Nitric oxide-sensitive hydrogels

Abstract

The present invention relates to a nitric oxide-sensitive hydrogel, which is formed by in situ hybridization at a target site through a click chemistry reaction and is prepared using a crosslinker sensitive to nitric oxide, and thus can effectively capture and eliminate nitric oxide overexpressed at the target site. Furthermore, a drug carried in the hydrogel can be locally and selectively released to a target site requiring treatment, and thus can be useful for preventing or treating inflammatory diseases caused by overexpression of nitric oxide.

Description

[Technical field]

[0001] The present invention relates to a nitric oxide-sensitive hydrogel, which is formed by in situ hybridization at a target site through a click chemistry reaction and is prepared using a crosslinker sensitive to nitric oxide, and thus can effectively capture and eliminate nitric oxide overexpressed at the target site. Furthermore, a drug carried in the hydrogel can be locally and selectively released to a target site requiring treatment, and thus can be useful for preventing or treating inflammatory diseases caused by overexpression of nitric oxide. [Background technology]

[0002] Nitric oxide is a highly reactive radical molecule with a short half-life of within a few seconds. It is produced in various cells of the human body and is known to play various roles in the human body, including neurotransmission, vasodilation, and anti-cancer effects, depending on its concentration.

[0003] Since the latter half of the 2010s, there have been many attempts to locally and selectively capture nitric oxide that is overproduced at the disease site in order to alleviate or treat inflammatory diseases caused by hypersecretion of nitric oxide.

[0004] In these attempts, nanometer-sized technologies have been developed to facilitate injection, but they have problems due to rapid diffusion and removal from other organs other than the injection site. For example, since nitric oxide plays an important role in the body under normal conditions, non-specific removal of nitric oxide can cause numerous side effects such as systemic toxicity, and in the case of rheumatoid arthritis, a chronic inflammatory disease, cartilage injections are administered up to 3 to 4 times a year, so technologies that may cause rapid diffusion and removal from other organs other than the disease site are undesirable.

[0005] On the other hand, rheumatoid arthritis is a chronic inflammatory disease of unknown cause characterized by polyarthritis. In the early stages, inflammation occurs in the synovial membrane surrounding the joints, but the inflammation gradually spreads to the surrounding cartilage and bones, causing joint destruction and deformation. It is a disease that can affect not only the joints but also the entire body, with extra-articular symptoms such as anemia, sicca syndrome, subcutaneous nodules, pulmonary fibrosis, vasculitis, and skin ulcers.

[0006] The exact cause of rheumatoid arthritis has yet to be elucidated, but autoimmune phenomena are known to be the main mechanism. Autoimmunity is a phenomenon in which the immune system, which protects the body from the outside, attacks the body itself instead. In general, genetic predisposition and bacterial or viral infections are believed to be the causes of rheumatoid arthritis.

[0007] Rheumatoid arthritis is an intractable autoimmune disease that is accompanied by swelling, inflammation, stiffness and pain in the joints and presents a clinical picture of systemic polyarthritis, causing great pain to patients suffering from the disease.

[0008] Therefore, there is a need for new technologies that can capture nitric oxide locally at target sites for a long period of time, and further deliver therapeutic agents in response to nitric oxide depending on the degree of disease progression, thereby alleviating or treating diseases associated with overexpression of nitric oxide. Summary of the Invention [Problem to be solved by the invention]

[0009] The present invention provides a nitric oxide-sensitive hydrogel that can locally capture and eliminate nitric oxide overproduced in the body, and can further locally and selectively release a drug carried in the hydrogel to an affected site according to the concentration of nitric oxide, thereby enabling the prevention or treatment of inflammatory diseases. [Means for solving the problem]

[0010] This will be described in detail below. Any combination of the various elements disclosed in the present invention belongs to the scope of the present invention. In addition, the following detailed description does not necessarily limit the scope of the present invention.

[0011] The present invention provides an in situ hybridization hydrogel prepared from compounds represented by the following chemical formulas 1 to 3.

[0012] [ka] [Chemical formula 1]

[0013] [ka] [Chemical formula 2]

[0014] [ka] [Chemical formula 3]

[0015] Where: R1 and R2 each independently represent a C1-C10 alkylene; The above n and m are integers of 1 to 1,000.

[0016] In the present invention, "Cx-Cy" (wherein x and y are integers of 1 or more) refers to the number of carbon atoms. For example, C1-C10 alkylene refers to alkylene having 1 to 10 carbon atoms, and C1-C10 alkyl refers to alkyl having 1 to 10 carbon atoms.

[0017] As used herein, "alkyl" refers to a straight or branched chain saturated hydrocarbon group, including methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl, n-nonyl, n-decanyl, and the like.

[0018] In the present invention, "alkylene" refers to a divalent functional group derived from the alkyl defined above.

[0019] As used herein, "in situ" can be used interchangeably with "in situ" or "in situ," and means "at the site of administration" or "at the target site."

[0020] In the present invention, "in situ hybridization" means that after a precursor material capable of forming a hydrogel is injected into a patient's body, gelation occurs at a target site within a desired tissue, organ or cavity within the body due to binding of the precursor material.

[0021] By "patient" in the present invention is meant any individual or subject in need of treatment for a particular condition or disease, which may be a mammal, preferably a human.

[0022] The in situ hybridization hydrogel of the present invention does not require a surgical procedure for implantation, and instead forms a hydrogel in the body by simply mixing precursor materials using minimally invasive techniques, providing comfort to patients undergoing treatment. In addition, the hydrogel can be formed locally and selectively at a target site in the body, providing excellent therapeutic effects, particularly for sites that require treatment.

[0023] The compound represented by Chemical Formula 1 in the present invention may act as a crosslinker in the formation of a hydrogel.

[0024] [ka] [Chemical formula 1] (wherein R1 and R2 are each independently a C1-C10 alkylene).

[0025] The term "crosslinker" as used herein means a substance for bonding compounds together to form a network structure. According to an embodiment of the present invention, the compound represented by Chemical Formula 1 may be, but is not limited to, a compound represented by the following formula DA-NOCCL, in which R1 and R2 are C2 alkylene.

[0026] [ka] [DA-NOCCL]

[0027] In the present invention, the compound represented by Chemical Formula 1 can capture and eliminate overproduced nitric oxide, and therefore the hydrogel of the present invention prepared using the compound represented by Chemical Formula 1 can capture and eliminate overproduced nitric oxide in the body.

[0028] In addition, the compound represented by Chemical Formula 1 of the present invention may be decomposed in response to nitric oxide, and the hydrogel of the present invention prepared from the compound represented by Chemical Formula 1 may be decomposed in response to nitric oxide.

[0029] In the present invention, "sensitivity" means sensitively reacting to certain environmental conditions, and in this specification, "sensitivity" and "reactivity" can be used interchangeably.

[0030] In the experimental examples described below, it was confirmed that the hydrogel of the present invention can absorb nitric oxide and expand, and can capture and eliminate nitric oxide overproduced in the body, thereby effectively treating diseases caused by overproduction of nitric oxide. It was also confirmed that the hydrogel of the present invention can be decomposed in response to nitric oxide, and it was confirmed that the micelle structure bound in the network structure of the hydrogel can be selectively released to the site where a disease has occurred, and a drug carried in the micelle structure can be delivered locally, selectively, and intensively to the site where a disease has occurred. It was also confirmed that the hydrogel of the present invention can be decomposed to a different degree depending on the concentration of nitric oxide (NO), and therefore drug release can be adjusted according to the severity of the disease.

[0031] In the present invention, the compound represented by Chemical Formula 2 is also called PLA-b-PEG-N3 block copolymer, and can be bonded to the alkyne group of the compound represented by Chemical Formula 1 by a click chemistry reaction via the azide group.

[0032] [ka] [Chemical formula 2] (wherein n and m are integers of 1 to 1000).

[0033] The compound represented by Formula 2 may form a micelle structure by self-assembly.

[0034] In the present invention, the term "micelle" refers to a system consisting of an inner hydrophobic region and an outer hydrophilic region in an aqueous solution. In the present invention, a micelle structure was formed using a PLA-b-PEG-N3 block copolymer, which is a compound represented by Chemical Formula 2.

[0035] In the present invention, the micelle structure carries a hydrophobic drug and is specifically released at the diseased site, enabling a combined treatment for treating diseases caused by overproduced nitric oxide, i.e., treatment by scavenging nitric oxide and treatment by a drug.

[0036] According to an embodiment of the present invention, the micelle structure may have a uniform diameter of 10 to 1000 nm, preferably 70 to 900 nm, and more preferably 70 to 500 nm. If the diameter is less than the above range, it may not be possible to carry a sufficient amount of drug to treat the disease to be treated, and if the diameter exceeds the above range, it may not be suitable in terms of ease of injection.

