Composition for wound healing or tissue regeneration

Abstract

The present invention may be able to accelerate the processes of wound healing and skin tissue damage improvement by irradiating LED light to a photosensitizer-bound hyaluronic acid hydrogel to induce reactive oxygen. In particular, the composition of the present invention may be used for wound healing and skin tissue regeneration by promoting angiogenesis and inducing collagen accumulation in the dermal layer through a combination of photosensitizer-bound hyaluronic acid and LED light irradiation. In addition, the composition of the present invention has an effective antibacterial effect against various microorganisms, and can therefore be usefully employed as an antibacterial agent.

Description

Composition for wound healing or tissue regeneration

[0001] The present invention relates to a method for wound healing, tissue regeneration, or antibacterial action using a complex in which a photosensitizer is combined with a hyaluronic acid-based hydrogel.

[0002]

[0003] The skin is the most extensive organ in the body and serves as a protective barrier against external environmental factors. When an injury occurs, a complex wound healing process begins, involving hemostasis, inflammation, proliferation, and tissue remodeling.

[0004] Hydrogels have been widely used because they promote faster healing due to their ability to maintain a moist environment at the wound site. As a biocompatible material, hyaluronic acid (HA) is a naturally occurring polysaccharide found in connective tissue and is a natural substance present in animal skin, known to help maintain skin hydration and elasticity. Hydrogel dressings are essential for promoting effective wound healing because they can create a moist environment that supports tissue regeneration. Composed of cross-linked HA molecules, these dressings possess excellent water absorption capabilities and are biocompatible and non-immunogenic, making them suitable for treating a wide range of wounds, including chronic wounds and burns.

[0005] The field of bioengineering is constantly evolving, primarily focusing on the creation of new materials and technologies to solve urgent medical problems. In the field of wound healing, continuous research is being conducted to discover new materials capable of promoting healing and protecting against infection. Since material selection plays a crucial role in wound healing by influencing recovery speed and infection prevention, there is a need to discover new materials capable of creating a favorable environment that promotes the body's natural healing processes during the wound healing process.

[0006] The inventors arrived at the present invention by discovering that combining a photosensitizer molecule with HA generates ROS when exposed to light, thereby enhancing antibacterial efficacy against various pathogenic bacteria while being highly effective for wound healing and tissue regeneration.

[0007]

[0008] Based on the observation that when a photosensitizer is activated by light, it absorbs photons and undergoes a series of photochemical reactions to generate singlet oxygen, which exhibits a potent antibacterial effect, the inventors have discovered a novel material that is highly biocompatible and possesses antibacterial effects for wound healing, tissue regeneration, and the management of biofilm-related infections.

[0009] Therefore, the objective of the present invention is to provide a composition for wound healing, antibacterial action, and tissue regeneration.

[0010] Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims, and drawings.

[0011]

[0012] According to one embodiment of the present invention, the present invention provides a wound healing composition comprising a hyaluronic acid (HA) hydrogel as an active ingredient.

[0013] According to another embodiment of the present invention, the present invention provides a method for preventing or treating a wound, comprising the step of administering a hyaluronic acid hydrogel to a subject.

[0014] In the present invention, the active ingredient is characterized by additionally having a photosensitizer molecule bonded to a hyaluronic acid hydrogel.

[0015] In the present invention, the photosensitizer is characterized by being bonded to the hyaluronic acid hydrogel through a polyethylene glycol-based crosslinking agent, and the polyethylene glycol-based crosslinking agent may be PEGDE (Poly(ethylene glycol) diglycidyl ether), but is not limited thereto.

[0016] In the present invention, the composition is characterized by being used in conjunction with irradiation of light onto a lesion, and the light may be an LED (Light Emitting Diode), but is not limited thereto.

[0017] The inventors have made diligent research efforts to develop a biocompatible material possessing wound healing, tissue regeneration, and antibacterial capabilities. As a result, they discovered that by irradiating LED light onto a hyaluronic acid hydrogel combined with the photosensitizer to accurately release ROS, it can be effectively applied to wound healing and tissue regeneration through photodynamic therapy (PDT).

[0018] In this specification, the term "hyaluronic acid" refers to a high-molecular-weight polysaccharide that naturally exists in various parts of animals, such as connective tissue, skin, joints, and eyes. This substance possesses excellent water-retaining capabilities and performs important physiological roles within the body. It is a type of glycosaminoglycan and generally does not form amide bonds in its pure state. It is composed of alternating D-glucuronic acid and N-acetyl-d-glucosamine units connected mainly by β(1→4) and β(1→3) glycosidic bonds, forming a linear structure in which N-acetyl-D-glucosamine and glucuronic acid are repeatedly bonded. It is known to be highly hydrophilic and capable of attracting water up to 300 to 1,000 times its own weight.

[0019] In this specification, the term "hydrogel" refers to a material composed of hydrophilic polymers with a three-dimensional network structure capable of containing a large amount of water. It has an intermediate state between liquid and solid and is utilized in various fields due to its physical and chemical properties. Hydrogels have a structure in which polymer chains are cross-linked and can absorb water and swell. They are widely known as materials suitable for medical and bio applications because they are soft, flexible, and possess properties similar to biological tissues.

[0020] In this specification, the term "photosensitive agent" refers to a substance that absorbs light, converts energy into another form, and transfers this energy to surrounding molecules or systems to induce specific chemical or biological reactions. These agents are primarily sensitive to light of specific wavelengths and are used in various applications. The operating principle of a photosensitive agent is that after absorbing light, its electronic state transitions to an excited state, transferring this energy to surrounding molecules or promoting chemical reactions. They can induce reactions that damage cells by generating reactive oxygen species (ROS). Generally, photosensitive agents transition from a singlet state to a triplet state, react with oxygen molecules to generate reactive oxygen species such as singlet oxygen, and oxidize cells or molecules to induce specific effects. Recently, photosensitive agents are being utilized in Photodynamic Therapy (PDT) by accumulating them in cancer tissue and irradiating them with light of specific wavelengths to generate reactive oxygen species, thereby selectively destroying cancer cells. However, the toxicity of some photosensitizers is a problem, and there are many issues to consider, such as the limitation of light penetration depth depending on the type of photosensitizer and the need for light of specific wavelengths, so research to solve these issues is being actively conducted.

