Nitric oxide-sensitive hydrogels

Abstract

The present application relates to a nitric oxide-sensitive hydrogel, which is formed by in situ hybridization at a target site by means of a click chemistry reaction, and can effectively capture and remove overexpressed nitric oxide at the target site because it is manufactured using a nitric oxide-sensitive crosslinking agent, and further, a drug loaded into the hydrogel can be locally and selectively released at a target site requiring treatment, and thus can be effectively applied to prevent or treat inflammatory diseases caused by overexpression of nitric oxide.

Description

Technical Field

[0001] This invention relates to a nitric oxide-sensitive hydrogel, which is formed at a target site via in situ hybridization through a click chemistry reaction. Because it is manufactured using a nitric oxide-sensitive crosslinking agent, it can effectively capture and remove overexpressed nitric oxide at the target site. Furthermore, the drug loaded into the hydrogel can be released locally and selectively at the target site requiring treatment. Therefore, it can be effectively applied to the prevention or treatment of inflammatory diseases caused by nitric oxide overexpression. Background Technology

[0002] Nitric oxide is a highly reactive free radical molecule with a half-life of only a few seconds. It is generated from various cells in the human body and can play a variety of roles depending on its concentration in the body, such as nerve transmission, vasodilation, and anti-cancer effects.

[0003] Since the late 2010s, efforts have been made to actively explore localized and selective nitric oxide capture in order to alleviate or treat diseases caused by excessive nitric oxide secretion.

[0004] Along with these attempts, nanoscale-based technologies that are easy to inject have been developed. However, these technologies have the problem of rapidly diffusing and clearing nitric oxide from the injection site to other organs. For example, because nitric oxide plays an important role in the body within normal ranges, non-specific clearance of nitric oxide may induce various side effects such as systemic toxicity. In the case of rheumatoid arthritis, a chronic inflammatory disease, where chondroitin injections are administered a maximum of 3-4 times per year, technologies that rapidly diffuse and clear nitric oxide from the disease site to other organs are not desirable.

[0005] Furthermore, rheumatoid arthritis is a chronic inflammatory disease of unknown cause characterized by polyarthritis. It initially occurs in the synovium surrounding the joints, but the inflammation gradually spreads to the surrounding cartilage and bone, leading to joint destruction and deformity. This is a potentially systemic disease, not limited to joint symptoms, but also potentially inducing extra-articular symptoms such as anemia, Sjögren's syndrome, subcutaneous nodular pulmonary fibrosis, vasculitis, and skin ulcers.

[0006] While the exact cause of rheumatoid arthritis is not yet fully understood, its primary mechanism is known to be autoimmune activity. Autoimmunity is a phenomenon where the body's immune system, which protects it from external threats, malfunctions and attacks the body itself. Generally, the causes of rheumatoid arthritis are understood to include genetic factors and bacterial or viral infections.

[0007] As described above, rheumatoid arthritis is accompanied by swelling, inflammation, stiffness, and pain in the joints. It is a refractory autoimmune disease that presents with symptoms of polyarthritis throughout the body, causing great suffering to patients.

[0008] Therefore, there is a need to develop a novel technology that can locally and permanently capture nitric oxide at a target site, thereby enabling the simultaneous delivery of therapeutic agents based on the severity of the disease through nitric oxide sensitivity, and thus treating patients or diseases associated with nitric oxide overexpression. Summary of the Invention

[0009] A nitric oxide-sensitive hydrogel is provided that can locally capture and remove excessive nitric oxide produced in the body, and then release drugs loaded in the hydrogel locally and selectively at the disease site according to the concentration of nitric oxide, thereby being used for the prevention or treatment of inflammatory diseases.

[0010] The following will provide a detailed description. All combinations of the various elements disclosed in this invention fall within the scope of this invention. Furthermore, the scope of this invention should not be considered limited by the specific description below.

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

[0012]

Chemical Formula 1

[0013]

[0014] [Chemical Formula 2]

[0015]

[0016]

Chemical Formula 3

[0017]

[0018] in,

[0019] R1 and R2 are each independently a C1-C10 alkylene group.

[0020] n and m are integers from 1 to 1000.

[0021] In this invention, "Cx-Cy" (where x and y are integers of 1 or more) refers to the number of carbon atoms. For example, C1-C10 alkylene refers to alkylene with a carbon number of 1 to 10, while C1-C10 alkyl refers to alkyl with a carbon number of 1 to 10.

[0022] In this invention, "alkyl" refers to a straight-chain or branched 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, and n-decyl, etc.