[0037] In the present invention, the micelle structure may be fixed in the network structure of a hydrogel. The micelle structure may be released from the hydrogel when the hydrogel is decomposed in response to nitric oxide, and the drug carried therein may be selectively delivered to the site where nitric oxide is overproduced.

[0038] The hyaluronic acid-based polymer chain, which is the compound represented by Chemical Formula 3 in the present invention, is also called HA-N3 and serves to form the backbone of the hydrogel in the present invention.

[0039] [ka] [Chemical formula 3] (wherein n is an integer from 1 to 1000)

[0040] The "hyaluronic acid" in the present invention is a linear polysaccharide consisting of glucuronic acid and acetylglucosamine, and is one of the glycosaminoglycans present in the extracellular matrix (ECM), joint lubricant, and supports constituting cartilage. Crosslinked hyaluronic acid can be used as a joint lubricant due to its viscoelastic properties, and is a material with excellent biocompatibility that can be used in tissue engineering and drug delivery systems because it does not cause any immune problems when applied in vivo.

[0041] In the present invention, the compound represented by Chemical Formula 3 can be bound to the alkyne group of the compound represented by Chemical Formula 1 via the azide group by a click chemistry reaction.

[0042] In the present invention, the hydrogel may be prepared by a click chemistry reaction between the compound represented by Chemical Formula 1 and the compounds represented by Chemical Formulas 2 and 3.

[0043] In the present invention, "click chemistry" refers to a modular approach to organic synthesis that combines quickly, effectively, and predictably under specific conditions, as a customized reaction that occurs in the complex environment of living organisms. For example, the azide-alkyne cycloaddition reaction has a very high thermodynamic driving force and can efficiently form bonds between azide compounds and alkyne compounds in high yields, so that it can form intermolecular bonds with high yields even in reactions with macromolecules such as oligomers and polymers.

[0044] According to an embodiment of the present invention, the alkyne group of the compound represented by Chemical Formula 1 and the azide (-N3) group of the compound represented by Chemical Formula 2, and the alkyne group of the compound represented by Chemical Formula 1 and the azide (-N3) group of the compound represented by Chemical Formula 3, respectively, form 1,2,3-triazoles by click chemistry reaction, thereby forming a nitric oxide reactive in situ hybridization hydrogel comprising the micelle structure of the present invention.

[0045] In the present invention, the hydrogel can further enhance the effect of treating inflammation or disease caused by overexpressed nitric oxide by including a bioactive substance. Such a bioactive substance may be a substance used for the treatment, healing, prevention, or diagnosis of a disease, for example, a protein or peptide such as cells, growth factors, and hormones, a nucleic acid, an extracellular matrix substance, and a drug having a medical therapeutic function, but is not limited thereto. If it is a bioactive substance known to be effective for a specific disease, it can be applied to the hydrogel without limitation and used to enhance the therapeutic effect. In order to prepare a hydrogel to include a bioactive substance, a solution is prepared so that the bioactive substance is contained in one solution, and the hydrogel can be formed by mixing the other solution at the target site. Alternatively, the two solutions containing the bioactive substance can be mixed at the target site using a syringe to form the hydrogel.

[0046] In the present invention, the hydrogel may further include a hydrophobic drug, and the hydrophobic drug may be carried in a micellar structure formed by self-assembly of the compound represented by Chemical Formula 2. The micellar structure is formed in water by self-assembly of a block copolymer having hydrophilic and hydrophobic portions, and since the inside of the micellar structure exhibits strong hydrophobicity, the hydrophobic drug is easily carried in the inside of the micellar structure, which is a physical phenomenon caused by both the drug and the inside of the micellar structure exhibiting hydrophobicity, and the drug content can be increased compared to drug delivery vehicles using chemical bonds.

[0047] In the present invention, "hydrophobic" means to include non-polar or low polar substances that are not miscible with water, but is not limited thereto, and any substance that can exist stably in the hydrophobic interior of the micelle structure of the present invention is included in the hydrophobic substance.

[0048] In the present invention, the hydrophobic drug can be selected from analgesics, anti-inflammatory agents, immunosuppressants, or combinations thereof, and preferably can be, but is not limited to, anti-inflammatory agents.

[0049] In the present invention, the term "anti-inflammatory agent", also called an anti-inflammatory agent, refers to a drug that has the property of eliminating inflammation or that is involved in the inflammatory process and suppresses it.

[0050] Examples of the anti-inflammatory agent include 21-acetoxypregnenolone, alclomethasone, alclomethasone dipropionate, algestone, amcinonide, beclomethasone, betamethasone, betamethasone dipropionate, betamethasone sodium phosphate, betamethasone valerate, budesonide, chloroprednisone, ciclesonide, clobetasol, clobetasol 17-propionate, clobetasone-17-butyrate, clobetasone, clocortolone, cloprednol, corticosterone, cortisone, cortisone acetate, cortivazolate, and the like. le, deflazacort, desonide, desoximetasone, dexamethasone, dexamethasone sodium phosphate, diflorasone, diflucortolone, difluprednate, enoxolone, fluazacort, flucloronide, flumethasone, fluocinonide, fluocinolone, fluocinolone acetonide, fluocinonide, fluocortin butyl, fluocortolone, fluocortolone caproate, fluocortolone pivalate, fluorometholone, flunisolide, fluperolone acetate, fluprednidene acetate, fluprednisolone, flurane Drenolide, Fluticasone Propionate, Formocortal, Halcinonide, Halobetasol Propionate, Halometasone, Halopredone Acetate, Hydrocortamate, Hydrocortisone, Hydrocortisone Acetate, Hydrocortisone-17-Aceponate, Hydrocortisone-17-Buteprate, Hydrocortisone-17-Butyrate, Hydrocortisone-17-Valerate, Loteprednol, Mazipredone, Medrysone, Meprednisone, Methylprednisolone, Mometasone, Mometasone Furoate, Paramethasone, Paramethazone and, according to an embodiment of the present invention, the anti-inflammatory agent may be, but is not limited to, one or more selected from the group consisting of tixocortol, tixocortol pivalate, triamcinolone, triamcinolone acetonide, triamcinolone alcohol, triamcinolone benetonide, and triamcinolone hexacetonide.

[0051] Furthermore, according to the embodiment of the present invention, the hydrophobic drug may be contained in an amount of 3 to 7% by weight based on the total weight of the micelle structure, but is not limited thereto.

[0052] The hydrogels of the present invention may further comprise a hydrophobic drug and a hydrophilic drug.

[0053] In the present invention, the hydrophobic drug and the hydrophilic drug can be simultaneously released from the target site, where the release of the hydrophobic drug can be by erosion-based release and the release of the hydrophilic drug can be by Fickian diffusion.

[0054] In the experimental examples described below, it was confirmed that the hydrogel of the present invention can carry a hydrophobic drug inside the micelle, which is the hydrophobic domain of the hydrogel, and a hydrophilic drug in the hydrophilic domain of the hydrogel, and the two drugs can be simultaneously released from the target site that requires treatment. In addition, the hydrogel of the present invention can vary the degree of drug release depending on the concentration of nitric oxide, which means that the release of the drug can be adjusted depending on the degree of deterioration of the disease, indicating that customized treatment according to the state of the disease to be treated is possible.

[0055] In the present invention, the compounds of Formulae 1 to 3 for preparing a hydrogel may be co-injected into a target site.

[0056] In the present invention, "co-injection" means that the compounds of Formulae 1-3 are injected together in a formulation suitable for injection, for example, in a liquid state, into a target site in a patient's body. According to an embodiment of the present invention, a liquid composition containing the compounds of Formulae 1-3 is mixed uniformly before exiting the tip of an injection needle and entering a target site in a patient's body, and then injected as a mixture into the target site, exiting the injection needle and gelling in the body. After being co-injected into a target site, the compounds of Formulae 1-3 of the present invention can spontaneously combine through a click chemistry reaction to form a hydrogel.

[0057] In the present invention, the co-injection can be achieved using a dual syringe system or any other suitable syringe system, preferably by a dual syringe system. According to an embodiment of the present invention, the compounds represented by Formulae 1 to 3 may be physically separated before the co-injection, e.g., before the series of steps of simultaneous ejection by injection, mixing, and injection through a needle occurs in the patient's body. According to an embodiment of the present invention, a composition including the compounds represented by Formulae 1 and 3 may be physically separated from a composition including the compound represented by Formula 2. According to an embodiment of the present invention, a composition including the compounds represented by Formulae 1 and 2 may be physically separated from a compound represented by Formula 3. According to an embodiment of the present invention, a composition including the compound represented by Formula 1 may be physically separated from the compounds represented by Formulae 2 and 3.