[0021] In this specification, the term "Photodynamic Therapy (PDT)" refers to a non-invasive treatment method that uses light, photosensitizers, and oxygen to treat specific diseases. It is primarily used to selectively destroy cancer cells or pathogens and is utilized in various fields, including skin diseases, cancer, and infectious diseases. It is attracting attention as an innovative treatment method that can complement or replace conventional surgery or radiation therapy, and its scope of application is gradually expanding through the development of new photosensitizers and technologies.

[0022] In this specification, the term "wound" refers to a state in which the anatomical continuity of skin or tissue is damaged by external force or stimulation, or a state in which skin tissue is damaged and the inherent biological functions of skin tissue, including barrier function, are degraded due to an excessive or unwanted immune response or inflammation caused by it. As seen in the examples described below, the composition of the present invention can effectively exhibit the effects of wound healing or tissue regeneration by reducing the size of skin lesions in a wound healing model, significantly improving the wound, and restoring it to a state similar to normal skin tissue.

[0023] In the present invention, the wound to be treated with the composition of the present invention may be selected from the group consisting of abrasions, bruises, lacerations, incisions, punctures, and burns, but is not limited thereto as long as it corresponds to a case where the skin structure composed of the epidermis, dermis, and subcutaneous tissue is damaged.

[0024] In this specification, the term "treatment" means (a) inhibition of the progression of a disease, illness, or symptom; (b) alleviation of a disease, illness, or symptom; or (c) elimination of a disease, illness, or symptom. When the composition of the present invention is administered to a subject and irradiated with light, reactive oxygen species (ROS) are generated. Immune cells respond to the reactive oxygen species by secreting pro-inflammatory cytokines, and fibroblasts and keratinocytes also respond to the reactive oxygen species to promote proliferation. Consequently, this serves to inhibit, eliminate, or alleviate the progression of symptoms caused by excessive immune and inflammatory responses in skin tissues. Therefore, the composition of the present invention may serve as a treatment for these diseases on its own, or it may be applied as an adjuvant for treatment of the said diseases when administered together with other pharmacological components. Accordingly, in this specification, the terms "treatment" or "therapeutic agent" include the meaning of "adjuvant for treatment" or "adjuvant for treatment."

[0025] In this specification, the terms "administration" or "to administer" refer to directly administering a therapeutically effective amount of the composition of the present invention to a subject so that an equal amount is formed within the subject's body.

[0026] In the present invention, the term "therapeutic effective amount" refers to the content of a composition in which the pharmacological component within the composition is contained in an amount sufficient to provide a therapeutic or preventive effect to an individual to whom the pharmaceutical composition of the present invention is to be administered, and includes the meaning of "preventive effective amount."

[0027] In this specification, the term "object" includes, without limitation, humans, mice, rats, guinea pigs, dogs, cats, horses, cattle, pigs, monkeys, chimpanzees, baboons, or rhesus monkeys. Specifically, the object of the present invention is a human.

[0028] In the present invention, the photosensitive agent may be at least one selected from the group consisting of chlorin, porphyrin, phthalocyanine, purpurin, texaphyrin, or derivatives thereof, specifically, it may be at least one selected from the group consisting of chlorin e6 (Ce6), bacteriochlorin, hematoporphyrin, benzoporphyrin, and protoporphyrin IX (PpIX), and more specifically, it may be chlorin e6 (Ce6) or bacteriochlorin, but is not limited thereto as long as it corresponds to a preparation that transitions its electronic state to an excited state after absorbing light, transfers this energy to surrounding molecules, or promotes a chemical reaction.

[0029] In the present invention, the composition is characterized by being used in conjunction with irradiation of LED (Light Emitting Diode) light.

[0030] In the present invention, the output of the LED light is 10 μW / cm² 2 Up to 50 mW / cm 2 It can be investigated as. Specifically, 50 μW / cm² 2 Up to 40 mW / cm 2 It can be investigated as, more specifically, 80 μW / cm² 2 Up to 30 mW / cm 2 It may be investigated as such, but is not limited to this.

[0031] In the present invention, the irradiation time of the LED light may be within 5 to 80 minutes. Specifically, it may be within 10 to 70 minutes, and more specifically, within 15 to 60 minutes, but is not limited thereto.

[0032] The composition of the present invention may be used as a pharmaceutical composition or a cosmetic composition depending on its use.

[0033] In the present invention, the pharmaceutical composition may be characterized as being in the form of a capsule, tablet, granule, injection, ointment, powder, or beverage.

[0034] The pharmaceutical composition of the present invention is not limited to these, but may be formulated and used in the form of oral formulations such as powders, granules, capsules, tablets, and aqueous suspensions, as well as topical preparations, suppositories, and sterile injectable solutions, according to conventional methods. The pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier. For oral administration, the pharmaceutically acceptable carrier may include binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, colorants, flavorings, etc. For injectable preparations, it may include buffers, preservatives, analgesics, solubilizers, isotonic agents, stabilizers, etc., in combination; and for topical administration, a base, excipients, lubricants, preservatives, etc. The formulation of the pharmaceutical composition of the present invention may be prepared in various ways by mixing with the pharmaceutically acceptable carrier described above. For example, for oral administration, it can be manufactured in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, etc., and for injectables, it can be manufactured in the form of unit dosing ampoules or multiple dosing ampoules. In addition, it can be formulated as a solution, suspension, tablet, capsule, sustained-release formulation, etc.

[0035] Meanwhile, examples of carriers, excipients, and diluents suitable for formulation include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, or mineral oil. Additionally, fillers, anticoagulants, lubricants, wetting agents, fragrances, emulsifiers, preservatives, etc. may be additionally included.

[0036] The routes of administration of the pharmaceutical composition according to the present invention are not limited to but include oral, intravenous, intramuscular, intra-arterial, intramedullary, intradural, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal, topical, sublingual, or rectal. Oral or parenteral administration is preferred.

