Hyaluronic acid hydrogel having skin wound healing effect by continuously releasing drug, and hyaluronic acid hydrogel capable of controlling drug release by means of light by using photo-cleavable linker

Abstract

The present invention relates to a hyaluronic acid hydrogel having a skin wound healing effect by continuously releasing a drug, wherein the hydrogel is prepared by chemically modifying an acrylate group according to the presence or absence of PEG in an epidermal growth factor through an NHS ester amine coupling reaction, which is a type of bioconjugation, and then crosslinking between hyaluronic acid chains is performed through a vitamin B2 derivative (riboflavin phosphate) photoinitiator and blue light irradiation. The present invention relates to a hyaluronic acid hydrogel capable of controlling drug release by means of light by using a photo-cleavable linker, and to a method for preparing a hyaluronic acid (HA)-based hydrogel, and a technique capable of controlling drug release by means of light stimulation.

Description

A hyaluronic acid hydrogel with a skin wound healing effect by continuously releasing drugs and a hyaluronic acid hydrogel with a photo-cleavable linker that enables controlled drug release by light.

[0001] The present invention relates to a hyaluronic acid hydrogel that has a skin wound healing effect by continuously releasing a drug, and is prepared by chemically modifying an acrylate group of epidermal growth factor (EGF) with or without PEG using an NHS ester amine coupling reaction, which is a type of bioconjugation, and then crosslinking hyaluronic acid chains using a vitamin B2 derivative (Riboflavin phosphate) photoinitiator and blue light irradiation to produce a hydrogel. Simultaneously, a bond between the acrylate of the modified EGF and the thiol group of hyaluronic acid is formed in a single step reaction, thereby delaying the release of EGF chemically bonded to the hydrogel and further enabling control of the drug delivery rate, and thereby providing a hyaluronic acid hydrogel that has a skin wound healing effect by continuously releasing a drug.

[0002] The present invention relates to a hyaluronic acid hydrogel capable of controlling drug release by light using a photocleavable linker, a method for producing a hyaluronic acid (HA)-based hydrogel, and a technology for controlling drug release by light stimulation. That is, a method for producing a hydrogel by exposing hyaluronic acid (SH-HA, MA-HA, respectively) having a thiol functional group and a methacrylate functional group bonded to blue visible light in the presence of a vitamin B derivative (Riboflavin Phosphate, RFP), which is a natural photoinitiator, and EGF bonded with a photocleavable linker having a photocleavable function is fixed within the hydrogel to enable controlled release by light stimulation. The invention relates to a hyaluronic acid hydrogel capable of controlling drug release by light using a photocleavable linker, which has a stimulus-responsive function for optimizing the effect and efficiency of proteins and drugs and can be utilized in the field of drug delivery systems, and the invention relates to a hyaluronic acid hydrogel capable of controlling drug release by light using a photocleavable linker.

[0003] Growth factors are soluble proteins secreted by cells and can regulate cell behavior, including proliferation, differentiation, and migration. Various growth factors are widely used as healing factors for tissue regeneration, and actual studies have confirmed significant healing effects. Epidermal growth factor (EGF), in particular, is one of the earliest discovered and long-used growth factors. It is a 6kDa molecule with 53 amino acid residues and three intramolecular disulfide bonds. It binds to the receptor EGFR, initiating signaling pathways related to cell division, apoptosis, and protein secretion, and is utilized as a therapeutic factor for skin wound healing in the epidermis and dermis.

[0004] Various synthetic and natural polymers are utilized as carriers for growth factors or drugs. Hyaluronic acid (HA), a natural polymer, is a viscoelastic substance that protects tissues and regulates moisture. It acts as a matrix, encapsulating and binding soluble growth factors. In addition to its role as a carrier, HA also protects surrounding tissues and retains moisture to accelerate healing. When used as a hydrogel or carrier, HA requires physical and chemical crosslinking via functional groups to prevent dissolution of the hydrophilic polymer. Hydrogels can be formed by specific stimuli, such as temperature, magnetism, light, and ions, depending on their properties. Previously developed, a visible-light-induced photocrosslinked HA hydrogel utilizing riboflavin phosphate (RFP) as a photoinitiator is injectable. The liquid precursor solution undergoes crosslinking via a thiol-ene reaction induced by visible light irradiation to form a hydrogel.

[0005] In the present invention, epidermal growth factors modified with specific functional groups are chemically bound to an HA hydrogel matrix, enabling prolonged drug release without burst release. This simple hydrogel manufacturing process allows drug binding simultaneously with matrix crosslinking, without the need for a separate process for additional matrix binding, by simply preparing growth factors or drugs modified with specific groups in advance. This allows for long-term use without side effects by modifying growth factors or drugs for specific diseases.

[0006]

[0007] Hyaluronic acid (HA) is a natural polymer that can regulate cellular processes by binding to cell surface receptors, making it highly biocompatible. Furthermore, it is easily amenable to functional groups, which can be crosslinked physically and chemically to form hydrogels. Hydrogels possess a three-dimensional porous polymer network, capable of absorbing large amounts of water, and are also useful as drug delivery vehicles. Furthermore, by varying the molecular weight of HA, the functional group modification, or the concentration, the elasticity and rigidity can be tailored to the specific tissue.

[0008] Several methods have been developed to address the short half-life of growth factors and enable controlled release using hydrogel matrices. Initially, attempts were made to encapsulate growth factors in hydrogels to achieve controlled release. However, due to the high molecular weight of hyaluronic acid, the initial release of relatively small growth factors was still high. Excessive initial release of growth factors can cause adverse effects on healthy tissues, disrupting normal wound healing and forming hypertrophic scars. Therefore, active research has been conducted in the past few years to immobilize growth factors in biomaterials using various chemical methods. While these methods have shown delayed release compared to unimmobilized growth factors, the precise timing of release remains unknown and difficult to control.

[0009] Various physicochemical stimuli exist for controlled release, including light, temperature, pH, and enzymes. Internal stimuli, such as enzymes, proteins, and pH, can be significantly influenced by the body's microenvironment and have limitations in spatially controlling them within the body. Even temperature, an external stimulus, requires considerable time to regulate and can negatively impact surrounding tissues. However, light stimulation, because it occurs outside the body, offers the advantages of being noninvasive, free from temporal and spatial constraints, and adjustable light intensity and irradiance. When light is used as a stimulus, biomaterials containing photoresponsive moieties can undergo specific photochemical reactions, such as cleavage or isomerization, which can alter the physical and chemical properties of the biomaterial and bioconjugate. Due to these characteristics, photoresponsive biomaterials are widely studied in the fields of controlled drug release and tissue engineering.

[0010] Therefore, there is an urgent need to develop a functional hydrogel technology that can delay the release of growth factors while controlling the timing of release by light stimulation.

[0011]

[0012] The present invention relates to a hyaluronic acid hydrogel that has a skin wound healing effect by continuously releasing a drug, and is prepared by chemically modifying an acrylate group of epidermal growth factor (EGF) with or without PEG using an NHS ester amine coupling reaction, which is a type of bioconjugation, and then crosslinking hyaluronic acid chains using a vitamin B2 derivative (Riboflavin phosphate) photoinitiator and blue light irradiation to produce a hydrogel. At the same time, the bond between the acrylate of the modified EGF and the thiol group of hyaluronic acid is a one-step reaction, thereby delaying the release of EGF chemically bonded to the hydrogel and further providing a hyaluronic acid hydrogel that has a skin wound healing effect by continuously releasing a drug, which is capable of controlling the drug delivery rate.

[0013] The present invention relates to a hyaluronic acid hydrogel capable of controlling drug release by light using a photocleavable linker, a method for producing a hyaluronic acid (HA)-based hydrogel, and a technology for controlling drug release by light stimulation. That is, a method for producing a hydrogel by exposing hyaluronic acid (SH-HA, MA-HA, respectively) having a thiol functional group and a methacrylate functional group bonded to blue visible light in the presence of a vitamin B derivative (Riboflavin Phosphate, RFP), which is a natural photoinitiator, and EGF bonded with a photocleavable linker having a photocleavable function is fixed within the hydrogel to enable controlled release by light stimulation. The invention aims to provide a hyaluronic acid hydrogel capable of controlling drug release by light using a photocleavable linker, which has a stimulus-responsive function for optimizing the effect and efficiency of proteins and drugs and can be utilized in the field of drug delivery systems.

[0014]

[0015] In order to solve the above-described problem, in one embodiment of the present invention, a hyaluronic acid hydrogel having a skin wound healing effect by continuously releasing a drug is provided, which comprises a matrix material including MA-HA (Methacrylated hyaluronic acid) and SH-HA (Thiolated hyaluronic acid); and a protein modified with acrylate cross-linked to the matrix material.

[0016] In one embodiment of the present invention, the hyaluronic acid hydrogel further includes a vitamin B2 derivative (RFP, Riboflavin phosphate) and can be gelled by irradiating with blue light.

[0017] In one embodiment of the present invention, the protein may include a protein having a growth factor.

[0018] In one embodiment of the present invention, the protein having the growth factor may include at least one of epidermal growth factor (EGF), fibroblast growth factor (FGF), keratinocyte growth factor (KGF), and nerve growth factor inducible (VGF).

[0019] In one embodiment of the present invention, the MA-HA and SH-HA may have a volume ratio of 0.5 to 2 to 2 to 0.5.

[0020] In order to solve the above problem, in one embodiment of the present invention, a method for producing a hyaluronic acid hydrogel having a skin wound healing effect by continuously releasing a drug, wherein the hyaluronic acid hydrogel comprises: a matrix material including MA-HA and SH-HA; and an acrylate-modified protein cross-linked to the matrix material; and the method for producing the hyaluronic acid hydrogel comprises: a step of preparing a protein solution; a step of dissolving acrylic acid NHS ester in dimethyl sulfoxide (DMSO) to produce an NHS solution; and a step of mixing the NHS solution and the protein solution to produce an acrylate-modified A-protein solution.

[0021] In one embodiment of the present invention, the method for producing the hyaluronic acid hydrogel may include the steps of: mixing the MA-HA and SH-HA to produce a first solution; dissolving the first solution in phosphate-buffered saline (PBS) to produce a second solution; mixing the second solution and the A-protein solution to produce a third solution; adding a vitamin B2 derivative (RFP, Riboflavin phosphate) to the third solution to produce a fourth solution; and exposing the fourth solution to blue light to form a gel.

[0022] In one embodiment of the present invention, the step of exposing the fourth solution to blue light to form a gel may be performed by exposing the solution to blue light for 10 to 30 seconds.

[0023] In one embodiment of the present invention, the A-protein solution may be mixed in a volume ratio of 90 to 110 and the NHS solution may be mixed in a volume ratio of 0.1 to 3.

[0024] In one embodiment of the present invention, the step of preparing the A-protein solution may include a step of incubating the A-protein solution at 1 to 10˚C for 1 to 3 hours; and a step of dialyzing the incubated A-protein solution at 1 to 10˚C for 10 to 30 hours.

[0025] In order to solve the above-described problem, in one embodiment of the present invention, a hyaluronic acid hydrogel that continuously releases a wound healing substance is provided, which comprises a matrix material including MA-HA (Methacrylated hyaluronic acid) and SH-HA (Thiolated hyaluronic acid); and a protein modified with acrylate PEG succinimidyl carboxymethyl ester cross-linked to the matrix material.

[0026] In one embodiment of the present invention, the hyaluronic acid hydrogel further includes a vitamin B2 derivative (RFP, Riboflavin phosphate) and can be gelled by irradiating with blue light.

[0027] In one embodiment of the present invention, the protein may include a protein having a growth factor.

[0028] In one embodiment of the present invention, the protein having the growth factor may include at least one of epidermal growth factor (EGF), fibroblast growth factor (FGF), keratinocyte growth factor (KGF), and nerve growth factor inducible (VGF).

[0029] In one embodiment of the present invention, the MA-HA and SH-HA may have a volume ratio of 0.5 to 2 to 2 to 0.5.

[0030] In order to solve the above problem, in one embodiment of the present invention,

[0031] The above hyaluronic acid hydrogel comprises a matrix material including MA-HA and SH-HA; and a protein modified with acrylate PEG succinimidyl carboxymethyl ester cross-linked to the matrix material; and a method for producing the hyaluronic acid hydrogel comprises the steps of: preparing a protein solution; dissolving the acrylate PEG succinimidyl carboxymethyl ester in dimethyl sulfoxide to produce an A-PEG solution; and mixing the protein solution with the A-PEG solution to produce a PEG-A-protein solution.

[0032] In one embodiment of the present invention, the method for producing the hyaluronic acid hydrogel may further include: a step of mixing the MA-HA and SH-HA to produce a first solution; a step of dissolving the first solution in phosphate buffered saline to produce a second solution; a step of mixing the second solution and a PEG-A protein solution to produce a fifth solution; and a step of adding a vitamin B2 derivative to the fifth solution to produce a sixth solution; and a step of exposing the sixth solution to blue light to gel.

[0033] In one embodiment of the present invention, the protein modified with the acrylate PEG succinimidyl carboxymethyl ester may be mixed in a volume ratio of 9 to 11 for the protein solution and 0.5 to 2 for the A-PEG solution.