[0058] The hydrogel of the present invention may be formed within 0.5 to 10 minutes, preferably 0.5 to 1 minute, after the compound of Formulae 1 to 3 is injected into the target site. If the hydrogel formation time (gelation time) is less than the above range, the compound of Formulae 1 to 3 may be mixed and then crosslinked early, which may prevent smooth injection into the target site. If the time exceeds the above range, the injected substance may diffuse into the surrounding tissues, which may not be suitable for local hydrogel formation at the target site. Existing in-situ injection-type hydrogels developed by conventional temperature or UV (infrared) irradiation have a problem of slow phase transition speed. The hydrogel of the present invention can be gelled in a short time by click chemistry reaction, thus solving the problems of the conventional technology.

[0059] The hydrogels of the present invention can be used for the prevention, alleviation or treatment of inflammatory diseases.

[0060] In the present invention, the "inflammatory disease" refers to a disease whose main lesion is inflammation, and the inflammatory disease in the present invention may be an inflammatory disease exhibiting excessive production of nitric oxide in the body.

[0061] The hydrogel of the present invention can capture and eliminate overproduced nitric oxide. When the immune system is disrupted due to stress or the like, and nitric oxide is overproduced in the body, it can cause autoimmune diseases or inflammatory diseases. Therefore, the hydrogel of the present invention, which can capture and eliminate overproduced nitric oxide, can effectively treat diseases caused by the overproduction of nitric oxide.

[0062] In addition, the inflammatory disease may be a disease caused by inflammation of the joints, such as, but not limited to, undifferentiated spondyloarthropathy, undifferentiated arthropathy, arthritis, chronic inflammatory diseases caused by viral or bacterial infection, rheumatoid arthritis, reactive arthritis, osteoarthritis, osteoporosis, etc. According to an embodiment of the present invention, the inflammatory disease may be, but is not limited to, rheumatoid arthritis.

[0063] Rheumatoid arthritis is an autoimmune disease caused by high concentrations of nitric oxide. Therefore, the hydrogel of the present invention can sequester nitric oxide in a high concentration nitric oxide environment, inhibit bone resorption by osteoclasts induced by cytokines, and prevent cell death in the joints, thereby alleviating or treating rheumatoid arthritis.

[0064] By "alleviation or treatment" in the present invention is meant a decrease in the severity of disease symptoms, an increase in the frequency or duration of disease symptom-free periods, or prevention of damage or disability due to the affliction of the disease.

[0065] The hydrogels of the present invention may reduce levels of the pro-inflammatory cytokines TNF-α and IL-6.

[0066] In the present invention, the term "pro-inflammatory cytokine" refers to a cytokine that is mainly produced by activated macrophages and promotes inflammation throughout the body. Examples of pro-inflammatory cytokines include TNF-α (tumor necrosis factor-α), IL-1, IL-2, IL-6, and IL-8, which appear together during an inflammatory reaction and can be used as an indicator of the inflammatory reaction.

[0067] In the experimental examples described below, it was confirmed that the hydrogel of the present invention can reduce the levels of inflammatory cytokines TNF-α and IL-6, and thus the hydrogel of the present invention can be useful for treating inflammatory diseases.

[0068] The hydrogels of the present invention can be used for visco-supplements.

[0069] In the present invention, the term "visco-supplement" refers to a substance in which hyaluronic acid in a gel state is injected into a joint site to replenish the viscous lubricating fluid.

[0070] In the experimental examples described below, it was confirmed that the hydrogel of the present invention can be useful as a viscosupplement, since it shows almost complete recovery after rupture in response to stress-induced flow. Without being bound by any particular theory, this is attributed to the intermolecular hydrogen bonds between HA (hyaluronic acid) chains and the entropy-driven reassociation of the micellar structure.

[0071] According to another aspect of the present invention, there is provided an in situ hybridization hydrogel precursor composition comprising compounds represented by the following chemical formulas 1 to 3:

[0072] [ka] [Chemical formula 1]

[0073] [ka] [Chemical formula 2]

[0074] [ka] [Chemical formula 3]

[0075] Where: R1 and R2 each independently represent a C1-C10 alkylene; The above n and m are integers of 1 to 1,000.

[0076] By "precursor" in the present invention is meant a compound that takes part in a chemical reaction to produce another compound.

[0077] The precursor composition of the present invention may further contain a hydrophobic drug, and the hydrophobic drug may be supported in a micelle structure formed by self-assembly of the compound represented by Chemical Formula 2.

[0078] The precursor composition of the present invention may further comprise a hydrophobic drug and a hydrophilic drug.

[0079] According to another aspect of the present invention, there is provided a method for preparing an in situ hybridization hydrogel, comprising the steps of:

[0080] a) preparing a first precursor composition comprising a compound represented by the following Chemical Formula 1 and a compound represented by the following Chemical Formula 3: b) preparing a second precursor composition comprising a micellar structure formed from a compound represented by the following formula 2: c) co-injecting the first and second precursor compositions.

[0081] [ka] [Chemical formula 1]

[0082] [ka] [Chemical formula 2]

[0083] [ka] [Chemical formula 3]

[0084] Where: R1 and R2 each independently represent a C1-C10 alkylene; The above n and m are integers of 1 to 1,000.

[0085] According to an embodiment of the present invention, the compound represented by Chemical Formula 1 may be included in an amount of 0.02 to 0.06 wt % based on the total mass of the first precursor composition; the compound represented by Chemical Formula 2 may be included in an amount of 1.00 to 3.00 wt % based on the total mass of the second precursor composition; and the compound represented by Chemical Formula 3 may be included in an amount of 1.00 to 1.50 wt % based on the total mass of the first precursor composition, but is not limited thereto.

[0086] In the preparation method of the present invention, the second precursor composition may further include a catalyst, and according to an embodiment of the present invention, the catalyst may be, but is not limited to, Cu(I).

[0087] In the manufacturing method of the present invention, the second precursor composition may further contain a hydrophobic drug, and the hydrophobic drug may be supported in a micelle structure formed by self-assembly of the compound represented by Chemical Formula 2.

[0088] In the preparation method of the present invention, the first and second precursor compositions may further include a hydrophobic drug and a hydrophilic drug. According to an embodiment of the present invention, the hydrophobic drug may be included in the second precursor composition, the hydrophilic drug may be included in the first precursor composition, or both the hydrophobic drug and the hydrophilic drug may be included in the first precursor composition or the second precursor composition, but is not limited thereto.

[0089] In the manufacturing method of the present invention, the co-injection may be by a dual syringe system.

[0090] In the manufacturing method of the present invention, the first and second precursor compositions may be in the form of a suspension, solution or emulsion suitable for parenteral administration, may be manufactured in a solid or semi-solid form, and may contain formulating agents such as suspending agents, stabilizing agents, solubilizing agents and / or dispersing agents.

[0091] According to an embodiment of the present invention, the first and second precursor compositions may be in a liquid state and may be sterilized, but are not limited thereto, and may be preserved against the contaminating effects of microorganisms, such as bacteria and fungi. The first and second precursor compositions may also be in a powder form and may be sterilized. In this case, they may be formulated into a liquid form using water for injection or the like before use.

[0092] The present invention provides a method for preventing, alleviating or treating an inflammatory disease comprising administering to an individual having the inflammatory disease a hydrogel of the present invention.

[0093] In the present invention, "administration" means introducing the hydrogel of the present invention into a patient by any suitable method, and the administration route of the hydrogel of the present invention may be any common route as long as it can reach the desired tissue, but may be preferably parenteral administration. The parenteral administration may be, but is not limited to, intramuscular, subcutaneous, intraarticular, intraperitoneal administration, etc. According to an embodiment of the present invention, the administration may be, but is not limited to, intraarticular administration.

[0094] An individual in the present invention includes any human or non-human animal. The term "non-human animal" may be a vertebrate animal, such as a non-human primate, sheep, dog, and rodent, such as a mouse, rat, and guinea pig. The individual may be preferably a human, and in particular a human with a particular disease. The term "individual" in this specification may be used interchangeably with "subject" or "patient."

[0095] The matters mentioned in the in situ hybridization hydrogel, precursor composition, preparation method and treatment method of the present invention are equally applicable unless they contradict each other, and repeated description is omitted to avoid excessive complexity of the specification. Effect of the Invention

[0096] The hydrogel of the present invention can locally and selectively capture and eliminate excessively produced nitric oxide both inside and outside the body, and therefore has excellent preventive, alleviating or therapeutic effects on inflammatory diseases caused by excessive production of nitric oxide.

[0097] In addition, the hydrogel of the present invention can carry hydrophobic and / or hydrophilic drugs and act as a drug delivery vehicle to a target site, and can have a combined therapeutic effect by scavenging nitric oxide and delivering drugs.

[0098] Furthermore, since the hydrogel of the present invention can be decomposed in response to nitric oxide, the crosslinking points dissociate in response to nitric oxide in an environment in which nitric oxide is present, allowing the drug carried in the hydrogel to be delivered locally and selectively to the target site.

[0099] Furthermore, the hydrogel of the present invention can be formed locally at a target site in a minimally invasive manner without the need for surgical procedures, and is therefore highly convenient for patients.