[0037] The pharmaceutical composition of the present invention may be administered parenterally, specifically by transdermal administration, subcutaneous administration, or topical application to the skin surface. More specifically, the pharmaceutical composition of the present invention may be a transdermal agent or a topical agent, but is not limited thereto.

[0038] The pharmaceutical composition of the present invention may vary depending on several factors including the activity of the specific compound used, age, body weight, general health, gender, diet, time of administration, route of administration, elimination rate, drug combination, and the severity of the specific disease to be prevented or treated, and the dosage of the pharmaceutical composition may be appropriately selected by a person skilled in the art, depending on the patient's condition, body weight, degree of disease, form of medication, route of administration, and duration, and may be administered at a dose of 0.0001 to 50 mg / kg or 0.001 to 50 mg / kg per day. Administration may be administered once a day or divided into several doses. The dosage does not limit the scope of the present invention in any way. The pharmaceutical composition according to the present invention may be formulated as a pill, coated tablet, capsule, liquid, gel, syrup, slurry, or suspension.

[0039] The pharmaceutical composition of the present invention may be prepared in a unit volume form or contained in a multi-volume container by formulation using a pharmaceutically acceptable carrier and / or excipient, according to a method that can be easily practiced by a person skilled in the art to which the invention pertains.

[0040] The pharmaceutical composition of the present invention is characterized by having a significantly superior wound healing effect.

[0041] The ingredients included in the cosmetic composition of the present invention may include ingredients commonly used in cosmetic compositions in addition to the hyaluronic acid hydrogel combined with the photosensitizer as an active ingredient, such as conventional auxiliary agents like antioxidants, stabilizers, solubilizers, vitamins, pigments, and fragrances, and carriers, but are not limited thereto.

[0042] The cosmetic composition of the present invention can be prepared in any formulation conventionally manufactured in the art, and may be formulated, for example, as a solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, surfactant-containing cleansing, oil, powder foundation, emulsion foundation, wax foundation, and spray, but is not limited thereto.

[0043] In the present invention, the cosmetic composition may be manufactured in the form of a lotion, nourishing lotion, nourishing essence, massage cream, beauty bath additive, body lotion, body milk, bath oil, baby oil, baby powder, shower gel, shower cream, sunscreen lotion, sunscreen cream, tanning cream, skin lotion, skin cream, UV protection cosmetic, cleansing milk, hair loss treatment cosmetic, face and body lotion, face and body cream, skin whitening cream, hand lotion, hair lotion, cosmetic cream, jasmine oil, bath soap, liquid soap, beauty soap, shampoo, hand sanitizer (hand cleaner), medicinal soap (non-medical use), cream soap, facial wash, body cleanser, scalp cleanser, hair rinse, cosmetic soap, teeth whitening gel, toothpaste, etc. To this end, the composition of the present invention may further include a solvent or a suitable carrier, excipient, or diluent that is commonly used in the manufacture of cosmetic compositions.

[0044] In the case where the formulation of the present invention is a paste, cream, or gel, animal oil, vegetable oil, wax, paraffin, starch, tracanth, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc, or zinc oxide may be used as a carrier component.

[0045] In the case where the formulation of the present invention is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, or polyamide powder may be used as a carrier component, and in particular, in the case of a spray, it may additionally include a propellant such as chlorofluorohydrocarbon, propane / butane, or dimethyl ether.

[0046] When the formulation of the present invention is a solution or an emulsion, a solvent, a solubilizing agent, or an emulsifying agent is used as a carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyl glycol oil, glycerol aliphatic ester, polyethylene glycol, or fatty acid ester of sorbitan.

[0047] In the case where the formulation of the present invention is a suspension, liquid diluents such as water, ethanol, or propylene glycol, ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester, and polyoxyethylene sorbitan ester, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar, or tracant may be used as carrier components.

[0048] In the case where the formulation of the present invention is a cleansing agent containing a surfactant, aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyl taurate, sarcosinate, fatty acid amide ether sulfate, alkylamidobetaine, aliphatic alcohol, fatty acid glyceride, fatty acid diethanolamide, vegetable oil, lanolin derivative, or ethoxylated glycerol fatty acid ester, etc. may be used as a carrier component.

[0049] The types of solvents that may be further added to the cosmetic composition of the present invention are not particularly limited, but, for example, water, saline solution, DMSO, or a combination thereof may be used. In addition, carriers, excipients, or diluents include, but are not limited to, purified water, oil, wax, fatty acid, fatty acid alcohol, fatty acid ester, surfactant, humectant, thickener, antioxidant, viscosity stabilizer, chelating agent, buffer, lower alcohol, etc. Additionally, whitening agents, moisturizers, vitamins, sunscreens, perfumes, dyes, antibiotics, antibacterial agents, and antifungal agents may be included as needed.

[0050] In addition, in the present invention, hydrogenated vegetable oil, castor oil, cottonseed oil, olive oil, palm oil, jojoba oil, and avocado oil may be used as the oil, and beeswax, spermaceti, carnauba, candelilla, montan, ceresin, liquid paraffin, and lanolin may be used as the wax.

[0051] In addition, in the present invention, stearic acid, linoleic acid, linolenic acid, and oleic acid may be used as the fatty acid, cetyl alcohol, octyl dodecanol, oleyl alcohol, pantenol, lanolin alcohol, stearyl alcohol, and hexadecanol may be used as the fatty acid alcohol, and isopropyl myristate, isopropyl palmitate, and butyl stearate may be used as the fatty acid ester. As surfactants, cationic surfactants, anionic surfactants, and nonionic surfactants known in the art may be used, and surfactants derived from natural products are preferred as much as possible. In addition, hygroscopic agents, thickeners, antioxidants, etc., widely known in the cosmetics field may be included, and the types and amounts thereof are as known in the art.

[0052]

[0053] According to another embodiment of the present invention, the present invention provides an antibacterial composition comprising a hyaluronic acid (HA) hydrogel as an active ingredient.

[0054] In the present invention, the active ingredient is characterized by the additional binding of a photosensitizer to a hyaluronic acid hydrogel.

[0055] The composition of the present invention may be used as a pharmaceutical composition or a cosmetic composition depending on its use.