[0034] In one embodiment of the present invention, the step of preparing the PEG-A-protein solution may include a step of incubating the PEG-A-protein solution at 1 to 10˚C for 1 to 3 hours; and a step of dialyzing the incubated PEG-A-protein solution at 1 to 10˚C for 10 to 30 hours.

[0035] In one embodiment of the present invention, the step of exposing the sixth solution to blue light to gel may be performed by exposing the solution to blue light for 10 to 30 seconds.

[0036]

[0037] In order to solve the above problem, in one embodiment of the present invention, a wound healing substance can be selectively released upon light irradiation, and a hyaluronic acid hydrogel can be provided, which comprises: a hyaluronic acid hydrogel matrix; a photocleavage unit bound to the hyaluronic acid hydrogel matrix; and a growth factor bound to the hyaluronic acid hydrogel matrix; and a wound healing substance can be selectively released upon light irradiation.

[0038] In one embodiment of the present invention, the hyaluronic acid hydrogel comprises: a step of preparing a protein solution by dissolving a protein in phosphate-buffered saline (PBS); a step of preparing an Azido-PC solution by dissolving Azido-PC-NHS ester, which is a photocleavable linker, in dimethyl sulfoxide (DMSO); a step of preparing an Azido-PC-protein solution by mixing the protein solution and the Azido-PC solution; a step of preparing an SH-HA (Thiolated hyaluronic acid) by dissolving SH-HA in phosphate-buffered saline; a step of preparing a DBCO solution by dissolving dibenzocyclotine-maleimide (DBCO-Maleimide) in dimethyl sulfoxide; a step of preparing a DBCO-HA solution by mixing the SH-HA solution and the DBCO solution; It can be manufactured through a step of mixing the Azido-PC-protein solution and DBCO-HA solution to prepare a protein-PC-HA solution; a step of mixing the protein-PC-HA solution with MA-HA (Methacrylated hyaluronic acid) solution and a vitamin B2 derivative (RFP, Riboflavin phosphate) to prepare a hydrogel precursor solution; and a step of irradiating the hydrogel precursor solution with blue light to form a gel.

[0039] In one embodiment of the present invention, the hyaluronic acid hydrogel matrix includes MA-HA (Methacrylated hyaluronic acid) and SH-HA (Thiolated hyaluronic acid), and the MA-HA and SH-HA may have a volume ratio of 0.5 to 2 to 2 to 0.5.

[0040] In one embodiment of the present invention, the optical cutter may include Azido-PC-NHS ester.

[0041] In one embodiment of the present invention, the growth factor may include at least one of epidermal growth factor (EGF), fibroblast growth factor (FGF), keratinocyte growth factor (KGF), and nerve growth factor inducible (VGF).

[0042] In one embodiment of the present invention, the protein may have a concentration of 0.01 to 1 mg / ml relative to the phosphate buffered saline solution.

[0043] In one embodiment of the present invention, the Azido-PC-NHS ester may have a concentration of 20 to 40 mg / ml relative to the dimethyl sulfoxide.

[0044] In one embodiment of the present invention, the Azido-PC-NHS ester can be diluted to a concentration of 50 to 150 times and mixed with the protein solution.

[0045] In one embodiment of the present invention, the step of preparing the Azido-PC-protein solution may further include a step of purifying the Azido-PC-protein solution at 1 to 7 °C for 20 to 30 hours.

[0046] In one embodiment of the present invention, the dibenzocyclotine-maleimide may have a concentration of 5 to 15 mg / ml relative to the dimethyl sulfoxide.

[0047] In order to solve the above problem, in one embodiment of the present invention, a method for producing a hyaluronic acid hydrogel from which a wound healing material can be selectively released upon light irradiation is provided, wherein the hyaluronic acid hydrogel comprises: a hyaluronic acid hydrogel matrix; a photocleavable group bound to the hyaluronic acid hydrogel matrix; and a growth factor bound to the hyaluronic acid hydrogel matrix; the method comprises: a step of producing a protein solution by dissolving a protein in PBS; a step of dissolving Azido-PC-NHS ester, which is a photocleavable linker, in dimethyl sulfoxide (DMSO) to produce an Azido-PC solution; a step of mixing the protein solution and the Azido-PC solution to produce an Azido-PC-protein solution; A step of preparing an SH-HA solution by dissolving SH-HA (Thiolated hyaluronic acid) in phosphate-buffered saline (PBS); a step of preparing a DBCO solution by dissolving dibenzocyclopentane-maleimide (DBCO-Maleimide) in dimethyl sulfoxide; a step of preparing a DBCO-HA solution by mixing the SH-HA solution and the DB CO solution; a step of preparing a protein-PC-HA solution by mixing the Azido-PC-protein solution and the DB CO-HA solution; a step of preparing a hydrogel precursor solution by mixing the protein-PC-HA solution with a MA-HA (Methacrylated hyaluronic acid) solution and a vitamin B2 derivative (RFP, Riboflavin phosphate); A method for producing a hyaluronic acid hydrogel, from which a wound healing substance can be selectively released upon light irradiation, can be provided, including a step of irradiating the hydrogel precursor solution with blue light to form a gel.

[0048]

[0049] According to one embodiment of the present invention, the effect of producing a matrix material can be achieved by mixing MA-HA and SH-HA.

[0050] According to one embodiment of the present invention, there is provided a technology for improving wound healing effects through the possibility of sustained release without burst release by modifying EGF with acrylate and acrylate-PEG so that it can be chemically bound to an HA hydrogel matrix through a thiol-ene reaction. The natural photoinitiator and blue visible light used in the hydrogel production and modified EGF binding process enable the production of a hydrogel with excellent biocompatibility, and the modified EGF can maintain physiological activity for a long period of time within the hydrogel through chemical bonding, and can exhibit an effect that can be used as an effective therapeutic factor through delayed release.

[0051] According to one embodiment of the present invention, by modifying a protein having a growth factor with acrylate or PEG, the protein having a growth factor is cross-linked to a hyaluronic acid hydrogel to release the protein in a delayed manner, thereby exerting a lasting healing effect on various parts of the human body.

[0052] According to one embodiment of the present invention, by gelling the first solution using a vitamin B2 derivative that is harmless to the human body, the gelled hydrogel can be safely used on the human body.

[0053]

[0054] According to one embodiment of the present invention, a photoresponsive hydrogel-based system for controlled release of EGF is designed to enable precise temporal control and exhibit an effect of enhancing therapeutic potential in wound healing.

[0055] According to one embodiment of the present invention, a growth factor that promotes wound healing can be cross-linked to a hyaluronic acid hydrogel that basically helps wound healing, thereby enhancing the wound healing ability.

[0056] According to one embodiment of the present invention, when crosslinking a growth factor to hyaluronic acid, an optical cutter can be used to control the amount of growth factor administered using light.

[0057] According to one embodiment of the present invention, a harmless effect on the human body can be achieved by using RFP, a substance suitable for the human body, as a derivative that induces gelation.

[0058]

[0059]

[0060] Figure 1 schematically illustrates the manufacturing steps of a hyaluronic acid hydrogel having a skin wound healing effect by continuously releasing a drug according to one embodiment of the present invention.

[0061] Figure 2 schematically illustrates the manufacturing steps of a hyaluronic acid hydrogel that continuously releases a wound healing substance according to one embodiment of the present invention.

[0062] FIG. 3 schematically illustrates a schematic diagram of modified EGF immobilized inside a hyaluronic acid hydrogel according to one embodiment of the present invention.

[0063] FIG. 4 schematically illustrates MALDI-TOF data (a) and (b) of modified acrylate-EGF and acrylate-PEG-EGF of hyaluronic acid hydrogel according to one embodiment of the present invention.

[0064] Figure 5 shows an MTT assay graph confirming the proliferation of fibroblasts according to the concentration of each group EGF solution according to one embodiment of the present invention.

[0065] Figure 6 shows photographs (15.0 kV 12.0 mm × 200) (a) to (d) and a graph (e) showing the pore size of each group HA hydrogel SEM image and matrix according to one embodiment of the present invention.

[0066] Figure 7 shows the storage modulus & loss modulus graphs (a) to (d) and the storage modulus average graph (e) of each group HA hydrogel according to one embodiment of the present invention.

[0067] Figure 8 shows a graph of the decomposition behavior of each group HA hydrogel according to one embodiment of the present invention (a) and a graph of the EGF release amount confirmed by ELISA (b).

[0068] Figure 9 shows an MTT assay graph confirming the proliferation of fibroblasts over time in each group HA hydrogel according to one embodiment of the present invention.

[0069] Figure 10 shows a photo (a) and a graph (b) of a scratch assay wound closure confirming the movement of fibroblasts over time in each group HA hydrogel according to one embodiment of the present invention.

[0070] Figure 11 shows a wound closure photograph (a), a graph (b), an H&E tissue staining photograph (c), a Masson's trichrome staining photograph (d), and an epidermal thickness graph (e) in a skin wound mouse model of each group HA hydrogel according to one embodiment of the present invention.

[0071]

[0072] Figure 12 schematically illustrates the manufacturing steps of hyaluronic acid according to one embodiment of the present invention.

[0073] FIG. 13 schematically illustrates a schematic diagram of the production of an HA hydrogel matrix in which a PC linker is modified and EGF is fixed according to one embodiment of the present invention.

[0074] FIG. 14 schematically illustrates MALDI-TOF spectra data of EGF and Azido-PC-EGF according to one embodiment of the present invention (a), evaluation of photocleavage efficiency according to UV irradiation time (b), and evaluation of cell viability of NIH / 3T3 fibroblasts according to EGF concentration (1-1000 ng / ml) (c).

[0075] Figure 15 schematically illustrates the FT-IR spectra (a) and UV-Vis spectra (b) results of SH-HA, DBCO-HA, HA Gel, and EGF-PC-HA Gel according to one embodiment of the present invention.

[0076] Figure 16 schematically illustrates (a) the release amount evaluation and (b) the binding schematic diagram of HA hydrogel using FITC-BSA according to one embodiment of the present invention.

[0077] Figure 17 schematically illustrates the photomask results (a) and (b) of HA hydrogel using FITC-BSA according to one embodiment of the present invention, and the gray scale value quantitative results (c) and (d).

[0078] Figure 18 schematically illustrates representative microscopic images (a) of cell proliferation ability evaluation and cell migration ability evaluation of a hydrogel manufactured according to one embodiment of the present invention, and (b) of the results of quantification of wound closure area (c).

[0079] Figure 19 schematically illustrates a schematic diagram (a) of the wound healing ability evaluation of the hydrogel produced through a rat wound skin experiment according to one embodiment of the present invention, a representative image of each wound (b), and a result of quantifying the wound closure area (c).

[0080] Figure 20 schematically illustrates the results of Hematoxylin & Eosin (a), Masson's trichrome (b) staining, and the results of granulation tissue thickness quantification (c) on the 10th day of a rat wound skin experiment according to one embodiment of the present invention.

[0081]

[0082] Hereinafter, various embodiments and / or aspects are now disclosed with reference to the drawings. In the following description, for purposes of explanation, numerous specific details are set forth to provide a thorough understanding of one or more aspects. However, it will be apparent to one skilled in the art that such aspects may be practiced without these specific details. The following description and the attached drawings detail specific exemplary aspects of one or more aspects. However, these aspects are exemplary, and it is to be understood that any of the various methods within the principles of the various aspects may be utilized, and the description is intended to encompass all such aspects and their equivalents.

[0083]

[0084] Additionally, various aspects and features will be presented by systems that may include a number of devices, components, and / or modules. It is also to be understood and appreciated that various systems may include additional devices, components, and / or modules, and / or may not include all of the devices, components, and modules discussed in connection with the drawings.

[0085] The terms "embodiment," "example," "aspect," and "example" used herein may not be construed as implying that any aspect or design described is better or advantageous than other aspects or designs. The terms "part," "component," "module," "system," and "interface" used below generally refer to computer-related entities, and may refer to, for example, hardware, a combination of hardware and software, or software.

[0086] Additionally, it should be understood that the terms "comprises" and / or "comprising" imply the presence of the features and / or components, but do not preclude the presence or addition of one or more other features, components and / or groups thereof.

[0087] Additionally, terms including ordinal numbers, such as first, second, etc., may be used to describe various components, but the components are not limited by these terms. These terms are used solely to distinguish one component from another. For example, without departing from the scope of the present invention, a first component may be referred to as a second component, and similarly, a second component may also be referred to as a first component. The term and / or includes a combination of a plurality of related described items or any of a plurality of related described items.

[0088] Additionally, in the embodiments of the present invention, unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Terms defined in commonly used dictionaries should be interpreted as having a meaning consistent with their meaning in the context of the relevant technology, and shall not be interpreted in an idealized or overly formal sense unless explicitly defined in the embodiments of the present invention.