[0100] In addition, the hydrogel of the present invention can be rapidly removed from the target site and prevented from spreading to other organs, and does not suffer from problems associated with the prior art, such as systemic toxicity.

[0101] In addition, the hydrogel of the present invention has excellent recovery of mechanical properties due to its self-healing ability, and can be effectively used as a visco-supplement. [Brief description of the drawings]

[0102] [Figure 1] 1 is a schematic diagram illustrating a process of forming a hydrogel of the present invention and a process in which the formed hydrogel reacts with nitric oxide to dissociate crosslinks. [Diagram 2] 1 shows a synthesis process for the compound represented by chemical formula 1. [Diagram 3]1H NMR data of the compounds obtained in each step of the synthesis of the compound represented by chemical formula 1. [Figure 4] FIG. 4a shows the mechanism by which the compound represented by Chemical Formula 1 reacts with nitric oxide and is decomposed. FIG. 4b shows 1H NMR data confirming whether the compound represented by Chemical Formula 1 reacts with nitric oxide and is decomposed. [Diagram 5] 1 shows FT-IR (a) and UV-Vis (ultraviolet-visible) absorption data (b) confirming whether the compound represented by Chemical Formula 1 reacts with nitric oxide and is decomposed. [Figure 6] 1 shows a synthesis process for the compound represented by chemical formula 2. [Figure 7] 1 shows 1H NMR spectrum data of the compounds obtained in each step of the synthesis of the compound represented by chemical formula 2. [Figure 8] FT-IR, GPC, TEM, and DLS data for the compound represented by formula 2. FIG. 8a shows FT-IR spectrum data for PLA-b-PEG-N3. FIG. 8b shows Gel Permeation Chromatography (GPC) data for PLA-b-PEG-N3. FIG. 8c shows a transmission electron microscope (TEM) image for PLA-b-PEG-N3. FIG. 8d shows the average hydrodynamic size determined by dynamic light scattering (DLS) for PLA-b-PEG-N3. [Figure 9] 1 shows a synthesis process for the compound represented by chemical formula 3. [Figure 10] 1H NMR data of a compound obtained in the synthesis process of a compound represented by chemical formula 3 is shown. [Figure 11]The properties of the M-NO gel of the present invention were analyzed. Figure 11a shows the cryogenic scanning electron microscopy image of the M-NO gel. Figure 11b shows the oscillatory rheological properties of the self-healing M-NO gel by dynamic transient test. [Figure 12] The properties of the M-NO gel of the present invention were analyzed. Figure 12a shows a schematic diagram of the self-healing of the M-NO gel. Figure 12b shows the frequency-dependent oscillatory rheological properties of the M-NO gel. Figure 12c shows the NO concentration-dependent oscillatory rheological properties of the M-NO gel and the control gel (non-NO reactive). [Figure 13] The properties of the M-NO gel of the present invention were analyzed. Figure 13a confirms the NO scavenging ability of the M-NO gel. Data are expressed as mean ± SD (n = 4). Figure 13b shows the relative swelling ratio of the M-NO gel over time at various concentrations. Data are expressed as mean ± SD (n = 4). Figure 13c shows the relative swelling ratio of the control gel (NO non-responsive) over time at various concentrations. Data are expressed as mean ± SD (n = 3). Figure 13d shows images of the M-NO gel and the control gel (NO non-responsive) after incubation in NO solutions of various concentrations for 2 hours. The scale bar is 1 cm. [Figure 14] The properties of the M-NO gel of the present invention were analyzed. Figure 14a shows a schematic diagram of simultaneous dual drug release in the M-NO gel. Figure 14b shows the BSA-FITC (hydrophilic cargo model) release profile of the M-NO gel. Figure 14c shows the BSA-FITC release profile of the control gel. Figure 14d shows the Nile Red (hydrophobic cargo model) release profile loaded on the micelle structure of the M-NO gel. Figure 14e shows the Nile Red release profile of the control gel. [Figure 15] The properties of the M-NO gel of the present invention were analyzed. Figure 15a shows the average hydrodynamic size of the PLA-b-PEG-N3 micellar structures before hydrogel formation and in the supernatant after incubating the M-NO gel with NO solution (250 μM) for 24 hours. Figure 15b shows the TEM image of the supernatant after erosion of the M-NO gel. The white box in the image indicates the morphology of the micellar structures before gel formation. [Figure 16] The reactivity and NO scavenging ability of M-NO gel after LPS treatment in LPS-treated RAW264.7 cells are shown in Fig. 16a. The NO concentration in RAW264.7 and NIH / 3T3 cell lines before and after 24 h of treatment with 5 μg / ml LPS is shown in Fig. 16b. The cell viability of RAW264.7 cell line treated with various concentrations of M-NO gel and control gel is shown in Fig. 16b. The data are shown as mean ± SD (n = 5). Fig. 16c shows the NO scavenging ability of M-NO gel. The NO level in the cell culture supernatant after 24 h of culture was confirmed by Griess assay after various concentrations of M-NO gel were treated in RAW264.7 cell line. Fig. 16d shows the confocal microscopy images of LPS-activated RAW264.7 cell line treated with M-NO gel and control gel. Intracellular NO and nuclei were stained with DAF-2DA (green) and DAPI (blue), respectively, and the scale bar is 100 μm. [Figure 17]Figure 17 shows the reactivity and NO scavenging ability of M-NO gel after LPS treatment in LPS-treated RAW264.7 cells. Figure 17a shows the quantification of TNF-α levels after treatment with M-NO gel and control gel. Figure 17b shows the quantification of IL-6 levels after treatment with M-NO gel and control gel. Figure 17c shows images of M-NO gel and control gel after overnight incubation on cultured RAW264.7 or NIH / 3T3 cell lines (stimulated or not stimulated with LPS) treated with M-NO gel and control gel. Figure 17d shows the relative swelling ratio after overnight incubation on cultured RAW264.7 or NIH / 3T3 cell lines (stimulated or not stimulated with LPS) treated with M-NO gel and control gel. Figure 17e shows a schematic diagram of in vitro NO-reactivity and scavenging ability of M-NO gel to reduce the levels of inflammatory cytokines. [Figure 18] FIG. 18 shows the therapeutic effect of the M-NO gel of the present invention on rheumatoid arthritis in collagen-induced arthritis (CIA) mice, an animal model of rheumatoid arthritis. FIG. 18a shows the timeline of the entire experiment. FIG. 18b shows the relative paw volume change in the RA mouse model after sample treatment. The relative paw volume was statistically analyzed by one-way analysis of variance (ONE-WAY ANOVA) by comparing the RA model mice treated with saline (B) with the other experimental groups (**p<0.01, ***p<0.001). [Figure 19] 1 shows the therapeutic effect of the M-NO gel of the present invention on rheumatoid arthritis in collagen-induced arthritis (CIA) mice, an animal model of rheumatoid arthritis. Representative images of mouse hind paws at 4, 6 and 8 weeks are shown. [Figure 20]20 shows the therapeutic effect of the M-NO gel of the present invention on rheumatoid arthritis in collagen-induced arthritis (CIA) mice, an animal model of rheumatoid arthritis. Representative images of mouse hind paws at 4, 6, and 8 weeks are shown. FIG. 20a shows the time-dependent mean clinical score of RA mice after sample treatment. Data are expressed as mean ± SD (n=8). FIG. 20b shows the mean clinical score of RA mice after sample treatment at 8 weeks. RA model mice treated with saline (B) were compared with other experimental groups and statistically analyzed using one-way analysis of variance (ONE-WAY ANOVA) (**p<0.01, ***p<0.001). FIG. 20c shows the fall latency of RA mice after sample treatment over time using rotarod analysis. Data are expressed as mean ± SEM (n=8). FIG. 20d shows the fall latency of RA mice after sample treatment at 8 weeks. The RA model mice treated with saline (B) were compared with the other experimental groups and statistically analyzed using one-way analysis of variance (**p<0.01, ***p<0.001). [Figure 21] FIG. 21 shows the therapeutic effect of the M-NO gel of the present invention on rheumatoid arthritis in collagen-induced arthritis (CIA) mice, an animal model of rheumatoid arthritis. FIG. 21 shows the therapeutic effect of the M-NO gel of the present invention on rheumatoid arthritis in collagen-induced arthritis (CIA) mice, an animal model of rheumatoid arthritis. FIG. 21a shows a micro-computed tomography (micro-CT) image of the front paw of a RA mouse model. The red arrows indicate the destruction and damage of the bone. FIG. 21b shows the histological analysis of the joint tissue after sample treatment. The scale bar is 100 μm. [Figure 22]The therapeutic effect of the M-NO gel of the present invention on rheumatoid arthritis was confirmed in collagen-induced arthritis (CIA) mice, an animal model of rheumatoid arthritis. Figure 22a shows the average histological scores of joint tissues determined using H&E staining, Masson's trichrome staining, and Safranin-O staining. The saline-treated group (B) of RA model mice was compared with the other experimental groups and statistically analyzed using one-way ANOVA (*p<0.05, ***p<0.001; ns no significant difference). Figure 22b shows the quantification of TNF-α levels in serum specimens of sample-treated RA mice. Figure 22c shows IL-6 levels in serum specimens of sample-treated RA mice. Data are presented as mean ± SD (n=4), # compared with other groups and statistically analyzed using one-way analysis of variance (ONE-WAY ANOVA) (*p<0.05, ***p<0.001; ns no significant difference). Figure 22d shows the quantification of TNF-α levels in paw tissue fluid of sample-treated RA mice. Figure 22e shows the quantification of IL-6 levels in paw tissue fluid of sample-treated RA mice. Data are presented as mean ± SD (n=4), # compared with other experimental groups and statistically analyzed using one-way analysis of variance (ONE-WAY ANOVA) (*p<0.05, ***p<0.001; ns no significant difference). Figure 22f shows the quantification of NO levels in paw tissue fluid of sample-treated RA mice at week 8. Data are expressed as mean ± SD (n = 4). The saline-treated RA model mice (B) were compared with the other experimental groups and statistically analyzed using one-way analysis of variance (*p < 0.05, ***p < 0.001; ns not significant). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0103] The present invention will be described in more detail below with reference to examples. However, these examples are merely illustrative of the present invention, and the scope of the present invention is not limited by these examples. In addition, terms not specifically defined in the present specification should be understood to have the meanings commonly used in the technical field to which the present invention belongs.