[0023] In this invention, "alkylene" refers to a divalent functional group derived from an alkyl group as defined above.

[0024] In this invention, "in situ" can be used interchangeably with "in place" or "in location," referring to "drug administration site" or "target site."

[0025] In this invention, "in situ hybridization" refers to the phenomenon where, after a precursor substance capable of forming a hydrogel is injected into the patient's body, gelation occurs at the target site in the desired tissue, organ, or body cavity due to the binding of the precursor substance.

[0026] In this invention, "patient" refers to any individual or object that needs treatment for a specific condition or disease, which may be a mammal, and preferably a human.

[0027] The in situ hybridization hydrogel of the present invention does not require surgical transplantation. Instead, it can be formed in vivo through a simple mixing of precursor substances using minimally invasive techniques. This can create comfort for the patient receiving treatment and can form hydrogels locally and selectively at the target site in vivo, thus achieving excellent therapeutic effects, especially at the site where treatment is needed.

[0028] In this invention, the compound represented by chemical formula 1 can act as a crosslinking agent when forming a hydrogel.

[0029]

Chemical Formula 1

[0030]

[0031] (R1 and R2 are each independently a C1-C10 alkylene group)

[0032] In this invention, "crosslinking agent" refers to a substance that forms a network structure by binding compounds together. In embodiments of this invention, the structure represented by chemical formula 1 may be a compound represented by DA-NOCCL where R1 and R2 are C2 alkylene groups, but is not limited thereto.

[0033] [DA-NOCCL]

[0034]

[0035] In this invention, the compound represented by Chemical Formula 1 can capture and remove excessive nitric oxide. Therefore, the hydrogel of this invention, made using the compound represented by Chemical Formula 1, can capture and remove excessive nitric oxide in the body.

[0036] Furthermore, the compound of the present invention represented by Chemical Formula 1 can be decomposed by means of nitric oxide sensitivity, and therefore the hydrogel of the present invention made from the compound represented by Chemical Formula 1 can be decomposed by means of nitric oxide sensitivity.

[0037] In this invention, "sensitivity" refers to reacting in a way that is sensitive to certain environmental conditions. In this specification, "sensitivity" can be used interchangeably with "reactivity".

[0038] In the experimental examples described later, it was verified that the hydrogel of the present invention can absorb and swell with nitric oxide, thereby confirming that it can capture and remove excessive nitric oxide produced in the body and effectively treat diseases caused by excessive nitric oxide production. Furthermore, it was verified that the hydrogel of the present invention can decompose by means of nitric oxide sensitivity, thereby confirming that microparticle structures bound within the hydrogel network structure can be selectively released to the disease site, and that the drug loaded in the microparticle structures can be locally, selectively, and concentratedly delivered to the disease site. Moreover, it was confirmed that the degree of decomposition of the hydrogel of the present invention depends on changes in NO concentration, thereby allowing for the adjustment of drug release according to the severity of the disease.

[0039] In this invention, the compound represented by chemical formula 2 is also referred to as PLA-b-PEG-N3 block copolymer, which can be combined with the alkynyl group of the compound represented by chemical formula 1 by means of a click chemistry reaction of the azide group.

[0040] [Chemical Formula 2]

[0041]

[0042] (where n and m are integers from 1 to 1000)

[0043] The compound represented by the chemical formula 2 can form microparticle structures by means of self-assembly.

[0044] In this invention, "microparticle" refers to a system consisting of an inner hydrophobic region and an outer hydrophilic region in an aqueous solution. In this invention, a compound represented by chemical formula 2, namely PLA-b-PEG-N3 block copolymer, is used to form the microparticle structure.

[0045] In this invention, the microparticle structure can carry hydrophobic drugs and release them specifically at the disease site, thereby enabling combined treatment of diseases caused by excessive nitric oxide, namely, treatment by removing nitric oxide and treatment by drugs.

[0046] In embodiments of the present invention, the microparticle structure may have a uniform diameter of 10 to 100 nm, preferably 70 to 900 nm, and more preferably 70 to 500 nm. A diameter less than this range may result in insufficient drug delivery for treating the disease, while a diameter exceeding this range may lead to inconvenience in injection.

[0047] In this invention, the microparticle structures can be anchored to the network structure of the hydrogel. These microparticle structures can be released from the hydrogel upon nitric oxide-sensitive decomposition, thereby selectively delivering the loaded drug to sites of excessive nitric oxide production.

[0048] In this invention, the compound represented by chemical formula 3, i.e., the polymer chain based on hyaluronic acid, is also referred to as HA-N3, which serves to form the backbone of the hydrogel in this invention.