[0056] Since the hyaluronic acid hydrogel, photosensitizer, pharmaceutical composition, cosmetic composition, etc. used in the present invention have already been described above, their description is omitted to avoid excessive duplication.

[0057] In this specification, the term "antimicrobial" refers to the action of inhibiting the growth and proliferation of microorganisms such as bacteria, fungi, and viruses, or killing them. It is a concept that includes both the inhibition of microbial proliferation (bacteriostatic effect), which prevents microorganisms from reproducing further so that the host's immune system can eliminate them, and the killing of microorganisms (bactericidal effect), which is achieved by directly killing microorganisms through the destruction of cell walls, inhibition of protein synthesis, damage to cell membrane function, etc.

[0058] The composition of the present invention may have antimicrobial activity against various microorganisms, and said microorganisms may include bacteria, fungi, and viruses, and among bacteria, may include Gram-positive bacteria, Gram-negative bacteria, and anaerobic bacteria, and may be at least one bacterium selected from the group consisting of Staphylococcus spp., Streptococcus spp., Enterococcus spp., Pseudomonas aeruginosa, Klebsiella pneumoniae, and Acinetobacter baumannii, and specifically may be Staphylococcus aureus or Pseudomonas aeruginosa, but is not limited thereto.

[0059]

[0060] According to another embodiment of the present invention, the present invention provides a tissue regeneration composition comprising a hyaluronic acid (HA) hydrogel as an active ingredient.

[0061] In the present invention, the active ingredient is characterized by the additional binding of a photosensitizer to a hyaluronic acid hydrogel.

[0062] The composition of the present invention may be used as a pharmaceutical composition or a cosmetic composition depending on its use.

[0063] Since the hyaluronic acid hydrogel, photosensitizer, pharmaceutical composition, cosmetic composition, etc. used in the present invention have already been described above, their description is omitted to avoid excessive duplication.

[0064] In this specification, the term "tissue regeneration" refers to the process of restoring the original structure and function by replacing or repairing damaged or lost tissue with new cells and tissues.

[0065] The composition of the present invention has the effect of tissue regeneration and may include epithelial tissue, connective tissue, muscle tissue, nerve tissue, and other special tissues. Specifically, it may be epithelial tissue or muscle tissue, and more specifically, it may be skin tissue or muscle tissue, but is not limited thereto.

[0066]

[0067] The present invention is effective for wound healing and skin tissue regeneration by inducing active oxygen through the promotion of angiogenesis and collagen accumulation in the dermis layer by irradiating LED light onto a hyaluronic acid hydrogel combined with a photosensitizer. Furthermore, the composition of the present invention has an effective antibacterial effect against various microorganisms and can be utilized as an antibacterial agent.

[0068]

[0069] FIG. 1a is a schematic diagram showing the preparation of a complex by combining hyaluronic acid and a photosensitizer, Chlorin e6 (Ce6), according to one embodiment of the present invention.

[0070] FIG. 1b is a schematic diagram of the entire wound healing process that promotes antibacterial action and cell growth by generating active oxygen through the use of light and a hydrogel combining hyaluronic acid and a photosensitizer according to one embodiment of the present invention.

[0071] Figure 2 is a figure showing the results of confirming cytotoxicity caused by the generation of reactive oxygen species (ROS) in NHDF (Normal Human Dermal Fibroblasts) cells by irradiating Ce6-hyaluronic acid with various LED intensities and times according to one embodiment of the present invention using an MTT assay.

[0072] FIGS. 3a and 3b are figures showing the results of confirming ROS generation by irradiating chlorine e6 with LED light using a DPBF (1,3-Diphenylisobenzofuran) assay according to one embodiment of the present invention and monitoring the decomposition of DPBF according to irradiation time and intensity.

[0073] Figures 4a and 4b show the results of confirming the antimicrobial effect against microorganisms by applying to Staphylococcus aureus or Pseudomonas aeruginosa according to one embodiment of the present invention.

[0074] FIGS. 5a to 5c are figures showing the results of measuring the secretion amounts of pro-inflammatory cytokines TNF-α, IL-1, and IL-6 in macrophages induced by ROS according to one embodiment of the present invention using ELISA.

[0075] Figures 6a and 6b show the results of measuring the secretion amounts of anti-inflammatory cytokines TGF-β and IL-10 in macrophages induced by ROS according to one embodiment of the present invention using ELISA.

[0076] FIG. 7 shows 100 μW / cm² according to an embodiment of the present invention. 2 This is a graph showing the change in the degree of fibroblast proliferation according to the irradiation time of LED light under certain conditions.

[0077] FIG. 8 shows 100 μW / cm² according to an embodiment of the present invention. 2This is a graph showing the change in the degree of keratinocyte proliferation according to the irradiation time of LED light under the conditions.

[0078] FIGS. 9a to 9c show 100 μW / cm² of LED light in vascular endothelial cells according to one embodiment of the present invention. 2 This is a diagram showing the results of confirming the effect of promoting neovascularization of vascular endothelial cells according to the 30-minute investigation condition.

[0079] FIGS. 10a and FIGS. 10b are figures showing the change in wound size over time using an in vivo wound healing model (full-thickness wound healing model) according to one embodiment of the present invention.

[0080] Figures 11a and 11b show the results of comparison with normal skin tissue using an in vivo wound healing model (full-thickness wound healing model) according to one embodiment of the present invention.

[0081]

[0082] The present invention will be described in more detail below through examples. These examples are intended solely to explain the invention more specifically, and it will be obvious to those skilled in the art that the scope of the invention is not limited by these examples according to the gist of the invention.