[0104] Preparation Example 1. Synthesis of compound of formula 1 To synthesize the clickable crosslinker DA-NOCCL that reacts with nitric oxide, the amine group at the C2 position of 4-nitro-o-phenylenediamine was first selectively Boc-protected using guanidine HCl as a catalyst. Then, 4-NO2 was reduced to 4-NH2, and 4-pentynoic acid was bonded to each amine group via an amide bond. Then, the synthesis was completed by Boc-deprotection under acidic conditions. The synthesis process of the crosslinker DA-NOCCL is shown in Figure 2, and the successful synthesis of each compound obtained in the synthesis process and the crosslinker DA-NOCCL are shown. 1 This was confirmed by 1 H NMR and is shown in Figure 3.

[0105] The specific steps of the synthesis are as follows:

[0106] Step 1: 4-nitro-o-phenylenediamine (1 g, 6.53 mmol) and guanidine HCl (95 mg, 0.91 mmol) were dissolved in 20 ml of ethanol, di-tert-butyl dicarbonate (2.85 g, 13.06 mmol) was added dropwise, and the reaction mixture was vigorously stirred at 25° C. for 24 hours. After the reaction was completed, the organic solvent in the reaction solution was evaporated under reduced pressure and suspended in distilled water. The aqueous solution was extracted three times with ethyl acetate (EA), and the organic portion was washed with Na2SO4 to obtain a red-yellow crude solid, which was purified by silica gel column chromatography (eluent: EA-hexane mixture) to obtain compound 1 (1.22 g, yield: 73.8%).

[0107] [ka] [Compound 1]

[0108] 1 H NMR (500MHz, DMSO-d6): δ=8.56 (brs, 1H, D2O exchangeable), 8.30 (d, J=1.6Hz, 1H), 7.78 (d d, J=9.0, 2.7Hz, 1H), 6.72(d, J=9.0Hz, 1H), 6.46(brs, 2H, D2O exchangeable), 1.48(s, 9H)

[0109] Step 2: To a solution of compound 1 (400 mg, 1.58 mmol) in 10 ml of anhydrous THF under nitrogen, 100 mg of palladium on activated carbon (10% Pd / C) was added, then hydrogen gas was pumped in until the pressure reached up to 40 psi, and the reaction mixture was stirred at 25° C. for 36 h. After the reaction was completed, the reaction mixture was filtered through a Celite 545 bed to remove Pd / C, and evaporated under reduced pressure to give a purple solid compound 2 (322 mg, yield: 91.2%).

[0110] [ka] [Compound 2]

[0111] 1 H NMR (500MHz, DMSO-d6): δ=8.08 (brs, 1H, D2O exchangeable), 6.64 (d, J=1.6Hz, 1H), 6.45 (d, J=8.3Hz, 1H), 6.16 (dd, J=8.3, 2.5Hz, 1H), 4.29 (brs, 2H, D2O exchangeable), 4.01 (brs, 2H, D2O exchangeable), 1.45 (s, 9H)

[0112] Step 3: Compound 2 (200 mg, 0.90 mmol), 4-pentynoic acid (200 mg, 2.04 mmol), HOBt (280 mg, 2.07 mmol) and triethylamine (TEA, 190 mg, 1.88 mmol) were completely dissolved in 10 ml of anhydrous DMF. After EDC (400 mg, 2.09 mmol) was added to the solution, the reaction mixture was vigorously stirred at 25 °C for 24 h. After the reaction was completed, the reaction mixture was evaporated under reduced pressure and suspended in CHCl. ​​The organic solvent was extracted with distilled water three times, and the organic portion was washed with NaSO to obtain a light brown crude-solid, which was purified by silica gel column chromatography (eluent: EA-hexane mixture) to obtain compound 3 (285 mg, 0.74 mmol, yield: 82.7%).

[0113] [ka] [Compound 3]

[0114] 1 H NMR (500MHz, DMSO-d6): δ=9.99 (brs, 1H, D2O exchangeable), 9.41 (brs, 1H, D2O exchangeable), 8.26 (brs, 1H, D2O exchangeable) ngeable)), 7.87(d, J=2.2Hz, 1H), 7.39(dd, J=8.7, 2.2Hz, 1H), 7.27(d, J=8.7Hz, 1H), 2.79(t, J=2.5Hz, 2H), 2.55-2.41(m, 8H), 1.47(s, 9H)

[0115] Step 4: 5 ml of trifluoroacetic acid (TFA) was added dropwise to compound 3 (200 mg, 0.52 mmol) suspended in 20 ml of CHCl at 0° C. Then the reaction mixture was stirred vigorously at 25° C. for 12 h. After the reaction was completed, the reaction mixture was evaporated under reduced pressure and neutralized with saturated sodium bicarbonate (NaHCO3) solution. The neutralized solution was extracted three times with 5% (v / v) methanol in CHCl2, and the organic portion was washed with NaSO4 to obtain the dark brown solid crosslinker DA-NOCCL (138 mg, 0.49 mmol, yield: 93.5%).

[0116] [ka] [DA-NOCCL]

[0117] 1 H NMR (500MHz, DMSO-d6): δ=9.70 (brs, 1H, D2O exchangeable), 9.06 (brs, 1H, D2O exchangeable), 7.08 (d, J=2.2Hz, 1H), 7.01 (d, J=8.5Hz, 1H), 6.70 (dd, J=8.5, 2.2Hz, 1H), 4.88 (brs, 2H, D2O exchangeable), 2.78 (dt, J=14.7, 2.4, 2H), 2.44 (m, 8H).

[0118] The mechanism by which the synthesized crosslinker DA-NOCCL reacts with nitric oxide and is decomposed was confirmed. 1 The 1 H NMR, FT-IR and UV-Vis (ultraviolet-visible) absorption data are shown in Figures 4 and 5.

[0119] As can be seen in Figures 4 and 5, after incubation in a saturated NO solution, the characteristic peaks of primary amines disappeared and benzotriazole groups were formed, confirming that the crosslinker DA-NOCCL reacted with NO and decomposed.

[0120] Preparation Example 2. Synthesis of compound of formula 2 To synthesize the crosslinker represented by Chemical Formula 1 and the clickable compound represented by Chemical Formula 2, PEG (polyethylene glycol) was used as the starting material, and one of the hydroxyl groups of PEG was selectively tosylated under silver oxide (Ag2O) catalyzed conditions, and the tosylate was then replaced with bromide. The PEG-Br formed was then subjected to ring-bond polymerization with D,L-lactide to form a block copolymer of PLA-b-PEG-Br. Finally, the bromide group was replaced with azide to complete the synthesis. 10 mg of the synthesized PLA-b-PEG-N3 was dissolved in 1 ml of THF, and 9 ml of water was added dropwise to form a micelle structure, and the remaining THF was removed by dialysis. The specific steps of the synthesis are as follows, and the synthesis process is shown in Figure 6. The successful synthesis of PLA-b-PEG-N3 was 1 The results were confirmed by 1 H NMR, FT-IR, and GPC, and are shown in Figures 7, 8a, and 8b, respectively.