[0089]

[0090] Epidermal growth factor (EGF) is a soluble factor. Commonly used recombinant EGF is rapidly degraded or denatured upon contact with enzymes or chemicals, resulting in a short half-life of approximately 2-3 hours. One way to overcome this problem is to physically or chemically entrap EGF within a matrix. Simply dispersing EGF within a carrier such as a hydrogel can result in burst release depending on the hydrogel's degradation behavior, preventing prolonged drug release. However, growth factors chemically bound to the matrix are stabilized against degradation and can maintain physiological function until released. Therefore, to maintain growth factor function while delivering it effectively over a long period of time, a method of immobilizing it within a matrix is ​​necessary.

[0091] HA, characterized by its high water retention capacity, is widely used in wound dressings. However, its stability is low and it is rapidly degraded by certain enzymes, making it difficult to function as a carrier. Therefore, for long-term drug and growth factor release, a hydrogel fabrication process utilizing HA's functional groups via chemical crosslinking is necessary. Representative chemical crosslinking methods utilizing UV and V photoinitiators offer the advantage of rapid reaction times, but their high wavelength energies can lead to biocompatibility issues.

[0092] The present invention aims to manufacture HA hydrogel through photoirradiation, and to promote gelation through a reaction with significantly improved stability by using blue light irradiation with low wavelength energy and riboflavin phosphate (RFP), a derivative of vitamin B2, as a photoinitiator. At the same time, the goal is to achieve long-term drug release through a one-step process in which EGF, which has low stability, is modified with a specific functional group to chemically bond EGF to the interior of the gel simultaneously with gelation upon photoirradiation. In addition, the precursor solution can be injected into the skin in solution form, enabling wide application to various drugs and diseases.

[0093]

[0094] Figure 1 schematically illustrates the manufacturing steps of a hyaluronic acid hydrogel having a skin wound healing effect by continuously releasing a drug according to one embodiment of the present invention.

[0095]

[0096] In one embodiment of the present invention, a hyaluronic acid hydrogel having a skin wound healing effect by continuously releasing a drug can be provided, the hyaluronic acid hydrogel including a matrix material including MA-HA (Methacrylated hyaluronic acid) and SH-HA (Thiolated hyaluronic acid); and a protein modified with acrylate cross-linked to the matrix material.

[0097] In one embodiment of the present invention, the hyaluronic acid hydrogel further includes a vitamin B2 derivative (RFP, Riboflavin phosphate) and can be gelled by irradiating with blue light.

[0098] In one embodiment of the present invention, the protein may include a protein having a growth factor.

[0099] In one embodiment of the present invention, the protein having the growth factor may include at least one of epidermal growth factor (EGF), fibroblast growth factor (FGF), keratinocyte growth factor (KGF), and nerve growth factor inducible (VGF).

[0100] In one embodiment of the present invention, the MA-HA and SH-HA may have a volume ratio of 0.5 to 2 to 2 to 0.5.

[0101] In order to solve the above problem, in one embodiment of the present invention, a method for producing a hyaluronic acid hydrogel having a skin wound healing effect by continuously releasing a drug, wherein the hyaluronic acid hydrogel includes a matrix material including MA-HA and SH-HA; and a protein modified with acrylate cross-linked to the matrix material; and the method for producing the hyaluronic acid hydrogel may include the steps of: preparing a protein solution; dissolving acrylic acid NHS ester in dimethyl sulfoxide (DMSO) to produce an NHS solution; and mixing the NHS solution and the protein solution to produce an A-protein solution modified with acrylate.

[0102] In one embodiment of the present invention, the method for producing the hyaluronic acid hydrogel may include the steps of: mixing the MA-HA and SH-HA to produce a first solution; dissolving the first solution in phosphate-buffered saline (PBS) to produce a second solution; mixing the second solution and the A-protein solution to produce a third solution; adding a vitamin B2 derivative (RFP, Riboflavin phosphate) to the third solution to produce a fourth solution; and exposing the fourth solution to blue light to form a gel.

[0103] In one embodiment of the present invention, the step of exposing the fourth solution to blue light to form a gel may be performed by exposing the solution to blue light for 10 to 30 seconds.

[0104] In one embodiment of the present invention, the A-protein solution may be mixed in a volume ratio of 90 to 110 and the NHS solution may be mixed in a volume ratio of 0.1 to 3.

[0105] In one embodiment of the present invention, the step of preparing the A-protein solution may include a step of incubating the A-protein solution at 1 to 10˚C for 1 to 3 hours; and a step of dialyzing the incubated A-protein solution at 1 to 10˚C for 10 to 30 hours.

[0106]

[0107] Figure 2 schematically illustrates the manufacturing steps of a hyaluronic acid hydrogel that continuously releases a wound healing substance according to one embodiment of the present invention.

[0108]

[0109] In order to solve the above-mentioned problem, in one embodiment of the present invention, a hyaluronic acid hydrogel that continuously releases a wound healing material may include a matrix material including MA-HA (Methacrylated hyaluronic acid) and SH-HA (Thiolated hyaluronic acid); and a protein modified with acrylate PEG succinimidyl carboxymethyl ester cross-linked to the matrix material.

[0110] In one embodiment of the present invention, the hyaluronic acid hydrogel further includes a vitamin B2 derivative (RFP, Riboflavin phosphate) and can be gelled by irradiating with blue light.

[0111] In one embodiment of the present invention, the protein may include a protein having a growth factor.

[0112] In one embodiment of the present invention, the protein having the growth factor may include at least one of epidermal growth factor (EGF), fibroblast growth factor (FGF), keratinocyte growth factor (KGF), and nerve growth factor inducible (VGF).

[0113] In one embodiment of the present invention, the MA-HA and SH-HA may have a volume ratio of 0.5 to 2 to 2 to 0.5.

[0114] In order to solve the above-described problem, in one embodiment of the present invention, the hyaluronic acid hydrogel comprises a matrix material including MA-HA and SH-HA; and a protein modified with acrylate PEG succinimidyl carboxymethyl ester cross-linked to the matrix material; and a method for producing the hyaluronic acid hydrogel, which continuously releases a wound healing material, comprises the steps of: preparing a protein solution; dissolving the acrylate PEG succinimidyl carboxymethyl ester in dimethyl sulfoxide to produce an A-PEG solution; and mixing the protein solution with the A-PEG solution to produce a PEG-A-protein solution.

[0115] In one embodiment of the present invention, the method for producing the hyaluronic acid hydrogel may further include: a step of mixing the MA-HA and SH-HA to produce a first solution; a step of dissolving the first solution in phosphate buffered saline to produce a second solution; a step of mixing the second solution and a PEG-A-protein solution to produce a fifth solution; and a step of adding a vitamin B2 derivative to the fifth solution to produce a sixth solution; and a step of exposing the sixth solution to blue light to gel.

[0116] In one embodiment of the present invention, the protein modified with the acrylate PEG succinimidyl carboxymethyl ester may be mixed in a volume ratio of 9 to 11 for the protein solution and 0.5 to 2 for the A-PEG solution.

[0117] In one embodiment of the present invention, the step of preparing the PEG-A-protein solution comprises:

[0118] The method may include a step of incubating the PEG-A-protein solution at 1 to 10˚C for 1 to 3 hours; and a step of dialyzing the incubated PEG-A-protein solution at 1 to 10˚C for 10 to 30 hours.

[0119] In one embodiment of the present invention, the step of exposing the sixth solution to blue light to gel may be performed by exposing the solution to blue light for 10 to 30 seconds.

[0120]

[0121] FIG. 3 schematically illustrates a schematic diagram of modified EGF immobilized inside a hyaluronic acid hydrogel according to one embodiment of the present invention.

[0122] In one embodiment of the present invention, epidermal growth factors modified with specific functional groups are chemically bound to an HA hydrogel matrix, enabling prolonged drug release without burst release. This simple hydrogel manufacturing process allows for drug binding simultaneously with matrix crosslinking, without the need for a separate process for additional matrix binding, by simply preparing growth factors or drugs modified with specific groups in advance. This enables long-term use without side effects by modifying growth factors or drugs for specific diseases.

[0123]

[0124] MA-HA according to the present specification and one embodiment of the present invention is methacrylated hyaluronic acid, which is hyaluronic acid modified with methacrylic acid (methacrylate) and is represented by the following chemical formula 1. SH-HA according to the present specification and one embodiment of the present invention is thiolated hyaluronic acid, which is hyaluronic acid modified with thiol and is represented by the following chemical formula 2:

[0125] [Chemical Formula 1]

[0126]

[0127] [Chemical Formula 2]

[0128]

[0129] In the above chemical formulas 1 and 2, m and n are each independently integers from 200 to 1500. In one embodiment, in the above chemical formulas 1 and 2, m and n are each 1000.

[0130]

[0131] The MA-HA solution according to the present specification and an embodiment of the present invention is a methacrylated hyaluronic acid solution, which is a hyaluronic acid solution modified with methacrylic acid (methacrylate) and includes MA-HA represented by the above chemical formula 1. The SH-HA solution according to the present specification and an embodiment of the present invention is a thiolated hyaluronic acid solution, which is a hyaluronic acid solution modified with a thiol and includes SH-HA represented by the above chemical formula 2. The descriptions of chemical formulas 1 and 2 are as described above.

[0132]

[0133] According to one embodiment of the present invention, the protein comprises EGF, and will be described as EGF hereinafter. Therefore, according to one embodiment of the present invention, the protein solution may be the same as an EGF solution, the A-protein solution may be the same as an A-EGF solution, and the PEG-A-protein solution may be the same as a PEG-A-EGF solution.

[0134] According to one embodiment of the present invention, A- may be the same as Acrylate-. That is, the A-protein solution may be an Acrylate-EGF solution, and the PEG-A-protein solution may be a PEG-acrylate-EGF solution.

[0135] Additionally, the vitamin B2 derivative according to one embodiment of the present invention may be RFP (Riboflavin phosphate).

[0136] Likewise, the blue light according to one embodiment of the present invention may be an electromagnetic wave having a wavelength of preferably 380 to 520 nm, and more specifically, may be blue light within the visible light range having a wavelength of 420 to 480 nm.

[0137] Meanwhile, HA according to one embodiment of the present invention is hyaluronic acid, and HA hydrogel or hydrogel below refers to the hyaluronic acid hydrogel of the present invention. In addition, modified EGF may refer to EGF modified with acrylate or PEG-Acrylate, i.e., A-EGF and PEG-A-EGF.

[0138]

[0139] Hereinafter, a more detailed method for producing the hyaluronic acid hydrogel of the present invention will be described.

[0140] First, the N-hydroxysucinimide (NHS) ester coupling method was used to introduce an acrylate group into EGF. The modification method of acrylate-EGF, in which an acrylate group is introduced into EGF without a linker, is as follows. The EGF solution was prepared to have a concentration of 1 mg / ml, and the acrylic acid NHS ester was dissolved in DMSO to have a concentration of 81.8 mg / ml, and then the EGF solution and the acrylic acid NHS ester solution were mixed at a volume ratio of 99:1.

[0141] Acrylate PEG succinimidyl carboxymethyl ester, which has a poly(ethylene glycol) (PEG) linker between the acrylate group and EGF, was modified as follows. The EGF solution was prepared at a concentration of 1 mg / ml, and acrylate PEG succinimidyl carboxymethyl ester was dissolved in DMSO at a concentration of 94.6 mg / ml. The EGF solution and acrylate PEG succinimidyl carboxymethyl ester solution were mixed at a volume ratio of 9:1. Both solutions were incubated at 4 °C for 2 h to allow the primary amine group of EGF to react with the NHS ester. After incubation, each solution was dialyzed at 4 °C for 24 h using MWCO 3500 Da, 6-8 kDa dialysis tubing.

[0142]

[0143] Meanwhile, MA-HA (Methacrylated hyaluronic acid) and SH-HA (Thiolated hyaluronic acid) were dissolved in phosphate-buffered saline (PBS) to a concentration of 10 mg / ml. The MA-HA and SH-HA solutions were mixed in a 1:1 volume ratio. Riboflavin phosphate (RFP) was prepared at 1% w / v in PBS and added to the mixed HA solution to a final concentration of 0.01% w / v. The final mixed HA solution was exposed to blue light for 20 seconds to induce gelation. In all experiments, the final concentration of EGF and modified EGF was prepared to be 100 ng / ml when fabricating the HA hydrogels for each group.

[0144]

[0145] Through the above process, plain EGF and modified EGF (acrylate-EGF, acrylate-PEG-EGF) were each incorporated into the HA hydrogel precursor solution to prepare a final EGF concentration of 100 ng / ml. 100 μl of the hydrogel was dispensed into SPL Insert Hanging 6-well and gelled under blue light for 20 s. The prepared hydrogel was degraded in 5 ml of PBS or 5 ml of hyaluronidase (400-1000 U / mg Solid) at 37 °C. Degraded extracts were obtained from the hydrogel on days 3, 5, 10, and 13, respectively. 5 ml of PBS or hyaluronidase (400-1000 U / mg Solid) was replaced each time an extraction was obtained, and the extraction was used to quantify the amount of HA and the amount of released EGF.