[0083]

[0084] Examples

[0085] Experimental method

[0086] Preparation of Ce6-HA Hydrogel

[0087] Sodium HA was purchased from Lifecore Biomedical (USA, 200 kDa), and EDC, NHS, selenocystamine dihydrochloride (C4H 12N2Se2·2HCl) and polyethylene glycol diglycidyl ether (PEGDE) were purchased from Sigma-Aldrich (St. Louis, Missouri, USA). Chlorin e6 (Ce6) was purchased from Santa Cruz Biotechnology (USA), and Dulbecco's phosphate-buffered saline (DPBS) was purchased from Biosesang (Korea). Ce6-HA was synthesized using carbodiimide coupling chemistry. Specifically, HA was dissolved in PBS buffer, then added to the HA solution containing EDC, NHS, and selenocystamine, and stirred at room temperature for 4 to 6 hours. Ce6 was dissolved in DMSO at 5 mg / ml, then 0.9 ml of the Ce6 solution was added to the mixture and left overnight. The mixed solution was then transferred to an ultrafiltration centrifuge tube (Pall Corporation, USA), and the sample was centrifuged at 4500 rpm for 20 minutes at 20 °C. The ultrafiltration centrifuge tube was rinsed three times with deionized water to remove unbound HA and Ce6, washed, and the crosslinking agent PEGDE was added. The mixture was then transferred to a Petri dish and dried at room temperature. The Ce6-HA hydrogel was 1 cm 2 A circle was formed, sterilized using 70% ethanol, and then washed with distilled water (DW) (see Fig. 1).

[0088]

[0089] [Schematic Diagram of Ce6-Hyaluronic Acid Synthesis Process]

[0090]

[0091] Evaluation of crosslinking agent cytotoxicity

[0092] 5X10 to evaluate the potential toxicity of Ce6-HA 4 / cm 2 Density of NHDF and 1X10 5 / cm 2 HaCaT cells of a certain density were seeded into well plates. After seeding the cells, cross-linked Ce6-HA (cross-linked with various molar ratios of PEGDE) was introduced into the wells and incubated at 37°C for 1 day. Cell viability was evaluated using MTT reagent, which was added to each well and incubated in the dark for 4 hours. Then, DMSO was added to lyse the cells, and purple formazan crystals were extracted. The resulting mixture was transferred to a 96-well plate, and the safety and toxicity of the drug were confirmed by measuring the absorbance at 570 nm using a microplate reader.

[0093]

[0094] Measurement of ROS generation changes via DPBF

[0095] The catalytic activity of Ce6-HA was evaluated using 1,3-diphenylisobenzofuran (DPBF; Sigma-Aldrich) as an active oxygen indicator. Ce6-HA samples were placed in a DPBF solution, and at a wavelength of 660 nm, 1, 5, 10, or 20 mW / cm² 2 Intensity or 50, 100, 250, 500, 750 μW / cm² 2 or 1 mW / cm² 2 It was exposed to LED lighting of a certain intensity at 5-minute intervals.

[0096]

[0097] Toxicological evaluation of ROS generated from Ce6-HA

[0098] MTT assay (cytotoxicity assessment)

[0099] 5×10 NHDF in a well plate 4 The cells were initially inoculated at a density of cells / well and cultured overnight at 37°C in a CO2 incubator with controlled humidity. Subsequently, Ce6-HA was added to each well, and the cells were treated with 5 to 30 mW / cm² 2Cells were exposed to red LED light at various intensities for 10 to 60 minutes. Cell viability after LED light exposure was measured using the MTT assay. MTT reagent was added to the wells and incubated for 4 hours in a dark CO2 environment. Subsequently, DMSO was added to extract formazan crystals, and absorbance was measured at 570 nm. The percentage of cell viability compared to the control group was used as the evaluation criterion.

[0100]

[0101] Ames test (genotoxicity assessment)

[0102] To determine whether genetic mutations were induced by ROS of LED illumination, Ce6 hydrogel, and Ce6-HA, the Ames test was performed using Salmonella typhimurium strains TA98, TA100, TA1535, TA1537, and Escherichia coli WP2uvrA (pKM101). These bacterial strains were obtained from Moltox and cultured in nutrient medium at 37 °C. Each bacterial strain was divided into three groups: a group combined with LED irradiation and HA, a group with Ce6-HA without LED irradiation, and a group combined with LED irradiation and Ce6-HA. The LED intensity was set to 10 mW / cm² for 20 minutes. 2The parameters were set as such, and mutagenicity was evaluated using a pre-culture method. For each assay, a negative control containing saline and a positive control containing NQNO, sodium azide, ICR191, or MMS were prepared. Subsequently, histidine / biotin or tryptophan solution was added to the bacterial suspension treated with these solutions and incubated in a shaking incubator at 37°C for 20 minutes. Next, top agar was mixed with these combinations and transferred to minimal agar plates. The resulting mixture was then placed in an incubator at 37°C for 48 hours, and the number of revertant colonies on each plate was counted. This process was repeated three times to verify the results.

[0103]

[0104] antibacterial effect

[0105] Colony count test

[0106] To evaluate the antimicrobial effects of HA and Ce6-HA upon exposure to LEDs under various conditions, Staphylococcus aureus (ATCC 14458) and Pseudomonas aeruginosa (ATCC 9027) were used. The bacteria were cultured at 37°C for 18 hours using the standard agar method. 5×10⁻⁶ in saline solution 6 Bacterial suspensions at a concentration of CFU / ml were prepared and treated with HA or Ce6-HA, and LED exposure was 5 mW / cm² for 10 to 30 minutes. 2 At an intensity of 10 mW / cm² for 10 to 20 minutes 2 It was treated with an intensity of [value]. Next, the bacterial suspension was diluted with saline solution, spread onto a plate counting medium, dried before incubation at 37°C overnight, and the number of colonies was measured after incubation.

[0107]

[0108] Fluorescence analysis

[0109] Fluorescent staining was used to determine the non-survival rates of viable S. aureus and P. aeruginosa after ROS treatment. S. aureus and P. aeruginosa were treated with HA or Ce6-HA, followed by treatment with 5 to 10 mW / cm² for 10 to 30 minutes. 2 The samples were exposed to LED light at an intensity of [indicated]. Then, suspensions of S. aureus and P. aeruginosa were stained using the BacLight LIVE / DEAD kit at room temperature for 15 minutes. The resulting mixture was transferred to a black 96-well plate and scanned using a microplate reader. Excitation was performed at a wavelength of 485 nm, and emission was recorded at 530 nm (green) or 630 nm (red).