[0121] The specific steps of the synthesis are as follows:

[0122] Step 1: Silver(I) oxide (1.74 g, 7.51 mmol) and potassium iodide (KI, 0.33 g, 1.99 mmol) were added to a solution of vacuum-dried 2 kDa PEG (10 g, 5.00 mmol) in 160 ml of anhydrous dichloromethane (CHCl), followed by p-toluenesulfonyl chloride (TsCl, 1 g, 5.25 mmol) in 20 ml of anhydrous CHCl and vigorously stirred at 0° C. overnight. After the reaction was completed, the reaction mixture was carefully filtered through a Celite 545 bed and evaporated under reduced pressure to obtain a colorless oily product. The obtained product was further purified by multiple CHCl / diethyl ether ((CH)O) recrystallization to obtain a white solid precipitate (monoTos-PEG, 9.72 g, yield: 90.9%).

[0123] Step 2: monoTos-PEG (4.0 g, 1.86 mmol) and potassium bromide (KBr, 1.1 g, 9.24 mmol) were both dissolved in 40 ml of anhydrous dimethylformamide (DMF) and reacted at 65° C. for 18 h under stirring. After the reaction was completed, the reaction mixture was evaporated under reduced pressure and redispersed in CH2Cl2. The product was passed through a short silica gel and further purified by multiple CH2Cl2 / (C2H5)2O recrystallization to obtain a white solid precipitate (monoBr-PEG, 3.59 g, yield: 93.5%).

[0124] Step 3: monoBr-PEG (1.5 g, 0.72 mmol) and 3,6-dimethyl-1,4-dioxane-2,5-dione (D,L-lactide, 2.1 g, 14.57 mmol) were dissolved in 5 ml of anhydrous toluene. After the solution was subjected to several freeze-thaw cycles, tin(II) 2-ethylhexanoate catalyst (Sn(Oct)2, 0.01 g, 0.02 mmol) was added to initiate the polymerization. The reaction mixture was vigorously stirred at 120°C for 36 h. After the reaction was completed, the mixture was evaporated under reduced pressure and redispersed in CHCl2. The product was then purified by multiple CHCl2 / (CH5)2O recrystallizations to obtain a white amorphous solid precipitate. The product was further purified (PLA-b-PEG-Br) by dialysis against distilled water for 2 days and subsequent lyophilization. The molecular weight was measured by gel permeation chromatography (GPC) in tetrahydrofuran (THF) (Mn=5540, PDI=1.50).

[0125] Step 4: The prepared PLA-b-PEG-Br (1.1 g, 0.19 mmol) and excess sodium azide (NaN3, 0.45 g, 6.92 mmol) were dissolved in 20 ml of anhydrous DMF, and the reaction mixture was vigorously stirred at 50° C. for 3 days. After the reaction was completed, the product was purified (PLA-b-PEG-N3) by dialysis against distilled water for 2 days and subsequent lyophilization. The molecular weight was measured by GPC in THF (Mn=5864, PDI=1.55).

[0126] In addition, the TEM and DLS data of the micellar structure formed by self-assembly of the prepared PLA-b-PEG-N3 are shown in Figures 8c and 8d, respectively. As can be seen in Figures 8c and 8d, it was confirmed that PLA-b-PEG-N3 can self-assemble under aqueous conditions to form a uniform micellar structure with a size of approximately 284.1 ± 7.8 nm.

[0127] Preparation Example 3. Synthesis of compound of formula 3 The hyaluronic acid-based polymer chain (HA-N3), a compound represented by Chemical Formula 3 that can be clicked with the compound represented by Chemical Formula 1 to form a hydrogel backbone, was synthesized by substituting the chloride group of 2-chloroethylamine with an azide group, and then binding the substituted 2-azidoethylamine to hyaluronic acid through an amide bond. The synthesis process of the hyaluronic acid-based polymer chain is shown in Figure 9, and the compounds obtained at each step of the synthesis are shown in Table 1. 1 The 1 H NMR spectrum is shown in FIG.

[0128] The specific steps of the synthesis are as follows:

[0129] Step 1: 2-Chloroethylamine hydrochloride (1.16 g, 100.00 mmol) and NaN3 (1.3 g, 20.00 mmol) were both dissolved in 30 ml of distilled water, and the reaction mixture was vigorously stirred at 80 °C for 12 h. After the reaction was completed, 1 M sodium hydroxide (NaOH) solution was added to adjust the pH of the solution to 10-11. The alkaline solution was extracted with CHCl3 times, and the organic portion was washed with anhydrous sodium sulfate (NaSO4) to obtain a pale yellow oily product (2-azidoethylamine, 0.62 g, yield: 72.0%). 1 H NMR (500MHz, D2O): δ=3.41 (t, J=5.7Hz, 1H), 2.80 (t, J=5.7Hz, 1H).

[0130] Step 2: 100 kDa hyaluronic acid (HA, 200 mg, 0.50 mmol (repeating unit)) was completely dissolved in 40 ml distilled water, and N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC, 144 mg, 0.75 mmol), 1-hydroxybenzotriazole (HOBt, 101 mg, 0.75 mmol) and 2-azidoethylamine (51.7 mg, 0.60 mmol) were added to the solution. After adjusting the pH to 6.8, the reaction mixture was vigorously stirred at 25°C for 1 day under dark conditions. After the reaction was completed, the product was purified by successive dialysis against 0.1 M sodium chloride (NaCl) and distilled water, and lyophilized to obtain HA-N3.

[0131] Preparation Example 4. Preparation of hydrogel The nitric oxide-sensitive hydrogel of the present invention (hereinafter, M-NO gel) was produced by the following method.

[0132] Specifically, the M-NO gel was prepared using a dual syringe system. One syringe of the dual syringe system contained the compounds represented by formula 1 and formula 3, and the other syringe contained the compound represented by formula 2 and a Cu(I) catalyst. The syringes containing formula 1 and formula 3 were prepared in 1% (v / v) DMSO, with respective concentrations of 0.04 wt% and 2.5 wt%. The syringes containing the compound represented by formula 2 and the Cu(I) catalyst were prepared in distilled water, with respective concentrations of 2 wt% and 0.02 wt%. Here, the Cu(I) catalyst was 15.5 mg of CuSO 4· The solution was prepared by mixing 5H2O and 22.0 mg of sodium ascorbate with 0.2 mL of distilled water.

[0133] In addition, the nitric oxide non-reactive hydrogel used as a control was prepared by replacing DA-NOCCL with DA-NONCL (dialkyne-functionalized non-cleavable cross-linker) in the preparation method of the M-NO gel.

[0134] Experimental Example 1. Morphological analysis of M-NO gel For morphological analysis of the M-NO gel of the present invention, the M-NO gel prepared with a dual syringe system was imaged by cryogenic scanning electron microscopy, which is shown in Figure 11a.

[0135] As can be seen in FIG. 11a, it was confirmed that the micelle structures were uniformly distributed and fixed within the hydrogel network.

[0136] Experimental Example 2. Mechanical property analysis of M-NO gel To confirm the use of the M-NO gel of the present invention as an artificial visco-supplement for joints, the self-healing properties of the M-NO gel after rupture were analyzed by a dynamic transient test.

[0137] Specifically, step-strain (ε = 0.5-200%, ωrad / s, 25°C) over multiple cycles was measured to confirm the presence or absence of self-healing of mechanical properties after rupture of the network structure at high strain, which is an important parameter for artificial viscous supplements, and the results are shown in Figures 11b and 12a.

[0138] As can be seen from the above results, the M-NO gel showed almost complete recovery in response to stress-induced flow, confirming that the M-NO gel of the present invention can be useful as a viscosupplement.

[0139] Experimental Example 3. Analysis of NO concentration-dependent changes in mechanical properties of M-NO gel To confirm the change in mechanical properties of M-NO gels depending on the NO concentration, the frequency-dependent rheological parameters were analyzed.

[0140] Specifically, the frequency-dependent oscillatory rheological properties (ε%, 25°C) and concentration-dependent rheological properties (ωrad / s to ωrad / s, ε%, 25°C) of the NO-nonreactive gel and the NO-reactive M-NO gel of the present invention were quantified and compared by measuring the angular frequency, and the results are shown in Figures 12b and 12c.

[0141] As shown in Figures 12b and 12c, the storage modulus of the NO-reactive M-NO gel gradually decreased with the concentration of NO, indicating a decrease in mechanical strength upon NO-mediated decomposition of the crosslinker, whereas the control NO-non-reactive gel showed no significant change in storage modulus even in high-concentration NO solutions. In other words, it was confirmed that the M-NO gel of the present invention exhibited a decomposition pattern with increasing NO concentration.