[0146] First, to quantify the amount of HA, a CTAB assay was performed using the method of a previous study. 2.5 mg of CTAB was dissolved in 100 ml of 2% w / v NaOH, and the samples of each group were dissolved in PBS. Fifty microliters of the sample solution and 100 μl of the CTAB reagent were added to a 96-well plate at a 1:2% v / v ratio and incubated at 37 °C for 10 min. The absorbance at 400 nm was measured to obtain standard curves, and the accumulated HA amount on each day was quantified. The amount of released EGF was quantified using an EGF ELISA Kit according to the manufacturer's protocol. The amount of EGF released on each day was determined by the absorbance at 450 nm displayed in a microplate, and the amount of EGF was quantified through a standard curve of EGF and modified.

[0147]

[0148] That is, the hyaluronic acid hydrogel of the present invention can be manufactured by mixing the EGF solution and the acrylate PEG succinimidyl carboxymethyl ester solution with a solution of MA-HA and SH-HA dissolved in PBS, adding RFP, and gelling with blue light.

[0149]

[0150] FIG. 4 schematically illustrates MALDI-TOF data (a) and (b) of modified hyaluronic acid hydrogel according to one embodiment of the present invention. Modified acrylate-EGF and acrylate-PEG-EGF.

[0151] Specifically, successful modification of acrylate and acrylate-PEG groups was confirmed by the difference in molecular weight of EGF using MALDI-TOF. Compared to the molecular weight of unmodified EGF, three peaks were observed with a difference of 54 Da, confirming the introduction of up to three acrylate groups into EGF. In the case of acrylate-PEG, two peaks were observed with a difference of 2000 Da compared to unmodified EGF, confirming the introduction of up to two acrylate-PEG groups.

[0152]

[0153] Figure 5 shows an MTT assay graph confirming the proliferation of fibroblasts according to the concentration of each group EGF solution according to one embodiment of the present invention.

[0154] The graph in Fig. 5 demonstrates the effects of HA hydrogel on fibroblast activity and proliferation / migration. MTT assay results demonstrated that modified EGF solutions induced cell proliferation at concentrations of 100–1,000 ng / ml, and scratch assays confirmed the ability of modified EGF-containing hydrogels to achieve sustained release of growth factors and maintain cell migration (wound closure) activity.

[0155]

[0156] Figure 6 shows photographs (a) to (d) and a graph (e) showing the pore size of each group HA hydrogel SEM image and matrix according to one embodiment of the present invention.

[0157] The surface properties of the hyaluronic acid hydrogel of the present invention were observed and analyzed using a scanning electron microscope (SEM). HA hydrogel samples were incubated in PBS at 4°C for 24 h, rapidly frozen in liquid nitrogen, lyophilized for 24 h, and then sectioned with forceps. 100 μl of precursor solution from each group was loaded onto the surface of a 25 mm diameter petri dish covered with parafilm, and gelation was then performed.

[0158] The physical properties of HA hydrogels containing modified EGF were investigated using SEM and rheometer. SEM results showed that the HA matrix was cross-linked in all hydrogel groups, forming a porous three-dimensional structure and that the pore size increased with acrylate modification. Rheometer analysis of the physical stiffness of the hydrogels confirmed that all groups maintained a stable form within the range of 57–99.19 Pa at body temperature. Additionally, the introduction of EGF and acrylate-EGF disrupted cross-linking between HA chains, resulting in lower stiffness than the baseline HA hydrogel. However, the presence of a PEG linker with high mechanical strength increased the stiffness.

[0159]

[0160] Figure 7 shows the storage modulus & loss modulus graphs (a) to (d) and the storage modulus average graph (e) of each group HA hydrogel according to one embodiment of the present invention.

[0161] The dynamic rheological behavior of the hydrogel was measured using a rheometer with a diameter of 25 mm, parallel geometry, and a flat bottom. After loading 100 μl of the gel precursor solution onto the parallel plates of the rheometer, gelation was initiated by irradiating blue light for 20 s. The storage (G') and loss (G'') moduli of the gel were measured by varying the frequency from 0.1 to 10 Hz during the sweep.

[0162]

[0163] Figure 8 shows a graph of the decomposition behavior of each group HA hydrogel according to one embodiment of the present invention (a) and a graph of the EGF release amount confirmed by ELISA (b).

[0164] In order to secure a long-term delayed release behavior of EGF without burst release according to one embodiment of the present invention and to evaluate the operation of EGF modified with an acrylate group, the release behavior according to the type of modification of EGF was measured along with the degradation of HA hydrogel, and the results were plotted in the degradation behavior graph of Fig. 6.

[0165] To control the HA hydrogel degradation behavior from 0 to 14 days, PBS and HA-degrading enzyme hyaluronidase were used, and it was confirmed that the degradation slope increased when the enzyme was used. While the gel degradation behavior of all groups was generally similar, the EGF release behavior was significantly delayed in the modified EGF groups compared to unmodified EGF, and long-term release of more than 2 weeks was confirmed.

[0166]

[0167] Figure 9 shows an MTT assay graph confirming the proliferation of fibroblasts over time in each group HA hydrogel according to one embodiment of the present invention.

[0168] NIH / 3T3 fibroblast cells were cultured in medium (DMEM supplemented with 10% v / v FBS and 1% v / v penicillin-streptomycin) at 37 °C, 5% CO2, and 95% air. The concentrations of modified and unmodified EGF groups were serially diluted to 1, 10, 100, and 1000 ng / mL. 5 × 103 cells were seeded in each well of a 96-well plate. After culturing for one day in cultured medium, the cell status was checked under a microscope. The cells were then replaced with serum-free medium and cultured for 12 h to create harsh conditions. The serum-free medium was removed, and the solution sample and new cultured medium were added at a ratio of 1:10 v / v% and cultured for 24 h. After removing the solution, 10% v / v MTT solution (6 mg / mL) was added and incubated at 37 °C for 2 h. The resulting formazan crystals were dissolved in DMSO. The absorbance was measured at 540 nm.

[0169] Cell bioactivity in gels containing modified and unmodified materials was evaluated using the MTT assay. 25 μl of gel was placed in a hanging insert, and sufficient medium was added. The gel was incubated for 2 h to adapt to the medium. After removal, the medium was replaced with fresh medium. 0.5 × 104 cells were seeded in each 24-well plate on days 1, 4, 7, and 10. The medium was replaced every other day. On days 1, 4, 7, and 10, the medium was removed, and a 10% v / v MTT solution (6 mg / ml) was added to the medium and incubated at 37 °C for 2 h. The resulting formazan crystals were dissolved with DMSO. The absorbance was measured at 540 nm.

[0170]

[0171] Figure 10 shows a photo (a) and a graph (b) of a scratch assay wound closure confirming the movement of fibroblasts over time in each group HA hydrogel according to one embodiment of the present invention.

[0172] A scratch assay was conducted to determine cell migration ability and proliferation. A scratch tool was inserted into each well of a 12-well plate, and 70 μl of NIH / 3T3 fibroblast cell stock (8.0 × 105 cells / ml) was seeded into each insert. The insert was incubated overnight with medium (DMEM supplemented with 10% v / v FBS and 1% v / v penicillin-streptomycin). The medium was removed, and the insert attached to the bottom of the plate was carefully removed using forceps. Gels for each group were prepared in the same manner as in the MTT preparation method, placed in the appropriate well plate for each group, and floated. Afterwards, 1 ml of DMEM containing 10% FBS was added to the inside and outside of the scratch tool and incubated for 24 hours. The following day, cell attachment was confirmed using an optical microscope. Cell migration through the gap created by the tool was assessed and images were captured after 6, 18, 24, and 30 hours. The wound closure percentage was quantified using the ImageJ program.

[0173]

[0174] Figure 11 shows a wound closure photograph (a), a graph (b), an H&E tissue staining photograph (c), a Masson's trichrome staining photograph (d), and an epidermal thickness graph (e) in a skin wound mouse model of each group HA hydrogel according to one embodiment of the present invention.

[0175] The wound healing ability of EGF Hydrogel was evaluated using a BALB / c mouse model. A 5-mm biopsy punch was used to create a wound, and a Grace bio-lebs 14 mm x 7 mm wound splint was used to secure the wound to prevent contraction. After fixation, the ring was sutured with a surgical needle to prevent movement. The gel of each group was applied to the wound in the form of a precursor solution (50 μl) and gelated under blue light for 40 sec. Afterwards, a 3M Tegaderm transparent waterproof film was applied. The Tegaderm was removed on days 0, 3, 6, and 10, and the wounds were captured, and the wound closure area was quantified using ImageJ.

[0176] Wound tissues were harvested on day 10. The skin tissues were fixed with 4% paraformaldehyde and simultaneously histologically analyzed. Hematoxylin and eosin (H&E) and Masson's trichrome staining were used to qualitatively compare the wound healing ability of the control group and the groups to which each hydrogel was applied. For histological analysis, skin tissue sections from the stained central scar area were observed under a microscope, and images were viewed on a computer using the 3D HISTECH Slide Center program. The stained images of all groups were fixed and mounted at 9.6 X. Scar length, percent closure of the panniculus carnosus (PC) muscle, and epidermal layer depth were measured and quantified using the ImageJ program at 3.6 X magnification.

[0177]

[0178] According to one embodiment of the present invention, there is provided a technology for improving wound healing effects through the possibility of sustained release without burst release by modifying EGF with acrylate and acrylate-PEG so that it can be chemically bound to an HA hydrogel matrix through a thiol-ene reaction. The natural photoinitiator and blue visible light used in the hydrogel production and modified EGF binding process enable the production of a hydrogel with excellent biocompatibility, and the modified EGF can maintain physiological activity for a long period of time within the hydrogel through chemical bonding, and can exhibit an effect that can be used as an effective therapeutic factor through delayed release.

[0179]

[0180] According to one embodiment of the present invention, the effect of producing a matrix material can be achieved by mixing MA-HA and SH-HA.

[0181] According to one embodiment of the present invention, there is provided a technology for improving wound healing effects through the possibility of sustained release without burst release by modifying EGF with acrylate and acrylate-PEG so that it can be chemically bound to an HA hydrogel matrix through a thiol-ene reaction. The natural photoinitiator and blue visible light used in the hydrogel production and modified EGF binding process enable the production of a hydrogel with excellent biocompatibility, and the modified EGF can maintain physiological activity for a long period of time within the hydrogel through chemical bonding, and can exhibit an effect that can be used as an effective therapeutic factor through delayed release.

[0182] According to one embodiment of the present invention, by modifying a protein having a growth factor with acrylate or PEG, the protein having a growth factor is cross-linked to a hyaluronic acid hydrogel to release the protein in a delayed manner, thereby exerting a lasting healing effect on various parts of the human body.

[0183] According to one embodiment of the present invention, by gelling the first solution using a vitamin B2 derivative that is harmless to the human body, the gelled hydrogel can be safely used on the human body.

[0184]

[0185] Although the embodiments have been described with limited examples and drawings, those skilled in the art will appreciate that various modifications and variations can be made based on the above teachings. For example, appropriate results can be achieved even if the described techniques are performed in a different order than described, and / or components of the described systems, structures, devices, circuits, etc. are combined or combined in a different manner than described, or are replaced or substituted with other components or equivalents. Therefore, other implementations, other embodiments, and equivalents of the claims also fall within the scope of the claims described below.

[0186]

[0187]

[0188] Hereinafter, various embodiments and / or aspects are now disclosed with reference to the drawings. In the following description, for purposes of explanation, numerous specific details are set forth to provide a thorough understanding of one or more aspects. However, it will be apparent to one skilled in the art that such aspects may be practiced without these specific details. The following description and the attached drawings detail specific exemplary aspects of one or more aspects. However, these aspects are exemplary, and it is to be understood that any of the various methods within the principles of the various aspects may be utilized, and the description is intended to encompass all such aspects and their equivalents.

[0189]

[0190] Additionally, various aspects and features will be presented by systems that may include a number of devices, components, and / or modules. It is also to be understood and appreciated that various systems may include additional devices, components, and / or modules, and / or may not include all of the devices, components, and modules discussed in connection with the drawings.

[0191] The terms "embodiment," "example," "aspect," and "example" used herein may not be construed as implying that any aspect or design described is better or advantageous than other aspects or designs. The terms "part," "component," "module," "system," and "interface" used below generally refer to computer-related entities, and may refer to, for example, hardware, a combination of hardware and software, or software.

[0192] Additionally, it should be understood that the terms "comprises" and / or "comprising" imply the presence of the features and / or components, but do not preclude the presence or addition of one or more other features, components and / or groups thereof.

[0193] Additionally, terms including ordinal numbers, such as first, second, etc., may be used to describe various components, but the components are not limited by these terms. These terms are used solely to distinguish one component from another. For example, without departing from the scope of the present invention, a first component may be referred to as a second component, and similarly, a second component may also be referred to as a first component. The term and / or includes a combination of a plurality of related described items or any of a plurality of related described items.

[0194] Additionally, in the embodiments of the present invention, unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Terms defined in commonly used dictionaries should be interpreted as having a meaning consistent with their meaning in the context of the relevant technology, and shall not be interpreted in an idealized or overly formal sense unless explicitly defined in the embodiments of the present invention.