[0110]

[0111] ROS-induced macrophage polarization

[0112] Analysis of inflammatory cytokines

[0113] RAW 264.7 cells were obtained from the American Type Culture Collection (ATCC®). Levels of TNF-α, IL-1β, IL-6, IL-10, and TGF-β were measured using an ELISA kit (R&D Systems). RAW 264.7 cells were 1 x 10⁻¹⁰ 5 Cells were cultured in a plate at a concentration of cells / ml until completely covered. Then, the hydrogel was applied to the cells, followed by 1 to 5 mW / cm² for 10 to 20 minutes. 2 LED light was irradiated at an intensity of . To confirm the effect of low-power LED stimulation, 100 μW / cm² was applied for 30 and 60 minutes. 2The intensity was applied. The cell suspension was centrifuged and the supernatant was collected to measure cytokine levels, and the concentrations of inflammatory cytokines (TNF-α, IL-1β, IL-6) and anti-inflammatory cytokines (TGF-β, IL-10) in the supernatant were evaluated using an ELISA kit according to the manufacturer's guidelines and standard curves.

[0114]

[0115] Confirmation of the effects of ROS on cell proliferation

[0116] Neonatal human skin fibroblasts (NHDF, Lonza, Switzerland), a type of fibroblast, were cultured in fibroblast growth medium (Lonza), and the culture medium was replaced with fresh medium every 3 to 4 days. NHDF was 1 × 10⁻¹⁰ 4 Cells / cm 2 The cells were seeded into 48-well plates at a density of [value] and incubated overnight at 37°C in a CO2 incubator for cell attachment. HaCaT (ATCC®) cells were used as a representative keratinocyte model and cultured in Dulbecco's Modified Eagle's Medium containing 10% fetal bovine serum and 1% antibiotics (Welgene, Seoul, Korea). HaCaT cells were seeded into 48-well plates at a density of 1 × 10 4 Dog cells / cm² 2Cells were inoculated at a density and incubated overnight at 37°C in a CO2 incubator for proper cell attachment. After incubation, each well was treated with either HA or Ce6-HA, and the plates were then exposed to red LED light of varying intensities for different durations. Cell viability was assessed using the Cell Counting Kit-8 (CCK-8) at 4, 24, and 72 hours after irradiation. After the specified exposure times, CCK-8 solution was added to each well, and the plates were incubated in the dark for 2 hours to prevent light interference. Absorbance was then measured at 450 nm using a spectrophotometer, and growth rates were presented in comparison to a control group in which cell viability was measured at 4 hours after HA treatment.

[0117]

[0118] angiogenesis assay

[0119] 3 x 10 HUVEC 4 cells / cm 2 Cells were seeded into Matrigel-coated well plates at a density. After seeding, 0.2-μm microporous membrane transwells were introduced, and cells were treated with HA or Ce6-HA. HUVECs were classified into several groups: a control group, a group treated only with HA, a positive control group treated with a combination of HA and VEGF, a group treated with HA followed by LED exposure, and a group treated with Ce6-HA followed by LED exposure. LED irradiation was 100 μW / cm² for 30 minutes. 2It was set as follows. After cell inoculation and treatment, viability / death assays were performed 6 hours later using the Endothelial Tube Formation Assay kit (Cell Biolabs, San Diego, USA) according to the manufacturer's instructions to evaluate the length of cell junctions and the number of formed junctions. VEGF treatment was used as a positive control. Tube formation was subsequently observed using a microscope and quantitatively analyzed using ImageJ software; the evaluated parameters included the number of junctions and the total tube length.

[0120]

[0121] In vivo research

[0122] animal testing approval

[0123] Animal studies were conducted in accordance with the guidelines specified in the Guide for the Care and Use of Laboratory Animals. The research protocol was approved by the Institutional Animal Care and Use Committee (IACUC) of the Yonsei Laboratory Animal Research Center. Animals were housed in a pathogen-free environment of YLARC. A full-thickness wound model was established using 5-week-old male Balb / c nude mice purchased from Orient Bio. Prior to the experiment, Balb / c nude mice underwent a 1-week acclimatization period, after which the mice were randomly assigned to experimental groups.

[0124] Surgery for creating full-thickness wounds

[0125] For in vivo irradiation, the subjects were divided into four groups: control group (CON), wounds treated with HA (HA), wounds treated with HA and LED irradiation (HAL), and wounds treated with Ce6-HA and LED irradiation (CHL). Mice were anesthetized by intraperitoneal injection of zoletil (35 mg / kg) and rompun (2 mg / kg). The surgical site was sterilized with a 70% ethanol solution, and two full-thickness skin wounds with a diameter of 8 mm were created on the dorsal side of the mice using a biopsy punch during anesthesia. Round silicone splints were placed around the wounds and secured to the skin with 4-0 nylon sutures to maintain their position. Hydrogel was applied after the silicone splints were sutured. The group designated for LED treatment received 200 μW / cm² for 30 minutes. 2 It was exposed to LED lighting at an intensity of 200 μW / cm² for 30 minutes in a preliminary experiment on LED illuminance. 2 It was shown that [condition] was the most effective for wound contraction and the promotion of angiogenesis, and these parameters were subsequently used in this study. After each procedure, an occlusive polyurethane dressing (Tegaderm, 3M) was applied to protect the wound site. Macroscopic evaluation of wound healing was performed by photographing the wound using a digital camera and recording the mouse weight at intervals of 3 to 4 days. Photographs were taken to monitor the healing process and evaluate changes in the wound site over time. Upon completion of the study, the animals were euthanized using a CO2 chamber, and skin tissue samples were collected from the center of each wound for further analysis.

[0126] pathological examination

[0127] Damaged skin tissue was collected and immediately fixed in 10% paraformaldehyde for 24 hours. After fixation, the tissue samples were embedded in paraffin and sectioned into 5 μm thick slices. The sections were stained using hematoxylin and eosin (H&E) and Mason's Trichrome (MT) staining techniques. The stained slides were then examined under a bright-field microscope (Olympus BX51; Olympus, Tokyo, Japan) to evaluate their histological features.