[0142] Experimental Example 4. Confirmation of NO scavenging ability of M-NO gel The NO scavenging ability of the M-NO gel of the present invention was determined by preparing a 25μM Py-NO solution using a synthetic NO donor Py-NO (N-diazeniumdiolates-incorporated pyrrolidine) capable of releasing NO under aqueous conditions, treating the M-NO gel and the NO non-reactive gel with the prepared 25μM Py-NO solution, incubating for 1 hour, and quantifying the residual NO level in the supernatant by Griess analysis (the absorbance at 548nm is compared with a standard curve plotted with NaNO2 to determine the NO concentration). The swelling level of the hydrogel was also measured and confirmed, and the relative swelling ratio was calculated according to the following formula, and the results are shown in Figures 13a, 13b, 13c, and 13d.

[0143] <Calculation formula> -Expansion ratio = (Mf-Mi) / Mi (Mf: weight of hydrogel swollen in NO solution, Mi: weight of dry hydrogel) -Relative expansion ratio = expansion ratio / expansion ratio with water at 0h

[0144] As can be seen in Figures 13a, 13b, 13c, and 13d, the M-NO gel of the present invention significantly reduced the amount of NO solution depending on the NO concentration, and was significantly expanded compared to the control, non-NO reactive gel.

[0145] In other words, the M-NO gel of the present invention effectively scavenges nitric oxide in a concentration-dependent manner and has expansion properties due to an increase in pore size caused by the cleavage of the hydrogel network structure in response to the NO concentration, making on-demand controlled delivery possible according to the NO concentration.

[0146] Experimental Example 5. Confirmation of simultaneous dual drug release from M-NO gel The NO-responsive simultaneous dual-stage release of the M-NO gel was confirmed by the following experiment.

[0147] Specifically, a hydrophilic drug model, BSA-FITC (fluorescein isothiocyanate-labeled bovine serum albumin), was dissolved in a solution containing HA-N3 to a final concentration of 1 wt% and encapsulated in the hydrophilic domain of the hydrogel, and a hydrophobic drug model, Nile Red, was encapsulated in the hydrophobic domain of PLA-b-PEG-N3 micelles. The hydrophilic and hydrophobic drug models were co-encapsulated in the M-NO gel during hydrogel formation.

[0148] Then, 200 μL of M-NO gel was incubated in 5 mL of NO solution (0, 2.5, 25, and 250 μM) at 25° C. The supernatant was replaced with NO solution at the designated time intervals, and the resulting supernatant was concentrated by lyophilization, and the fluorescence (excitation / emission peaks at 485 / 510 nm) and absorbance at 585 nm were compared with standard curves to investigate the time-dependent simultaneous dual drug release profiles of BSA-FITC and Nile Red in response to NO.

[0149] The schematic diagram of dual drug release is shown in Figure 14a and the results are shown in Figures 14b, 14c, 14d and 14e.

[0150] As can be seen in Figures 14b, 14c, 14d, and 14e, the M-NO gel of the present invention was able to rapidly release hydrophilic molecules with Fickian diffusion release characteristics and all hydrophobic molecules with erosion-based mechanism release characteristics at high NO concentrations (Figures 14b and 14d), while the control NO-non-reactive gel did not show any special release characteristics despite the increase in NO concentration (Figures 14c and 14e).

[0151] In addition, whether or not hydrophobic drugs can be released from the micellar structure of the M-NO gel of the present invention based on erosion was confirmed by comparing the average hydrodynamic size of the PLA-b-PEG-N3 micellar structure before hydrogel formation with the average hydrodynamic size of the PLA-b-PEG-N3 micellar structure observed in the supernatant after incubating the M-NO gel with an NO solution (250 μM) for 24 hours, and the results are shown in Figures 15a and 15b.

[0152] As shown in Figures 15a and 15b, micellar structures with hydrodynamic size and morphology similar to those of the PLA-b-PEG-N3 micellar structures before hydrogel formation were observed in the supernatant of the M-NO gel incubated with NO solution, confirming that hydrophobic drugs can be released by an erosion-based mechanism.

[0153] Experimental Example 6. Confirmation of cytotoxicity of M-NO The dose-dependent cytotoxicity of M-NO of the present invention and the control group was confirmed by MTT assay.

[0154] Specifically, RAW264.7 cells were cultured in a 96-well plate at 1 × 10 4The cells were seeded at an initial density of cells / well and cultured at 37° C. in DMEM medium containing 10% FBS, 100 U / mL penicillin, and 100 μg / mL streptomycin.

[0155] After 24 hours of incubation, the medium was replaced with fresh medium containing the sample and incubated again for 40 hours. The cells were then washed and incubated in medium containing 0.5 mg / mL MTT solution for 4 hours. Finally, the medium was replaced with 200 μL DMSO and the cell viability was examined by measuring the absorbance at 570 nm. The results are shown in Figure 16b.

[0156] As can be seen in FIG. 16b, the M-NO gel of the present invention had almost no effect on cell viability, confirming the safety of the M-NO of the present invention.

[0157] Experimental Example 7. Confirmation of NO scavenging ability of M-NO The NO-scavenging ability of the compositions of the present invention and associated cytokine levels were confirmed using the LPS-stimulated NO-releasing RAW264.7 macrophage cell line.

[0158] (1) Confirmation of NO scavenging ability depending on concentration 5 x 10 RAW264.7 cell line 4 The cells were seeded into 12-well plates at a density of 100 cells / well and cultured at 37° C. in DMEM medium containing 10% FBS, 100 U / mL penicillin, and 100 μg / mL streptomycin.

[0159] After overnight incubation, the medium was replaced with fresh medium containing the sample and 5 μg / mL LPS, and the cells were again incubated for 24 hours.

[0160] Finally, the medium was centrifuged (3,000 rpm, 10 min, 4° C.) to obtain the supernatant, which was then transferred to a 96-well plate. The remaining NO levels in the supernatant were determined by Griess assay, which is shown in FIG. 16c.

[0161] As can be seen in FIG. 16c, the M-NO gel of the present invention was able to significantly reduce the NO level in LPS-stimulated RAW264.7 cell line by 25%, which was similar to the value observed in LPS-untreated RAW264.7.

[0162] (2) Confirmation of NO scavenging ability using confocal microscopy Intracellular NO levels were imaged by confocal microscopy to assess NO scavenging ability.

[0163] Specifically, 1 × 10 cells 5 The cells were seeded onto cover glasses arranged in 6-well plates at a density of 10 cells / well and cultured at 37°C in DMEM medium containing 10% FBS, 100 U / mL penicillin, and 100 μg / mL streptomycin.

[0164] After overnight incubation, the medium was replaced with fresh medium containing the samples (25 μg / mL for crosslinker and 250 μg / mL for gel) and 5 μg / mL LPS, and the cells were again incubated for 24 h.

[0165] Thereafter, the sample medium was replaced with fresh medium containing 5 μ m DAF-2DA and incubated again for 40 min, after which the cells were washed with DPBS and incubated with fresh medium for another 20 min.

[0166] Then, the tissue was washed with DPBS, treated with 10% neutral formalin (NBF) under dark conditions at room temperature, and observed by confocal microscopy after 30 minutes. The results are shown in FIG. 16d.

[0167] As can be seen in FIG. 16d, high fluorescence intensity was observed in the RAW264.7 cell line stimulated with LPS, but the signal was significantly reduced after treatment with M-NO gel, confirming that the M-NO of the present invention can efficiently scavenge NO.

[0168] (3) Quantification of cytokine levels NO is an important pro-inflammatory mediator, and its overproduction increases the levels of representative cytokines such as TNF-α and IL-6 in vitro. Therefore, we examined whether the M-NO gel of the present invention can reduce the levels of cytokines TNF-α and IL-6.

[0169] Specifically, LPS-stimulated RAW264.7 cells were treated with M-NO gel or control gel (250 μg / mL) and cultured for 24 hours. The inflammatory cytokines TNF-α and IL-6 in the cell culture supernatant were then quantified by ELISA, and the results are shown in Figures 17a and 17b.

[0170] As can be seen in Figures 17a and 17b, the levels of NO-induced inflammatory cytokines TNF-α and IL-6 were significantly reduced after treatment with M-NO gel, confirming that the M-NO gel of the present invention can effectively scavenge overproduced NO, thereby reducing inflammation and treating various inflammatory diseases.

[0171] (4) Expansion of M-NO gel by NO capture Cultured RAW264.7 or NIH / 3T3 cells, either stimulated or not stimulated with LPS, were treated with M-NO gel or control gel and then cultured overnight to determine whether the M-NO gel expanded due to NO capture, and the results are shown in Figures 17c and 17d.

[0172] As can be seen in Figures 17c and 17d, the M-NO gel was found to be able to significantly swell in response to NO in the LPS-stimulated RAW264.7 cell line, confirming that the M-NO gel of the present invention has favorable conditions for on-demand release.

[0173] Experimental Example 6. Confirmation of therapeutic effect for rheumatoid arthritis (1) The therapeutic effect of the M-NO gel of the present invention on rheumatoid arthritis (RA) was confirmed in collagen-induced arthritis (CIA) mice, an animal model of RA (FIG. 18a).