[0195]

[0196] The present invention aims to provide a method for manufacturing a hydrogel by modifying a photocleavable linker having an azide group and an NHS ester group through the amine group of epidermal growth factor, reacting it with hyaluronic acid modified with a DBCO group to fix it, and using riboflavin phosphate (RFP), a non-toxic natural photoinitiator, and blue light. This not only delays the drug release behavior, but also allows the epidermal growth factor to be released at a desired time and space by UV light of a specific wavelength, thereby enhancing the therapeutic potential of epidermal growth factor in wound healing applications.

[0197]

[0198] Figure 12 schematically illustrates the manufacturing steps of hyaluronic acid according to one embodiment of the present invention.

[0199]

[0200] A method for producing a hyaluronic acid hydrogel from which a wound healing material can be selectively released upon light irradiation according to one embodiment of the present invention, the hyaluronic acid hydrogel comprising: a hyaluronic acid hydrogel matrix; a photocleavable group bound to the hyaluronic acid hydrogel matrix; and a growth factor bound to the hyaluronic acid hydrogel matrix; the method comprising: preparing a protein solution by dissolving a protein in PBS; preparing an Azido-PC solution by dissolving Azido-PC-NHS ester, which is a photocleavable linker, in dimethyl sulfoxide (DMSO); preparing an Azido-PC-protein solution by mixing the protein solution and the Azido-PC solution; A wound healing material can be selectively released upon light irradiation, comprising: a step of preparing an SH-HA solution by dissolving SH-HA (Thiolated hyaluronic acid) in phosphate-buffered saline (PBS); a step of preparing a DBCO solution by dissolving dibenzocyclopentane-maleimide (DBCO-Maleimide) in dimethyl sulfoxide; a step of preparing a DBCO-HA solution by mixing the SH-HA solution and the DBCO solution; a step of preparing a protein-PC-HA solution by mixing the Azido-PC-protein solution and the DBCO-HA solution; a step of preparing a hydrogel precursor solution by mixing the protein-PC-HA solution with a MA-HA (Methacrylated hyaluronic acid) solution and a vitamin B2 derivative (RFP, Riboflavin phosphate); and a step of gelling the hydrogel precursor solution by irradiating the hydrogel precursor solution with blue light.

[0201] In one embodiment of the present invention, the hyaluronic acid hydrogel matrix includes MA-HA (Methacrylated hyaluronic acid) and SH-HA (Thiolated hyaluronic acid), and the MA-HA and SH-HA may have a volume ratio of 0.5 to 2 to 2 to 0.5.

[0202] In one embodiment of the present invention, the optical cutter may include Azido-PC-NHS ester.

[0203] In one embodiment of the present invention, the growth factor may include at least one of epidermal growth factor (EGF), fibroblast growth factor (FGF), keratinocyte growth factor (KGF), and nerve growth factor inducible (VGF).

[0204] In one embodiment of the present invention, the protein may have a concentration of 0.01 to 1 mg / ml relative to the phosphate buffered saline solution.

[0205] In one embodiment of the present invention, the Azido-PC-NHS ester may have a concentration of 20 to 40 mg / ml relative to the dimethyl sulfoxide.

[0206] In one embodiment of the present invention, the Azido-PC-NHS ester can be diluted to a concentration of 50 to 150 times and mixed with the protein solution.

[0207] In one embodiment of the present invention, the step of preparing the Azido-PC-protein solution may further include a step of purifying the Azido-PC-protein solution at 1 to 7 °C for 20 to 30 hours.

[0208] In one embodiment of the present invention, the dibenzocyclotine-maleimide may have a concentration of 5 to 15 mg / ml relative to the dimethyl sulfoxide.

[0209]

[0210] MA-HA according to the present specification and one embodiment of the present invention is methacrylated hyaluronic acid, which is hyaluronic acid modified with methacrylic acid (methacrylate) and is represented by the following chemical formula 1. SH-HA according to the present specification and one embodiment of the present invention is thiolated hyaluronic acid, which is hyaluronic acid modified with thiol and is represented by the following chemical formula 2:

[0211] [Chemical Formula 1]

[0212]

[0213] [Chemical Formula 2]

[0214]

[0215] In the above chemical formulas 1 and 2, m and n are each independently integers from 200 to 1500. In one embodiment, in the above chemical formulas 1 and 2, m and n are each 1000.

[0216]

[0217] The MA-HA solution according to the present specification and an embodiment of the present invention is a methacrylated hyaluronic acid solution, which is a hyaluronic acid solution modified with methacrylic acid (methacrylate) and includes MA-HA represented by the above chemical formula 1. The SH-HA solution according to the present specification and an embodiment of the present invention is a thiolated hyaluronic acid solution, which is a hyaluronic acid solution modified with a thiol and includes SH-HA represented by the above chemical formula 2. The descriptions of chemical formulas 1 and 2 are as described above.

[0218]

[0219] According to one embodiment of the present invention, a protein comprises a growth factor, and since the growth factor comprises EGF, the protein will be described as EGF hereinafter. Accordingly, according to one embodiment of the present invention, a protein solution may be identical to an EGF solution, an Azido-PC-protein solution may be identical to an Azido-PC-EGF solution, and a protein-PC-HA solution may be identical to an EGF-PC-HA.

[0220] Additionally, the vitamin B2 derivative according to one embodiment of the present invention may be RFP (Riboflavin phosphate).

[0221] Likewise, the blue light according to one embodiment of the present invention may be an electromagnetic wave having a wavelength of preferably 380 to 520 nm, and more specifically, may be blue light within the visible light range having a wavelength of 420 to 480 nm.

[0222] In addition, the optical cutter according to one embodiment of the present invention may include a PC linker, which may be identical to the -PC- of the Azido-PC-protein solution. Hereinafter, it will be described as a PC-linker.

[0223]

[0224] Specifically, the PC linker included in the photoconductor is represented by the chemical formula 4 part in the PC-Azido-NHS Ester represented by the chemical formula 3 below in one embodiment. At this time, when photocleavage by UV occurs, the PC-Azido-NHS Ester represented by the chemical formula 3 below is divided into radical compounds represented by the chemical formula 5 below and the chemical formula 6 below.

[0225] [Chemical Formula 3]

[0226]

[0227] [Chemical Formula 4]

[0228]

[0229] [Chemical Formula 5]

[0230]

[0231] [Chemical Formula 6]

[0232]

[0233]

[0234] Specifically, the process of EGF modifying a PC linker according to one embodiment of the present invention will be examined.

[0235] The process of EGF modifying a PC linker according to one embodiment of the present invention may include a step of preparing the protein solution, a step of preparing an Azido-PC solution, and a step of preparing an Azido-PC-protein solution.

[0236] First, EGF with an amine group was modified with PC-linker through PC-Azido-NHS Ester and NHS ester coupling reaction. EGF was dissolved in PBS to 0.1 mg / ml. Azido-PC-NHS ester was used as a photocleavable linker. This linker is efficiently photocleaved when irradiated with UV light (365 nm, 1-5 mW / cm2). PC linker was dissolved in DMSO to 31 mg / ml. The amount of PC linker was added in a 10-fold excess of the molar number of EGF.

[0237]

[0238] [Formula 1]

[0239] The concentration of Azido-PC-NHS ester in the final EGF determined according to the above [Formula 1] was 0.31 mg / ml. 31 mg / ml was diluted 100-fold and added to the EGF solution and mixed. The mixed solution was purified using a D-Tube™ Dialyzer Maxi, MWCO 6-8 kDa at 4 °C for 24 hours to remove unmodified Azido-PC-NHS ester and impurities. Purification was performed by replacing PBS 1X every 1, 2, 3, and 4 hours, and the obtained Azido PC modified EGF was aliquoted into 100 uL aliquots and stored frozen. In this paper, the following synthesized material is referred to as Azido-PC-EGF.

[0240] The molecular weight difference between Azido-PC-EGF (0.1 mg / ml) synthesized in this way and EGF was investigated using a matrix-assisted laser desorption and ionization time-of-flight (MALDI-TOF) mass spectrometer (Autoflex maX, Bruker, Billerica, MA, USA) located at Gyeonggido Business & Science Accelerator, South Korea, and the result is shown in Fig. 3a, which will be described later.

[0241] Meanwhile, according to one embodiment of the present invention, diluting a substance by n times means a dilution factor, and the dilution factor means the total volume of the final substance / volume of the substance.

[0242] That is, according to one embodiment of the present invention, the meaning that the Azido-PC-NHS ester is diluted to a concentration of 50 to 150 times and mixed with the protein solution means that the concentration of the Azido-PC-NHS ester is diluted to 1 / 150 to 1 / 50 of the initial concentration by adding 49 to 149 times the volume of the solvent to the Azido-PC-NHS ester.

[0243] Meanwhile, HA according to one embodiment of the present invention is hyaluronic acid, and HA hydrogel or hydrogel below means the hyaluronic acid hydrogel of the present invention.

[0244]

[0245] Next, we will look at the step of fixing EGF modified with PC linker to the HA Hydrogel matrix.

[0246] The step of fixing EGF modified with a PC linker to the HA Hydrogel matrix according to one embodiment of the present invention may include a step of preparing the SH-HA solution, a step of preparing a DBCO solution, and a step of preparing a protein-PC-HA solution.

[0247] Specifically, SH-HA (10 mg / mL) was dissolved in PBS (pH 7.4). DBCO-Maleimide was dissolved in DMSO at a concentration of 9.48 mg / mL. The two solutions were mixed by vortexing at a volume ratio of 99:1. DBCO-Maleimide was added in a 10 molar excess over SH-HA to ensure smooth conjugation. The mixed solution was purified using a D-Tube™ Dialyzer Maxi, MWCO 6-8 kDa at 4 °C for 24 h to remove unmodified DBCO-Maleimide and impurities, and protected from light. Purification was performed by replacing the solution with 1X PBS every 1, 2, 3, and 4 hours. In this paper, the following synthesized material is referred to as DBCO-HA.

[0248] DBCO-HA (10 mg / mL) synthesized through the above process was added to Azido-PC-EGF (0.01 mg / mL) after 10-fold dilution. The final concentration of EGF in the gel was 0.001 mg / mL. The mixed solution was reacted for 100 minutes at room temperature in a lightless environment. To determine whether the reaction between DBCO and the azide group occurred successfully, the DBCO absorbance peak appearing at 290–310 nm was analyzed for UV-Vis spectra using Nanodrop™ (2000 / 2000c Spectrophotometer) according to the reaction time (0, 10, 15, 30, 60, and 100 minutes), and the results are shown in Fig. 3b. The material synthesized in this way is referred to as EGF-PC-HA.

[0249]

[0250] Finally, we will examine the manufacturing steps of HA Hydrogel containing Azido-PC-EGF.

[0251] The manufacturing step of HA Hydrogel including Azido-PC-EGF according to one embodiment of the present invention may include a step of preparing the hydrogel precursor solution and a step of gelling by irradiating with blue light.

[0252] Specifically, the synthesized EGF-PC-HA (10 mg / ml) was mixed with MA-HA (10 mg / ml) at a 1:1 vol% ratio. Riboflavin (1%) was used as a photoinitiator and added to the gel solution at a 99:1 ratio to prepare a hydrogel precursor solution. The precursor solution was dispensed as 25 μL or 50 μL on parafilm and exposed to blue light (Blue Dental Wireless / Cordless LED Cure Curing Light Lamp, spectrum 420-480 nm) for 10 s, forming an EGF-PC-HA gel. The fabricated hydrogel was used to evaluate cell viability and wound healing ability, and the release profile within the hydrogel was replaced by FITC-Albumin instead of EGF.

[0253] Crosslinking of HA hydrogels was confirmed using Fourier transform infrared spectroscopy (FT-IR). All samples were lyophilized and their transmittance was measured using KBr pellets in the range of 3000–500 cm-1 at room temperature using an iS50 spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). A detailed description of this is provided later in Figure 4.

[0254]

[0255] Meanwhile, the step of preparing the Azido-PC-protein solution may further include a step of purifying the Azido-PC-protein solution at 1 to 7 °C for 20 to 30 hours.

[0256] Specifically, to evaluate the release behavior from HA hydrogel using PC Linker-modified FITC-BSA, FITC-Albumin (10 mg / ml) was dissolved in PBS (pH 7.4). PC-Azido-NHS ester was dissolved in DMSO at 29.1 mg / ml and diluted 10-fold to obtain an 8-fold molar excess of FITC-Albumin (10 mg / ml) to obtain a final concentration of 2.91 mg / ml of PC-Azido-NHS ester. To remove unmodified Azido-PC-NHS ester and impurities, purification was performed at 4 °C for 24 h using D-Tube™ Dialyzer Maxi, MWCO 6-8 kDa. Purification was performed by replacing PBS 1X every 1, 2, 3, and 4 hours, and the obtained Azido PC modified FITC-Albumin (10 mg / ml) was aliquoted into 100 μL portions and stored frozen. In this paper, the following synthesized material is referred to as Azido-PC-BSA.

[0257]

[0258] FIG. 13 schematically illustrates a schematic diagram of the production of an HA hydrogel matrix in which a PC linker is modified and EGF is fixed according to one embodiment of the present invention.