[0128]

[0129] Statistical analysis

[0130] Statistical analysis was performed using SPSS 26.0 (SPSS Inc.). Data were analyzed using one-way analysis of variance (ANOVA) and Student's t-tests for within-group individual comparisons. Statistical significance was defined as a P-value of less than 0.05, and data were presented as mean ± standard deviation.

[0131]

[0132] Experimental results

[0133] Confirmation of biocompatibility

[0134] We aimed to investigate the cytotoxicity of fibroblasts caused by the generation of reactive oxygen species (ROS) when Ce6-hyaluronic acid was irradiated with LED. The experimental results showed a maximum of 5 mW / cm² 2 At 10 to 30 minutes, 10 mW / cm² 2 It was confirmed that there was no significant toxicity in normal fibroblasts after 10 to 20 minutes (see Fig. 2). In addition, when examining whether genotoxicity occurred when Ce6-hyaluronic acid was irradiated with LED, it was confirmed that no genotoxicity was observed in response to hyaluronic acid, LED, Ce6-hyaluronic acid, or ROS (see Table 1 below). Here, the LED irradiation condition was 10 mW / cm² 2 It was maintained for 20 minutes.

[0135] TA98TA100TA1535TA1537E.coliHA negative26 ± 2.865.3 ± 3.28.3 ± 1.59 ± 3.5111 ± 22.5HA positive93.7 ± 9.0810.7 ± 91.0821.7 ± 68.21390.7 ± 164.12066.0 ± 258.2HA irradiation20.5 ± 3.555.0 ± 7.514.7 ± 4.510 ± 4.4120.7 ± 20.6CH non irradiation12.7 ± 5.965.3 ± 8.613.0 ± 2.67.3 ± 2.595.7 ± 11.5CH irradiation20.7 ± 6.572 ± 4.4 10.3 ± 3.8 10.7 ± 6.5 10 3.0 ± 6.0

[0136]

[0137] As can be seen from the results above, it was demonstrated that the composite material produced through the chemical bonding of Ce6 and hyaluronic acid does not exhibit cytotoxicity or genotoxicity. This ensures the safety and biocompatibility for such bio-applications and can enhance the potential for utilization in the medical field.

[0138]

[0139] Controlled generation of reactive oxygen species (ROS)

[0140] Since precise control of the amount of reactive oxygen species during the treatment process plays a crucial role in maximizing the effect of pathogens while simultaneously minimizing damage to host tissues, a method was devised to control the generation of ROS using a specific LED light wavelength with a hyaluronic acid hydrogel. Figures 3a and 3b show the results of confirming ROS generation by irradiating Chlorine e6 with LED light using a DPBF (1,3-Diphenylisobenzofuran) assay and monitoring the degradation of DPBF according to irradiation time and intensity. It can be seen that ROS generation is proportional to the LED irradiation time and output intensity.

[0141]

[0142] Sterilization effect on various microorganisms

[0143] Reactive oxygen species exert a bactericidal effect by acting directly on various types of microorganisms, and are particularly effective against representative pathogens such as Staphylococcus aureus (S. aureus) and Pseudomonas aeruginosa (P. aeruginosa). To verify this, a bacterial suspension was 5 x 10 6 After preparing at a concentration of CFU / ml, LED irradiation was performed following treatment with hyaluronic acid or Ce6-hyaluronic acid; the LED irradiation was set to 5 to 10 mW / cm² based on cytotoxicity tests. 2 It was performed at 10-minute intervals, and the control group was not irradiated with LED.

[0144] The experimental results showed that the hyaluronic acid group irradiated with LEDs did not differ significantly from the control group under any conditions, confirming that there was no antibacterial effect. However, in the group exposed to LED light and Ce6-hyaluronic acid, a significant decrease in bacterial viability was observed compared to the control group. Ce6-hyaluronic acid at 10 mW / cm² 2 After treatment with LEDs for 20 minutes, both S. aureus and P. aeruginosa showed a survival rate of less than 50%, indicating that ROS generated by LED light contributed to the reduction in bacterial survival rate. This confirmed that the Ce6-hyaluronic acid hydrogel irradiated with LED light has potential as an antimicrobial agent (see Figures 4a and 4b). This suggests the possibility of treatment against various pathogens, including pathogens.

[0145]

[0146] Inflammation control

[0147] Reactive oxygen species support host defense mechanisms against pathogens by not only eliminating microorganisms but also regulating inflammation and activating immune responses. Immune cells, such as macrophages, secrete pro-inflammatory cytokines in response to reactive oxygen species, which can thereby enhance the microbial elimination process. To verify these effects, RAW 264.7 cells were treated with hyaluronic acid or Ce6-hyaluronic acid, and subsequently, LEDs were applied at 1, 2, and 5 mW / cm² 2 As a result of irradiation for 10 to 20 minutes at an intensity of 10 μW / cm², it was confirmed that the Ce6-hyaluronic acid treatment group showed significantly higher concentrations of inflammatory cytokines such as TNF-α, IL-1, and IL-6 compared to the hyaluronic acid treatment group (see Figs. 5a to 5c), which indicates a change in the M1 phenotype of macrophages. The groups did not show differences in the concentrations of the anti-inflammatory cytokines TGF-β and IL-10, and at 100 μW / cm² 2 It was found that there was no difference in inflammatory and anti-inflammatory cytokines between the hyaluronic acid-treated group and the Ce6-hyaluronic acid-treated group during low-intensity ROS irradiation for 30 to 60 minutes (see Figures 6a and 6b). These results suggest that ROS induction promotes increased production of inflammatory cytokines by macrophages, which acts as a host defense mechanism against invading pathogens. In particular, low levels of ROS did not affect macrophages, which confirmed that the ROS response of macrophages may vary depending on the amount of ROS.

[0148]

[0149] Confirmation of proliferative cell response

[0150] To determine whether the Ce6-hyaluronic acid complex promotes the proliferation of fibroblasts and keratinocytes and aids in the neovascularization of vascular endothelial cells, 1X10 4 cell / cm 2After treating a fibroblast suspension prepared at a certain concentration with hyaluronic acid or Ce6-hyaluronic acid, 100 μW / cm² 2 LEDs were irradiated at an intensity of 20 to 60 minutes. As a result of the experiment, while there was no notable difference in proliferation between hyaluronic acid treatment and Ce6-hyaluronic acid treatment in the control group that was not irradiated with light, it was confirmed that the growth rate of cells treated with Ce6-hyaluronic acid increased by 15% on the third day after being exposed to light for 30 minutes compared to the group that received light irradiation after hyaluronic acid treatment. In addition, changing the light irradiation period to be shorter or longer than 30 minutes did not affect cell growth (see Fig. 7).

