₂ ). A coacervate (COD ₂ E) according to claim 1, characterized in that the coacervate is formed in the form of microdroplet formation. A collagen sponge comprising the coacervate (COD ₂ E) of claim 1. A drug delivery system comprising the coacervate (COD ₂ E) of claim 1. A pharmaceutical composition for preventing or treating antioxidant-related diseases, comprising the coacervate (COD ₂ E) of claim 1. 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. A health functional food composition for preventing or improving antioxidant-related diseases, comprising the coacervate (COD ₂ E) of claim 1. A health functional food composition according to claim 9, characterized in that the health functional food composition is an inner beauty food. An antioxidant cosmetic composition comprising the coacervate (COD ₂ E) of claim 1. An adhesive composition comprising the coacervate (COD ₂ E) of claim 1. 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).