[0259]

[0260] A method for producing a hyaluronic acid hydrogel from which a wound healing material can be selectively released upon light irradiation according to one embodiment of the present invention, the hyaluronic acid hydrogel comprising: a hyaluronic acid hydrogel matrix; a photocleavable group bound to the hyaluronic acid hydrogel matrix; and a growth factor bound to the hyaluronic acid hydrogel matrix; the method comprising: preparing a protein solution by dissolving a protein in PBS; preparing an Azido-PC solution by dissolving Azido-PC-NHS ester, which is a photocleavable linker, in dimethyl sulfoxide (DMSO); preparing an Azido-PC-protein solution by mixing the protein solution and the Azido-PC solution; A wound healing material can be selectively released upon light irradiation, comprising: a step of preparing an SH-HA solution by dissolving SH-HA (Thiolated hyaluronic acid) in phosphate-buffered saline (PBS); a step of preparing a DBCO solution by dissolving dibenzocyclopentane-maleimide (DBCO-Maleimide) in dimethyl sulfoxide; a step of preparing a DBCO-HA solution by mixing the SH-HA solution and the DBCO solution; a step of preparing a protein-PC-HA solution by mixing the Azido-PC-protein solution and the DBCO-HA solution; a step of preparing a hydrogel precursor solution by mixing the protein-PC-HA solution with a MA-HA (Methacrylated hyaluronic acid) solution and a vitamin B2 derivative (RFP, Riboflavin phosphate); and a step of gelling the hydrogel precursor solution by irradiating the hydrogel precursor solution with blue light.

[0261] In one embodiment of the present invention, the hyaluronic acid hydrogel matrix includes MA-HA (Methacrylated hyaluronic acid) and SH-HA (Thiolated hyaluronic acid), and the MA-HA and SH-HA may have a volume ratio of 0.5 to 2 to 2 to 0.5.

[0262] In one embodiment of the present invention, the optical cutter may include Azido-PC-NHS ester.

[0263] In one embodiment of the present invention, the growth factor may include at least one of epidermal growth factor (EGF), fibroblast growth factor (FGF), keratinocyte growth factor (KGF), and nerve growth factor inducible (VGF).

[0264] In one embodiment of the present invention, the protein may have a concentration of 0.01 to 1 mg / ml relative to the phosphate buffered saline solution.

[0265] In one embodiment of the present invention, the Azido-PC-NHS ester may have a concentration of 20 to 40 mg / ml relative to the dimethyl sulfoxide.

[0266] In one embodiment of the present invention, the Azido-PC-NHS ester can be diluted to a concentration of 50 to 150 times and mixed with the protein solution.

[0267] In one embodiment of the present invention, the step of preparing the Azido-PC-protein solution may further include a step of purifying the Azido-PC-protein solution at 1 to 7 °C for 20 to 30 hours.

[0268] In one embodiment of the present invention, the dibenzocyclotine-maleimide may have a concentration of 5 to 15 mg / ml relative to the dimethyl sulfoxide.

[0269]

[0270] A hyaluronic acid hydrogel according to one embodiment of the present invention may be a hyaluronic acid-based hydrogel in which a photocleavable linker is modified into an epidermal growth factor and fixed to hyaluronic acid, and the hyaluronic acid is crosslinked to form a hydrogel, thereby enabling the release of an epidermal growth factor to be induced by UV.

[0271]

[0272] FIG. 14 schematically illustrates MALDI-TOF spectra data of EGF and Azido-PC-EGF according to one embodiment of the present invention (a), evaluation of photocleavage efficiency according to UV irradiation time (b), and evaluation of cell viability of NIH / 3T3 fibroblasts according to EGF concentration (1-1000 ng / ml) (c).

[0273] Figure 14a shows the results of confirming the difference in molecular weight through matrix-assisted laser desorption and ionization time-of-flight (MALDI-TOF) mass spectrometer (Autoflex maX, Bruker, Billerica, MA, USA) after modifying epidermal growth factor with a photocleavable linker (Azido-PC-NHS Ester).

[0274] According to one embodiment of the present invention, Azido-PC-EGF (0.1 mg / ml) synthesized was analyzed for molecular weight difference with EGF using a matrix-assisted laser desorption and ionization time-of-flight (MALDI-TOF) mass spectrometer (Autoflex maX, Bruker, Billerica, MA, USA) located at Gyeonggido Business & Science Accelerator, South Korea, as shown in Figure 3a.

[0275]

[0276] Figure 14b confirms the successful introduction of a photocleavable linker into epidermal growth factor (Azido-PC-EGF). Furthermore, UV-Vis spectra measurements show the photocleavage efficiency of Azido-PC-EGF as a function of UV irradiation time.

[0277] Figure 14c shows the results of MTT Assay confirming that the bioactivity of EGF is maintained regardless of the modification of the PC linker and UV irradiation.

[0278] That is, whether the synthesized Azido-PC-EGF undergoes cleavage of the PC linker by UV stimulation was confirmed through UV-Vis spectroscopy (Fig. 14b). The PC linker is based on o-nitrobenzyl ester and is decomposed into carboxylic acid and o-nitrobenzaldehyde by UV. The UV absorbances of o-nitrobenzaldehyde and carboxylic acid are 270 and 210 nm, respectively, and the changes in the two groups were measured. As the UV irradiation time increased from 0 to 30 min, the absorption peak of carboxylic acid at 210 nm and the absorption peak of o-nitrobenzaldehyde at 270 nm gradually increased.

[0279]

[0280] Specifically, the bioactivity changes of modified Azido-PC-EGF were evaluated using the MTT assay. NIH / 3T3 Fibroblasts (Korean Cell Line Bank, Seoul, Korea) were cultured in culture medium (DMEM supplemented with 10% v / v FBS and 1% v / v penicillin-streptomycin) and incubated at 37°C in 5% CO2 and 95% air. These conditions are standard conditions generally used when culturing cells. The medium was changed twice a week, and when approximately 80% of the cells were obtained, subculturing was performed at a 1:3 ratio. NIH / 3T3 cells (5 × 103) were seeded in 96-well plates and cultured for 4 h under standard conditions. After 4 hours, the medium was changed to low FBS medium (DMEM supplemented with 1% v / v FBS and 1% v / v penicillin-streptomycin) for cell starvation and cultured for 24 hours. After 24 hours, EGF solution (1-1000 ng / ml) including Azido-PC-EGF was added at a 1:10 ratio to standard condition medium and cultured for 24 hours. After 24 hours, the medium was removed, and 10% v / v MTT solution (mg / mL) was added together with the medium and incubated at 37°C for 2 hours. The formed formazan crystals were dissolved with DMSO. The absorbance at 540 nm was measured using a microplate reader (Agilent Technologies, CA, USA) and is shown in Figure 14c.

[0281]

[0282] Figure 15 schematically illustrates the FT-IR spectra (a) and UV-Vis spectra (b) results of SH-HA, DBCO-HA, HA Gel, and EGF-PC-HA Gel according to one embodiment of the present invention.

[0283] Prior to conjugating Azido-PC-EGF to hyaluronic acid, a step of modifying thiolated hyaluronic acid (SH-HA) with DBCO-Maleimide (DBCO-MAL) to produce DBCO-HA was performed to ensure a successful reaction. The success of the modification was confirmed using Fourier transform infrared spectroscopy (FT-IR), and the results are shown in Fig. 4a.

[0284] According to one embodiment of the present invention, EGF-PC-HA is mixed with MA-HA and irradiated with blue light in the presence of RFP to produce a hydrogel. Crosslinking of the hyaluronic acid hydrogel is illustrated in Fig. 4a using Fourier transform infrared spectroscopy (FT-IR).

[0285]

[0286] In addition, DBCO-HA according to one embodiment of the present invention is combined with Azido-PC-EGF to produce EGF-PC-HA. In order to determine whether the reaction between DBCO and the azide group occurred well, the UV-Vis spectra of the DBCO absorbance peak appearing at 290 to 310 nm were analyzed using Nanodrop™ (2000 / 2000c Spectrophotometer) according to the reaction time (0, 10, 15, 30, 60, 100 min), and the results are shown in Fig. 4b.

[0287]

[0288] Figure 16 schematically illustrates (a) the release amount evaluation and (b) the binding schematic diagram of HA hydrogel using FITC-BSA according to one embodiment of the present invention.

[0289] Figure 16 schematically illustrates the results of evaluating the release behavior in HA hydrogel using FITC-BSA modified with PC Linker.

[0290] Specifically, FITC-Albumin (10 mg / ml) was dissolved in PBS (pH 7.4). PC-Azido-NHS ester was dissolved in DMSO at 29.1 mg / ml and diluted 10-fold to obtain an 8-fold molar excess of FITC-Albumin (10 mg / ml) so that the final concentration of PC-Azido-NHS ester was 2.91 mg / ml. To remove unmodified Azido-PC-NHS ester and impurities, purification was performed at 4 °C for 24 hours using a D-Tube™ Dialyzer Maxi, MWCO 6-8 kDa. Purification was performed by replacing PBS 1X every 1, 2, 3, and 4 hours, and the obtained Azido PC modified FITC-Albumin (10 mg / ml) was aliquoted into 100 μL aliquots and stored frozen. In this paper, the following synthesized material is referred to as Azido-PC-BSA.

[0291] The synthesized Azido-PC-BSA (10 mg / ml) was mixed with DBCO-HA (10 mg / ml) at a volume ratio of 10:1 and reacted for 100 minutes. Then, MA-HA (10 mg / ml) was added in the same volume ratio as DBCO-HA, and 0.01% riboflavin was added to prepare a hydrogel precursor solution. 50 μL of the precursor solution was dispensed onto parafilm and exposed to blue light for 10 seconds to form a BSA-PC-HA gel. The prepared hydrogel was floated on a 48-well plate for degradation and incubated in 1 ml of PBS 1X buffer or 1 ml of hyaluronidase from bovine testes (400-1000 U / mg Solid) at 37°C. To quantify FITC-BSA released from the gel, extracts were obtained on Days 3, 4, 5, 6, 7, and 8 and replaced with fresh PBS. The obtained extracts were measured for fluorescence intensity using a Multidetection Microplate Reader (TriStar2 LB 942), and the quantification was performed through interpolation using a calibration curve of PC-Azido-BSA from 0 to 1 mg / ml. Fluorescence microscopic images of the BSA-PC-HA Gel were captured on each day, and the intensity changes were quantified using Image J, and the results are shown in Fig. 5a.

[0292]

[0293] In one embodiment of the present invention, the hydrogel was prepared by replacing EGF with FITC-Albumin to determine the release profile. As shown in Fig. 16a, the BSA / HA Gel group initially showed a large amount of release, releasing more than about 50% of the BSA on the third day. On the other hand, the release amount in the BSA-PC-HA Gel group bound with a PC linker was less than about 20% until the third day, which was significantly lower than that of the BSA / HA Gel group and was within about 20%. In the BSA-PC-HA Gel that was not irradiated with UV, about 10% of the BSA was released from the third to the eighth day. In the BSA-PC-HA Gel that was irradiated with UV, 20% of the BSA was released from the third to the eighth day. In particular, it was confirmed that the increase in the release measured the day after UV irradiation was greater compared to the case where UV was not irradiated. Therefore, when the PC linker was introduced, it was confirmed that the linker was decomposed by UV irradiation and BSA was released outside the hydrogel.

[0294]

[0295] Figure 17 schematically illustrates the photomask results (a) and (b) of HA hydrogel using FITC-BSA according to one embodiment of the present invention, and the gray scale value quantitative results (c) and (d).

[0296] Figure 17 schematically illustrates the results of an experiment to induce selective release from HA hydrogel using a photomask.

[0297] Specifically, for the selective release-induced experiment using a photomask, the gel was prepared as follows. After preparing the hydrogel precursor solution as in the degradation and release experiment, 10 μL was dispensed into a mini petri dish. To make the gel into a flat film, a cover glass platform was placed on the dispensed gel and blue light was irradiated for 10 seconds. After irradiation, the cover glass was carefully removed and incubated with 3 ml of PBS for 1 hour to remove RFP that can be affected by UV. After removing the PBS, the fluorescence microscopic images of the hydrogel were captured using an optical microscope (EVOS M5000 Imaging System, Thermo Fisher Scientific, Waltham, MA, USA) before UV irradiation. The photomask (100 μm) was fixed with tape and UV light (Altis UVcure, 365 nm, 1-5 mW / cm2) was exposed for 0, 5, 10, and 15 minutes, respectively. After removing the photomask and washing 10 times with 1 ml of PBS, the hydrogel was degraded in 3 ml of PBS at 37°C for 24 hours. After 24 hours, fluorescence microscopic images of the hydrogel were captured (Fig. 16.a, b). The obtained images were converted to grayscale values ​​along a 1200 pixel line using Image J, and the patterned hydrogel was plotted graphically in Fig. 17c and Fig. 17d.