[0151] Keratinocytes were also treated in the same way at 100 μW / cm² 2 As a result of confirming proliferation by irradiating with LED at an intensity of 20 to 60 minutes, it was confirmed that keratinocytes showed a 20% increase in growth compared to hyaluronic acid-treated cells when irradiated with Ce6-hyaluronic acid for 20 and 30 minutes (see Fig. 8).

[0152] Vascular endothelial cells were seeded onto Matrigel for verification, and experiments were conducted using fibroblast and keratinocyte proliferation conditions, applying identical LED light exposure conditions. The subjects were classified into a control group, a hyaluronic acid-only treatment group, a positive control group using hyaluronic acid and vascular endothelial growth factor (VEGF) in combination, a group exposed to LED after hyaluronic acid treatment, and a group exposed to LED after Ce6-hyaluronic acid treatment. As a result, while the control group, the hyaluronic acid treatment group, and the group exposed to LED after hyaluronic acid treatment showed similar results, the positive control group treated with hyaluronic acid and VEGF and the group exposed to LED after Ce6-hyaluronic acid treatment showed significantly increased values ​​(see Figs. 9a to 9c). 100 μW / cm² 2It was confirmed that the group treated with Ce6-hyaluronic acid under 30 minutes of LED conditions showed a significant increase in junction number and total length compared to other experimental groups, which suggests that LED exposure to Ce6-hyaluronic acid induces reactive oxygen species in vascular endothelial cells and significantly promotes angiogenesis at a level similar to VEGF.

[0153]

[0154] Animal experiment results

[0155] As a result of examining the change in wound size over time using an in vivo full-thickness wound healing model, 200 μW / cm² 2 Under 30 minutes of LED conditions, it was confirmed that the wound size of the group treated with Ce6-hyaluronic acid significantly decreased starting from day 7. In other words, it can be seen that the experimental group that generated reactive oxygen species by applying LEDs to a hyaluronic acid hydrogel containing a photosensitizer exhibited the most superior wound healing effect (see Figures 10a and 10b). This indicates excellent results in terms of the speed and quality of wound healing, suggesting that it will enable faster recovery than existing methods. The superior wound healing effect observed with the combination of Ce6-hyaluronic acid and LED irradiation suggests that reactive oxygen induction has potential as a therapeutic strategy for wound closure and tissue regeneration promotion, and that utilizing the reactive oxygen induction ability of Ce6-hyaluronic acid can strengthen the physiological mechanisms of wound healing and accelerate the healing process.

[0156] For histological evaluation, hematoxylin and eosin staining (H&E) and Masson's Trichrome staining (MT) were performed in an in vivo wound healing model. The results confirmed that the experimental group with the appearance most similar to normal skin on day 21 was the group that received LED irradiation on Ce6-hyaluronic acid treated with ROS (see Figs. 11a and 11b). As shown in the schematic diagram in Fig. 1b, LED irradiation on Ce6-hyaluronic acid exhibits excellent effects on wound healing and induces a recovered epidermal structure, enhanced angiogenesis, and collagen accumulation in the dermis. Therefore, it is expected to be utilized in a variety of ways, ranging from wound treatment to immune modulation.

[0157]

[0158] Foregoing, specific parts of the present invention have been described in detail. It is evident to those skilled in the art that such specific descriptions are merely preferred embodiments and do not limit the scope of the invention. Accordingly, the actual scope of the invention is defined by the appended claims and their equivalents.

Claims

1. A pharmaceutical composition for wound treatment comprising hyaluronic acid (HA) hydrogel as an active ingredient.

2. In Paragraph 1, A composition characterized by additionally binding a photosensitizer molecule to the hyaluronic acid hydrogel.

3. In Paragraph 1, A composition in which the above-mentioned photosensitizer is at least one selected from the group consisting of chlorin, porphyrin, phthalocyanine, purpurin, texaphyrin, or derivatives thereof.

4. In Paragraph 3, A composition in which the above-mentioned photosensitizer is chlorin e6 (Ce6) or bacteriochlorin.

5. In Paragraph 3, A composition in which the above-mentioned photosensitizer is at least one selected from the group consisting of hematoporphyrin, benzoporphyrin, and protoporphyrin IX (PpIX).

6. In Paragraph 2, A composition in which the above-mentioned photosensitizer is bonded to the above-mentioned hyaluronic acid hydrogel through a polyethylene glycol-based crosslinking agent.

7. In Paragraph 6, A composition in which the above polyethylene glycol-based crosslinking agent is PEGDE (Poly(ethylene glycol) diglycidyl ether).

8. In Paragraph 1, A composition characterized by being used in conjunction with irradiation of light onto a lesion.

9. In Paragraph 8, A composition in which the light is an LED (Light Emitting Diode).

10. In Paragraph 8, The above light is 10 μW / cm² 2 Up to 50 mW / cm 2 A composition characterized by being investigated as.

11. In Paragraph 8, A composition characterized by the above light being irradiated for 5 to 80 minutes.

12. An antimicrobial pharmaceutical composition comprising hyaluronic acid (HA) hydrogel as an active ingredient.

13. In Paragraph 12, A composition characterized by additionally bonding a photosensitizer to the above hyaluronic acid hydrogel.

14. A pharmaceutical composition for skin regeneration comprising hyaluronic acid (HA) hydrogel as an active ingredient.

15. In Paragraph 14, A composition characterized by additionally bonding a photosensitizer to the above hyaluronic acid hydrogel.

16. A cosmetic composition for skin regeneration comprising hyaluronic acid (HA) hydrogel as an active ingredient.

17. In Paragraph 16, A composition characterized by additionally bonding a photosensitizer to the above hyaluronic acid hydrogel.

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