[0174] (1) Preparation of collagen-induced arthritis mouse model Primary immunization was performed by subcutaneously injecting 100 μL of an emulsified solution (1:1, v / v) of a mixture of collagen type II (CII, 2 mg / mL) and complete Freund's adjuvant (CFA, 1 mg / mL) dissolved in 10 mM acetic acid into the tail of 6-week-old DBA / 1j mice.

[0175] Two weeks later, 100 μL of an emulsified solution (1:1, v / v) of CII (2 mg / mL) and IFA (incomplete Freud's adjuvant, 1 mg / mL) mixed in 10 mM acetic acid was subcutaneously injected into the tail of the mice. Then, 4 weeks after the primary immunization, the RA model mice were randomly distributed into eight groups, and 20 μL of each sample was injected intra-articularly into the CIA mice. In situ hybridization Hydrogels were injected using a dual-syringe system (one syringe contained HA-N3 (2.5 wt%) and crosslinker (0.04 wt%), and the other syringe contained micelles (2 wt%) and Cu(I) (0.02 wt%)). The dexamethasone-loaded M-NO gel was diluted two-fold to match the hydrogel system at 1 wt % (loading: 5.02%, dose: 0.5 mg / kg).

[0176] The experimental groups are classified as follows: (A) Non-immunized healthy mouse (B) RA model mice treated with saline (C) Group of RA model mice treated with dexamethasone-loaded micelles (D) RA model mice treated with DA-NOCCL (E) RA model mice treated with M-NO gel (F) RA model mice treated with control gel (non-NO reactive). (G) RA model mice treated with dexamethasone-loaded M-NO gel (H) RA model mice treated with dexamethasone-loaded control gel (non-NO reactive).

[0177] (2) Confirmation of changes in paw volume and evaluation of clinical scores for arthritis The changes in paw volume over time in the RA mouse model were statistically analyzed in each RA mouse model experimental group relative to the saline-treated group (B), and the results are shown in Figure 18b. Representative images of mouse hind paws at weeks 4, 6, and 8 are shown in Figure 19.

[0178] As can be seen in FIG. 18b and FIG. 19, the groups treated with the M-NO gel of the present invention and the M-NO gel loaded with dexamethasone showed a significant decrease in paw volume.

[0179] In addition, the clinical scores of arthritis over time were evaluated by blind testing according to a standard scoring method (scale 0 to 5) while monitoring the clinical scores for 4 to 8 weeks, and the results are shown in Figures 20a and 20b. The clinical scores and evaluation criteria for arthritis evaluation are as follows.

[0180] 0: No clinical signs 1: Minimal diffuse erythema / edema (affecting 1 or 2 toes) 2: Mild diffuse erythema / edema (affecting 3 or more toes) 3: Moderate diffuse erythema / edema (entire leg) 4: Marked diffuse erythema / edema (entire foot and ankle) 5: Severe diffuse erythema / erythromatosis (entire foot), inability to bend toes (joint adhesions)

[0181] As can be seen in Figures 20a and 20b, the groups treated with the M-NO gel of the present invention and the M-NO gel loaded with dexamethasone showed a significant decrease in clinical scores.

[0182] From the above results, it was confirmed that the M-NO gel of the present invention and the M-NO gel loaded with dexamethasone can not only delay the progression of the disease but also alleviate the symptoms of arthritis itself. In particular, the M-NO gel loaded with dexamethasone showed almost complete alleviation of the symptoms of arthritis.

[0183] (3) Motility evaluation To assess locomotor activity in the RA mouse model, a rotarod assay was performed.

[0184] Specifically, before the primary immunization, mice were trained to stay on the rotarod for 3 days to reach a stable latency to fall. Then, the latency to fall was recorded at a fixed speed of 10 rpm for 120 s, and the results are shown in Figures 20c and 20d.

[0185] As can be seen in Figures 20c and 20d, the fall latency increased in the groups treated with the M-NO gel of the present invention and the M-NO gel loaded with dexamethasone, and in particular, the group treated with the M-NO gel loaded with dexamethasone showed a significant increase in fall latency, confirming a gradual improvement in the mobility of the mice.

[0186] Experimental Example 7. Confirmation of the therapeutic effect of rheumatoid arthritis (2) To investigate the therapeutic effect of the M-NO gel of the present invention on rheumatoid arthritis intensively, a series of additional experiments were carried out at week 8.

[0187] (1) Confirmation of bone and joint morphology Bone and joint morphology was confirmed by microcomputed tomography (micro-CT) imaging of representative forepaws from each experimental group treated with samples, and the results are shown in Figure 21a.

[0188] As can be seen in FIG. 21a, compared to other experimental groups, the groups treated with the M-NO gel of the present invention and the M-NO gel loaded with dexamethasone showed less bone erosion and clearer bone boundaries could be confirmed.

[0189] This indicates that the hydrogel of the present invention can prevent oxidative / nitrative stress and upregulation of osteoclasts by scavenging nitric oxide overproduced in RA lesions.

[0190] (2)Histological analysis Histological assays of the joint tissues of each experimental group treated with samples were performed by staining the joint tissues of the mice sacrificed at week 8 with hematoxylin and eosin (H&E), Masson's trichrome, and Safranin-O. The results of the analysis and the degree of cartilage damage evaluated according to a standard scoring method (scale 0-4) by blind test are shown in Figures 21b and 22a (B: bone, C: cartilage). The clinical scores and evaluation criteria are as follows:

[0191] 0: No damage, normal activity 1: Minimal erosion limited to a single area 2: Mild / moderate erosion in limited areas 3: Severe / widespread erosion 4: Mostly damaged

[0192] As can be seen from FIG. 21b and FIG. 22a, it was confirmed that the M-NO gel of the present invention and the M-NO gel loaded with dexamethasone were able to restore damaged cartilage and joint cavity.

[0193] (3) Confirmation of TNF-α and IL-6 levels and NO concentration Levels of the inflammatory cytokines TNF-α and IL-6 in serum and paw tissue fluid were quantified by ELISA according to the manufacturer's protocol at week 8. NO levels were determined by Griess assay and the results are shown in Figures 22b-22e.

[0194] 22b to 22e, the levels of inflammatory cytokines in both serum and paw tissue fluid were significantly reduced in the groups treated with the M-NO gel of the present invention and the M-NO gel loaded with dexamethasone, and in particular, the group treated with the M-NO gel loaded with dexamethasone was significantly reduced to the normal state.

[0195] In addition, the data of quantification of NO levels in the paw tissues of the RA mice treated with the samples at week 8 are shown in Fig. 22f. As shown in Fig. 22f, it was confirmed that the NO concentration in the paw tissue fluid was significantly reduced in the groups treated with the M-NO gel of the present invention and the M-NO gel loaded with dexamethasone.

Claims

1. An in situ hybridization hydrogel prepared from compounds represented by the following formulas 1 to 3: 【Chemistry 1】 [Chemical formula 1] 【Chemistry 2】 [Chemical formula 2] 【Chemistry 3】 [Chemical formula 3] Where: The R 1 and R 2 are each independently a C1-C10 alkylene; The above n and m are each an integer of 1 to 1,000.

2. The R 1 and R 2 The in situ hybridization hydrogel of claim 1 , wherein is a C2 alkylene.

3. The compound represented by Chemical Formula 2 forms a micelle structure by self-assembly, and the micelle structure has a uniform diameter of 10 to 1000 nm. The in situ hybridization hydrogel according to claim 1.

4. The hydrogel according to claim 1, wherein the hydrogel is prepared by a click chemistry reaction between the compound represented by Chemical Formula 1 and the compounds represented by Chemical Formulas 2 and 3.

5. The in situ hybridization hydrogel of claim 1, wherein the hydrogel further comprises a hydrophobic drug, a hydrophilic drug, or a combination thereof.

6. The in situ hybridization hydrogel according to claim 5, wherein the hydrophobic drug is supported in a micelle structure formed by self-assembly of the compound represented by Chemical Formula 2.

7. The in situ hybridization hydrogel of claim 5, wherein the hydrophobic drug is an anti-inflammatory agent.

8. The in situ hybridization hydrogel according to claim 5, wherein the hydrophobic drug is contained in an amount of 3 to 7% by weight based on the total weight of the micelle structure.

9. 6. The in situ hybridization hydrogel of claim 5, wherein the hydrophobic drug and the hydrophilic drug are released simultaneously from the target site.

10. The in situ hybridization hydrogel of claim 1, wherein the hydrogel is for treating an inflammatory disease.

11. The in situ hybridization hydrogel according to claim 10, wherein the inflammatory disease is a disease exhibiting excessive production of nitric oxide in the body.

12. 2. The in situ hybridization hydrogel of claim 1, wherein the hydrogel reduces the levels of TNF-α and IL-6.

13. The in situ hybridization hydrogel of claim 1, wherein the hydrogel is for use as a viscosupplement.

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