[0298] In one embodiment of the present invention, a photomask was used to enable light emission only in a selective area irradiated with light in a hydrogel. A BSA-PC-HA Gel group that can be photocleaved by UV and a BSA / HA Gel group that does not photoreact were set as controls and irradiated with UV over time. As shown in Fig. 17a, the BSA / HA Gel was not affected by UV irradiation, and it was visually confirmed that BSA was rapidly released from the gel after 24 hours of degradation in PBS, but the fluorescence intensity decreased. In addition, Fig. 17c shows the gray scale value results of each Fig. 17a image, and it was confirmed that there was no intensity change along the pixel line. On the other hand, looking at the BSA-PC-HA Gel group in Fig. 17b, in the area exposed to light, the PC linker was cleaved, releasing BSA from the gel after 24 hours of degradation, while in the area that was not exposed to light, the BSA was still bound to the HA matrix even after degradation, showing a fluorescence intensity similar to the initial one. Therefore, looking at Fig. 17d, which is the gray scale value result of the image of Fig. 17b, it shows the variation of fluorescence intensity as if intensity is patterned along the pixel line. As a result, a photo pattern is formed as the areas that were illuminated and those that were not illuminated by the photomask are repeated. This means that the molecule linked with the PC linker can control emission spatiotemporally and as needed by UV stimulation.

[0299]

[0300] Figure 18 schematically illustrates representative microscopic images (a) of cell proliferation ability evaluation and cell migration ability evaluation of a hydrogel manufactured according to one embodiment of the present invention, and (b) of the results of quantification of wound closure area (c).

[0301] Figure 18 schematically illustrates the results of evaluating the proliferation and migration ability of NIH / 3T3 fibroblasts in the fabricated hydrogel.

[0302] NIH / 3T3 Fibroblasts (Korean Cell Line Bank, Seoul, Korea) were cultured in culture medium (DMEM supplemented with 10% v / v FBS and 1% v / v penicillin-streptomycin) and incubated at 37°C in 5% CO2 and 95% air. These conditions are generally standard conditions used when culturing cells. The medium was changed twice a week, and when approximately 80% cell confluency was obtained, subculturing was performed at a 1:3 ratio. The MTT assay was performed to evaluate the cell viability of the fabricated hydrogel. NIH / 3T3 cells (5 × 103) were seeded in four 24-well plates (days 3, 5, 7, and 10) and cultured for 4 hours under standard conditions. Hydrogels were prepared in 25 μL aliquots in 24-well plates according to the previously described method, and incubated with 500 μL of medium (DMEM supplemented with 10% v / v FBS and 1% v / v penicillin-streptomycin) at 37°C in 5% CO2 and 95% air for 2 h to adapt to the medium. After incubation, the gels were separated from the parafilm and placed in the cell-seeded plate using SPLInsert™ Hanging 24-well. The medium was replaced every two days. On days 3, 5, 7, and 10, the medium was removed, and 10% v / v MTT solution (mg / mL) was added together with the medium, followed by incubation at 37°C for 2 h. The formed formazan crystals were dissolved by DMSO.The absorbance was measured at 540 nm using a microplate reader (Agilent Technologies, CA, USA) (Fig. 18a).

[0303] To evaluate the wound healing ability of the PC-EGF hydrogel, a scratch assay was performed. NIH / 3T3 fibroblasts (7 × 105 cells / ml) were seeded in 24-well plates equipped with a scratch tool (ibidi culture-insert 2-well, ibidi GmbH, Martinscried, Germany). After culturing overnight in medium (DMEM containing 10% v / v FBS and 1% w / w penicillin-streptomycin) under standard conditions, the insert was removed to create a scratch between the cells. The hydrogel was prepared using the same method as the MTT assay and incubated in medium. The gel was then placed onto the cell-seeded plate using the SPLInsert™ Hanging 24-well. The created scratches were captured with an optical microscope (EVOS M5000 Imaging System, Thermo Fisher Scientific, Waltham, MA, USA) at 0, 12, 24, 36, and 48 h (Fig. 18b), and in the group with UV treatment conditions, UV light (365 nm, 1-5 mW / cm2) was exposed for 5 min at 12 h. The wound area was measured using Image J software (version 1.40; National Institutes of Health, Bethesda, MD, USA), and the wound closure percentage was calculated using the following [Equation 2] and graphically depicted in Fig. 18c.

[0304]

[0305] [Formula 2]

[0306] That is, when a PC linker was introduced into EGF and conjugated within the hydrogel, the effect of the release profile change on NIH / 3T3 fibroblasts was evaluated. The biocompatibility and proliferation ability of the fabricated hydrogels were evaluated through the MTT assay (Fig. 18a). In the case of the EGF / HA Gel group, in which EGF was physically loaded, a high proliferation rate was shown until the 7th day due to the rapid release in the early stage, but in the case of the EGF-PC-HA Gel group, which was immobilized on the HA gel, an increase in the cell proliferation rate was confirmed after UV irradiation, and it was confirmed that the steady release of EGF by periodic light irradiation had a positive effect on cell proliferation until the 10th day. A similar pattern was confirmed in the results of the scratch assay (Fig. 7b,c). In the case of the EGF / HA Gel group, the highest wound closure rate was shown until 36 hours due to the rapid release in the early stage, but the bioactivity of EGF decreased thereafter, and there was relatively no significant increase thereafter. On the other hand, the EGF-PC-HA Gel group showed a low wound closure rate up to 24 hours, but in the EGF-PC-HA Gel group irradiated with UV after 12 hours, EGF separated from the matrix was released, and the closure rate increased the most up to 48 hours. Consequently, it was demonstrated that the release of EGF from hydrogels fabricated through UV irradiation can be controlled and can affect cell migration and proliferation.

[0307]

[0308] Figure 19 schematically illustrates a schematic diagram (a) of the wound healing ability evaluation of the hydrogel produced through a rat wound skin experiment according to one embodiment of the present invention, a representative image of each wound (b), and a result of quantifying the wound closure area (c).

[0309] Figure 19 shows the results of tissue staining and wound healing evaluation of rat skin for the fabricated hydrogel.

[0310] Specifically, 6-week-old balc / b mice (Orient Bio, Seongnam, Korea) were used as a model to evaluate wound healing ability. The mice were kept in a stable environment at 20°C and 40%–60% humidity for 1 week before being used in the experiment. Two wounds were created on the dorsal surface of the mice, one on each side of the midline, using a 5-mm biopsy punch. To simulate the wound healing process in the center without contraction, the wounds were secured using a donut-shaped Grace Bio-Lebs 14 mm x 7 mm wound splint. After securing, the ring was sutured with a surgical needle to prevent movement. Each hydrogel precursor solution was applied to the wounds at a volume of 50 μL and gelled by irradiating with blue light for 20 seconds. To prevent wound damage and gel detachment, a 3M™ Tegaderm™ Transparent Film Dressing was wrapped around the mice's back. Wound photographs were captured on Days 0, 3, 5, 7, and 10 after removing the Teaderm (Fig. 7b). On Days 3, 5, and 7, UV light (365 nm, 1-5 mW / cm2) was irradiated onto the dorsal surface of the mice where the gel was placed. The captured images were quantified as wound closure percent using Image J according to the wound splint size, and are shown in Fig. 19c.

[0311] The tissue staining method was as follows. Wound tissue was fixed with 4% paraformaldehyde, embedded in paraffin, and cut into 5-μm-thick slices. The slices were mounted on slides for H&E (Haematoxylin-Eosin) and Masson's trichrome staining according to the manufacturer's instructions.

[0312] The effect of EGF release by PC linker cleavage on skin wound healing was investigated through mouse skin regeneration. On day 0, a wound was made on the dorsal skin of the mice, and a gel was formed over the wound. On days 3, 5, and 7, UV was irradiated for 5 minutes each for the UV-treated group (Fig. 19a). On day 10, tissue samples were obtained from the wound area and stained with hematoxylin and eosin (H&E) and Masson's trichrome (MT) to observe the structure of the healed tissue, evaluate the degree of epidermal formation, and measure the thickness of the granulation tissue. As shown in the results of Fig. 19c, the EGF / HA Gel group showed the highest wound closure due to the initial release of large amounts of EGF due to diffusion, but there was little change on days 7 and 10.

[0313]

[0314] Figure 20 schematically illustrates the results of Hematoxylin & Eosin (a), Masson's trichrome (b) staining, and the results of granulation tissue thickness quantification (c) on the 10th day of a rat wound skin experiment according to one embodiment of the present invention.

[0315] As shown in the staining results in Fig. 20, the thickness of the recovered epidermal layer of the EGF / HA Gel group was the thickest (Fig. 20a), but the structure of the formed tissue and the distribution of collagen were incomplete (Fig. 20b). Therefore, although the initial release of a large amount of EGF appeared to accelerate wound healing in the early stages, it was far from normal healing, such as forming an uneven and bumpy wound surface. On the other hand, the EGF-PC-HA Gel (UV O) group prevented the initial rapid release and induced steady release through UV irradiation on days 3, 5, and 7. As a result, the wound closure rate on day 10 was 56.1 ± 7.3%, showing the best wound closure (Fig. 19c). The staining results observed the fastest recovery of the dermal layer, including the formation of blood vessels (Fig. 20a). Although the collagen fibers, which appeared in blue, did not reach that of the normal group, they were evenly and widely distributed with the highest density compared to the other groups (Fig. 20b). Figure 20c shows the results of quantifying the thickness of granulation tissue. Granulation tissue is an indicator of the active wound healing stage, and both the EGF / HA Gel group and the EGF-PC-HA Gel group showed the most active wound healing. In addition, in the Damaged (UV O) group, where only UV was irradiated on the wounded tissue, significantly increased granulation tissue thickness was observed along with a reduced distribution of inflammatory cells compared to the Damaged group. Although UV irradiation is generally considered a negative factor in wound healing, appropriate use of UVA with a wavelength of 365 nm can promote wound healing through sterilization or mild immune action. In this study, UV was used only for the purpose of inducing linker cleavage, but the possibility of using it together with light therapy was confirmed.

[0316]

[0317] That is, in the hyaluronic acid hydrogel according to one embodiment of the present invention, it is true that EGF has a wound healing effect, but depending on the amount and timing of application, the effect may have minimal impact on overall wound healing. Therefore, in this study, EGF was fixed in the HA hydrogel matrix to induce healing through continuous cell stimulation with sustained release, and the PC linker was used to control the release through continuous external stimulation, thereby inducing successful skin wound healing, thereby completing the present invention.

[0318]

[0319] According to one embodiment of the present invention, a photoresponsive hydrogel-based system for controlled release of EGF is designed to enable precise temporal control and exhibit an effect of enhancing therapeutic potential in wound healing.

[0320] According to one embodiment of the present invention, a growth factor that promotes wound healing can be cross-linked to a hyaluronic acid hydrogel that basically helps wound healing, thereby enhancing the wound healing ability.

[0321] According to one embodiment of the present invention, when crosslinking a growth factor to hyaluronic acid, an optical cutter can be used to control the amount of growth factor administered using light.

[0322] According to one embodiment of the present invention, a harmless effect on the human body can be achieved by using RFP, a substance suitable for the human body, as a derivative that induces gelation.

[0323]

[0324] Although the embodiments have been described with limited examples and drawings, those skilled in the art will appreciate that various modifications and variations can be made based on the above teachings. For example, appropriate results can be achieved even if the described techniques are performed in a different order than described, and / or components of the described systems, structures, devices, circuits, etc. are combined or combined in a different manner than described, or are replaced or substituted with other components or equivalents. Therefore, other implementations, other embodiments, and equivalents of the claims also fall within the scope of the claims described below.

Claims

1. As a hyaluronic acid hydrogel that continuously releases drugs and has a skin wound healing effect, A matrix material comprising hyaluronic acid modified with methacrylate and hyaluronic acid modified with thiol; and A hyaluronic acid hydrogel having a skin wound healing effect by continuously releasing a drug, comprising a protein modified with acrylate cross-linked to the above matrix material.

2. In claim 1, The above hyaluronic acid hydrogel, A hyaluronic acid hydrogel that contains a vitamin B2 derivative (RFP, Riboflavin phosphate) and is gel-formed when irradiated with blue light, and has a skin wound healing effect by continuously releasing drugs.

3. In claim 1, The above protein is, A hyaluronic acid hydrogel that contains a protein with growth factors and has a wound healing effect by continuously releasing drugs.

4. In claim 3, Proteins having the above growth factors are A hyaluronic acid hydrogel having a skin wound healing effect by continuously releasing a drug, comprising at least one of epidermal growth factor (EGF), fibroblast growth factor (FGF), keratinocyte growth factor (KGF), and nerve growth factor inducible (VGF).

5. In claim 1, The hyaluronic acid modified with the above methacrylic acid and the hyaluronic acid modified with the above thiol, A hyaluronic acid hydrogel having a volume ratio of 0.5 to 2 to 2 to 0.5, which continuously releases a drug and has a skin wound healing effect.

6. A method for manufacturing a hyaluronic acid hydrogel that continuously releases drugs and has a skin wound healing effect, The above hyaluronic acid hydrogel, A matrix material comprising hyaluronic acid modified with methacrylic acid and hyaluronic acid modified with thiol; and A protein modified with an acrylate cross-linked to the above matrix material; The method for manufacturing the above hyaluronic acid hydrogel is as follows: Step of preparing a protein solution; A step of preparing an NHS solution by dissolving acrylic acid NHS ester in dimethyl sulfoxide (DMSO); A method for producing a hyaluronic acid hydrogel having a skin wound healing effect by continuously releasing a drug, comprising the step of mixing the NHS solution and the protein solution to produce an A-protein solution modified with acrylate.

7. In claim 6, The method for manufacturing the above hyaluronic acid hydrogel is as follows: A step of preparing a first solution by mixing the hyaluronic acid modified with the above methacrylic acid and the hyaluronic acid modified with the above thiol; A step of preparing a second solution by dissolving the first solution in phosphate-buffered saline (PBS); A step of preparing a third solution by mixing the second solution and the A-protein solution; A step of preparing a fourth solution by adding a vitamin B2 derivative (RFP, Riboflavin phosphate) to the third solution; and A method for producing a hyaluronic acid hydrogel having a skin wound healing effect by continuously releasing a drug, comprising a step of exposing the fourth solution to blue light to form a gel.

8. In claim 7, The step of exposing the fourth solution to blue light to form a gel; A method for producing a hyaluronic acid hydrogel having a skin wound healing effect by continuously releasing a drug by exposing it to blue light for 10 to 30 seconds.

9. In claim 6, The above A-protein solution is, A method for producing a hyaluronic acid hydrogel having a skin wound healing effect by continuously releasing a drug, wherein the protein solution is mixed in a volume ratio of 90 to 110 and the NHS solution is mixed in a volume ratio of 0.1 to 3.

10. In claim 6, The step of preparing the above A-protein solution is: A step of incubating the above A-protein solution at 1 to 10˚C for 1 to 3 hours; A method for producing a hyaluronic acid hydrogel having a skin wound healing effect by continuously releasing a drug, comprising the step of dialyzing an incubated A-protein solution at 1 to 10˚C for 10 to 30 hours.

11. As a hyaluronic acid hydrogel that continuously releases wound healing substances, A matrix material comprising hyaluronic acid modified with methacrylic acid and hyaluronic acid modified with thiol; and A hyaluronic acid hydrogel that continuously releases a wound healing material, comprising a protein modified with acrylate PEG succinimidyl carboxymethyl ester cross-linked to the matrix material.

12. In claim 11, The above hyaluronic acid hydrogel, A hyaluronic acid hydrogel that continuously releases wound healing substances and further contains a vitamin B2 derivative (RFP, Riboflavin phosphate) and becomes gel-like when irradiated with blue light.

13. In claim 11, The above protein is, A hyaluronic acid hydrogel that continuously releases wound healing material containing proteins with growth factors.

14. In claim 13, Proteins having the above growth factors are A hyaluronic acid hydrogel that continuously releases a wound healing agent, comprising at least one of epidermal growth factor (EGF), fibroblast growth factor (FGF), keratinocyte growth factor (KGF), and nerve growth factor inducible (VGF).

15. In claim 11, The hyaluronic acid modified with the above methacrylic acid and the hyaluronic acid modified with the above thiol, A hyaluronic acid hydrogel that continuously releases a wound healing agent with a volume ratio of 0.5 to 2 to 2 to 0.

5.

16. A method for manufacturing a hyaluronic acid hydrogel that continuously releases a wound healing substance, The above hyaluronic acid hydrogel, A matrix material comprising hyaluronic acid modified with methacrylic acid and hyaluronic acid modified with thiol; and A protein modified with acrylate PEG succinimidyl carboxymethyl ester cross-linked to the above matrix material; The method for manufacturing the above hyaluronic acid hydrogel is as follows: Step of preparing a protein solution; A step of preparing an A-PEG solution by dissolving the above acrylate PEG succinimidyl carboxymethyl ester in dimethyl sulfoxide; A method for producing a hyaluronic acid hydrogel that continuously releases a wound healing substance, comprising the step of producing a PEG-A-protein solution by mixing the protein solution into the A-PEG solution.

17. In claim 16, The method for manufacturing the above hyaluronic acid hydrogel is as follows: A step of preparing a first solution by mixing the hyaluronic acid modified with the above methacrylic acid and the hyaluronic acid modified with the above thiol; A step of preparing a second solution by dissolving the first solution in phosphate buffered saline; A step of preparing a fifth solution by mixing the second solution and the PEG-A-protein solution; and A step of preparing a sixth solution by adding a vitamin B2 derivative to the fifth solution; A method for producing a hyaluronic acid hydrogel that continuously releases a wound healing substance, further comprising a step of exposing the sixth solution to blue light to cause gelation.

18. In claim 16, The protein modified with the above acrylate PEG succinimidyl carboxymethyl ester is, A method for producing a hyaluronic acid hydrogel that continuously releases a wound healing substance, wherein the protein solution is mixed in a volume ratio of 9 to 11 and the A-PEG solution is mixed in a volume ratio of 0.5 to 2.

19. In claim 16, The step of preparing the above PEG-A-protein solution is: A step of incubating the above PEG-A-protein solution at 1 to 10˚C for 1 to 3 hours; A method for producing a hyaluronic acid hydrogel that continuously releases a wound healing substance, comprising the step of dialyzing an incubated PEG-A-protein solution at 1 to 10˚C for 10 to 30 hours.

20. In claim 17, The step of exposing the above 6th solution to blue light to gel it is as follows: A method for producing a hyaluronic acid hydrogel having a skin wound healing effect by continuously releasing a drug by exposing it to blue light for 10 to 30 seconds.

21. A hyaluronic acid hydrogel from which a wound healing agent can be selectively released upon light irradiation. Hyaluronic acid hydrogel matrix; A photodetector coupled to the hyaluronic acid hydrogel matrix; and A hyaluronic acid hydrogel comprising a growth factor bound to the hyaluronic acid hydrogel matrix, wherein a wound healing material can be selectively released upon light irradiation.

22. In claim 21, The above hyaluronic acid hydrogel, A step of preparing a protein solution by dissolving the protein in phosphate-buffered saline (PBS); A step of preparing an Azido-PC solution by dissolving Azido-PC-NHS ester, which is a photocleavable linker, in dimethyl sulfoxide (DMSO); A step of preparing an Azido-PC-protein solution by mixing the above protein solution and Azido-PC solution; A step of preparing a thiol-modified hyaluronic acid solution by dissolving thiol-modified hyaluronic acid in phosphate buffered saline; A step of preparing a DBCO solution by dissolving dibenzocyclopentane-maleimide (DBCO-Maleimide) in dimethyl sulfoxide; A step of preparing a DBCO-HA solution by mixing the above thiol-modified hyaluronic acid solution and the DBCO solution; A step of preparing a protein-PC-HA solution by mixing the above Azido-PC-protein solution and DBCO-HA solution; A step of preparing a hydrogel precursor solution by mixing the above protein-PC-HA solution with a hyaluronic acid solution modified with methacrylic acid and a vitamin B2 derivative (RFP, Riboflavin phosphate); A hyaluronic acid hydrogel capable of selectively releasing a wound healing agent upon light irradiation, the hyaluronic acid hydrogel being manufactured through a step of gelling by irradiating the hydrogel precursor solution with blue light.

23. In claim 21, The above hyaluronic acid hydrogel matrix, Containing hyaluronic acid modified with methacrylic acid and hyaluronic acid modified with thiol, The hyaluronic acid modified with the above methacrylic acid and the hyaluronic acid modified with the above thiol, A hyaluronic acid hydrogel having a volume ratio of 0.5 to 2 to 2 to 0.5, wherein a wound healing agent can be selectively released upon light irradiation.

24. In claim 21, The above optical cutter, A hyaluronic acid hydrogel comprising Azido-PC-NHS ester, from which a wound healing agent can be selectively released upon light irradiation.

25. In claim 21, The above growth factors are, A hyaluronic acid hydrogel comprising at least one of epidermal growth factor (EGF), fibroblast growth factor (FGF), keratinocyte growth factor (KGF), and nerve growth factor inducible (VGF) that can selectively release a wound healing agent upon light irradiation.

26. In claim 22, The above protein is, A hyaluronic acid hydrogel having a concentration of 0.01 to 1 mg / ml relative to the above phosphate buffered saline solution, from which a wound healing agent can be selectively released upon light irradiation.

27. In claim 22, The above Azido-PC-NHS ester is, A hyaluronic acid hydrogel having a concentration of 20 to 40 mg / ml relative to the above dimethyl sulfoxide, from which a wound healing agent can be selectively released upon light irradiation.

28. In claim 22, The above Azido-PC-NHS ester is, A hyaluronic acid hydrogel, which is diluted to a concentration of 50 to 150 times and mixed with the protein solution, from which a wound healing agent can be selectively released upon light irradiation.

29. In claim 22, The step of preparing the above Azido-PC-protein solution is as follows: A hyaluronic acid hydrogel from which a wound healing agent can be selectively released upon light irradiation, further comprising a step of purifying the Azido-PC-protein solution at 1 to 7 °C for 20 to 30 hours.

30. In claim 22, The above dibenzocyclotine-maleimide is, A hyaluronic acid hydrogel having a concentration of 5 to 15 mg / ml relative to the above dimethyl sulfoxide, from which a wound healing agent can be selectively released upon light irradiation.

31. A method for producing a hyaluronic acid hydrogel from which a wound healing substance can be selectively released upon light irradiation, The above hyaluronic acid hydrogel, Hyaluronic acid hydrogel matrix; A photodetector coupled to the hyaluronic acid hydrogel matrix; and A growth factor bound to the hyaluronic acid hydrogel matrix; The above method, A step for preparing a protein solution by dissolving the protein in PBS; A step of preparing an Azido-PC solution by dissolving Azido-PC-NHS ester, which is a photocleavable linker, in dimethyl sulfoxide (DMSO); A step of preparing an Azido-PC-protein solution by mixing the above protein solution and Azido-PC solution; A step of preparing a thiol-modified hyaluronic acid solution by dissolving thiol-modified hyaluronic acid in phosphate-buffered saline (PBS); A step of preparing a DBCO solution by dissolving dibenzocyclopentane-maleimide (DBCO-Maleimide) in dimethyl sulfoxide; A step of preparing a DBCO-HA solution by mixing the above thiol-modified hyaluronic acid solution and the DBCO solution; A step of preparing a protein-PC-HA solution by mixing the above Azido-PC-protein solution and DBCO-HA solution; A step of preparing a hydrogel precursor solution by mixing the above protein-PC-HA solution with a hyaluronic acid solution modified with methacrylic acid and a vitamin B2 derivative (RFP, Riboflavin phosphate); A method for producing a hyaluronic acid hydrogel, wherein a wound healing agent can be selectively released upon light irradiation, comprising: a step of irradiating the hydrogel precursor solution with blue light to form a gel; 32. In claim 31, The above hyaluronic acid hydrogel matrix, Containing hyaluronic acid modified with methacrylic acid and hyaluronic acid modified with thiol, The hyaluronic acid modified with the above methacrylic acid and the hyaluronic acid modified with the above thiol, A method for producing a hyaluronic acid hydrogel having a volume ratio of 0.5 to 2 to 2 to 0.5, from which a wound healing agent can be selectively released upon light irradiation.

33. In claim 31, The above optical cutter, A method for producing a hyaluronic acid hydrogel comprising Azido-PC-NHS ester, from which a wound healing agent can be selectively released upon light irradiation.

34. In claim 31, The above growth factors are, A method for producing a hyaluronic acid hydrogel, wherein a wound healing agent can be selectively released upon light irradiation, the hyaluronic acid hydrogel comprising at least one of epidermal growth factor (EGF), fibroblast growth factor (FGF), keratinocyte growth factor (KGF), and nerve growth factor inducible (VGF).

35. In claim 31, The above protein is, A method for producing a hyaluronic acid hydrogel having a concentration of 0.01 to 1 mg / ml relative to the above phosphate buffered saline solution, from which a wound healing agent can be selectively released upon light irradiation.

36. In claim 31, The above Azido-PC-NHS ester is, A method for producing a hyaluronic acid hydrogel having a concentration of 20 to 40 mg / ml relative to the above dimethyl sulfoxide, from which a wound healing agent can be selectively released upon light irradiation.

37. In claim 31, The above Azido-PC-NHS ester is, A method for producing a hyaluronic acid hydrogel from which a wound healing agent can be selectively released upon light irradiation, the method comprising mixing the hyaluronic acid hydrogel with the protein solution by diluting it 50 to 150 times.

38. In claim 31, The step of preparing the above Azido-PC-protein solution is as follows: A method for producing a hyaluronic acid hydrogel from which a wound healing agent can be selectively released upon light irradiation, further comprising a step of purifying the Azido-PC-protein solution at 1 to 7 °C for 20 to 30 hours.

39. In claim 31, The above dibenzocyclotine-maleimide is, A method for producing a hyaluronic acid hydrogel having a concentration of 5 to 15 mg / ml relative to the above dimethyl sulfoxide, from which a wound healing agent can be selectively released upon light irradiation.

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