[0049]

Chemical Formula 3

[0050]

[0051] (where n is an integer from 1 to 1000)

[0052] In this invention, the "hyaluronic acid" is a linear polysaccharide composed of glucuronic acid and acetylglucosamine, and is one of the glucosamine glucans found in the carriers of the extracellular matrix (ECM), joint lubricating fluid, and cartilage. Cross-linked hyaluronic acid can be used as a joint lubricating fluid due to its viscoelastic properties, and it also does not present immunological problems when used in vivo. Therefore, it is an excellent biocompatible material suitable for tissue engineering and drug delivery systems.

[0053] In this invention, the compound represented by Formula 3 can be combined with the alkynyl group of the compound represented by Formula 1 by means of a click chemistry reaction of the azide group.

[0054] In this invention, the hydrogel can be manufactured by means of a click chemical reaction between a compound represented by chemical formula 1 and compounds represented by chemical formula 2 and chemical formula 3.

[0055] In this invention, "click chemistry" refers to targeted reactions occurring in the complex environment of a living organism, and is a modular approach related to organic synthesis that can be rapidly, efficiently, and predictably combined under specific conditions. For example, the azide-alkyne cycloaddition reaction, due to its extremely high thermodynamic driving force, can very efficiently form a combination of azide and alkyne compounds in high yields, and therefore can also form intermolecular bonds in high yields in reactions with polymers such as oligomers and polymers.

[0056] In embodiments of the present invention, the alkyne group of the compound represented by Formula 1 and the azide (-N3) group of the compound represented by Formula 2, and the alkyne group of the compound represented by Formula 1 and the azide (-N3) group of the compound represented by Formula 3, respectively, will form 1,2,3-triazole by means of click chemistry, thereby forming the nitric oxide reactive in situ hybridization hydrogel containing microparticle structures of the present invention.

[0057] In this invention, because the hydrogel contains bioactive substances, it can further enhance the therapeutic effect on inflammation or diseases caused by overexpression of nitric oxide. The bioactive substances are substances used for the treatment, cure, prevention, or diagnosis of diseases, such as cells, growth factors, hormones, proteins or peptides, nucleic acids, extracellular matrix substances, and drugs with therapeutic functions, but are not limited to these. Any bioactive substance known to be effective against a specific disease can be applied to the hydrogel without restriction to enhance the therapeutic effect. To ensure that the manufactured hydrogel contains bioactive substances, a solution containing the bioactive substances can be prepared and then mixed with other solutions at the target site to form a hydrogel. Alternatively, a syringe can be used to mix two solutions containing bioactive substances at the target site to form a hydrogel.

[0058] In this invention, the hydrogel may further comprise a hydrophobic drug that can be loaded into a microparticle structure formed by the self-assembly of a compound represented by Chemical Formula 2. The microparticle structure is formed in water by the self-assembly of a block copolymer having both hydrophilic and hydrophobic portions, and possesses strong internal hydrophobicity. Therefore, the hydrophobic drug can be easily loaded into the interior of the microparticle structure due to the physical phenomenon caused by the hydrophobicity of both the drug and the interior of the microparticle structure, which can increase the drug content compared to chemically bound drug delivery systems.

[0059] In this invention, "hydrophobic" means non-polar or low-polar substances that do not mix with water, but it is not limited to this. Any substance that can stably exist within the hydrophobic interior of the microparticle structure of this invention is included in the hydrophobic substance.

[0060] In this invention, the hydrophobic drug may be selected from analgesics, anti-inflammatory agents, immunosuppressants, or combinations thereof, preferably anti-inflammatory agents, but is not limited thereto.

[0061] In this invention, "anti-inflammatory agent" is also referred to as anti-inflammatory agent, which means a drug that has the property of eliminating inflammation or inhibiting it by participating in the inflammatory process.

[0062] The anti-inflammatory agent may be derived from, for example, 21-acetoxypregnenolone, aclomethasone, aclomethasone dipropionate, alpha-pregnane, ancinonide, beclomethasone, betamethasone dipropionate, betamethasone sodium phosphate, betamethasone valerate, budesonide, cloprednisolone, ciroxonide, clobetasol, clobetasol 17-propionate, clobetasol 17-butyrate, clobetasol, cloxetine, cloprednisolone, corticosteroids, cortisone, cortisone acetate, etc. Corivazole, Defucoxetine, Desoxind, Deoxymethasone, Dexamethasone, Dexamethasone Sodium Phosphate, Difluralasone, Diflucorone, Difluprednisolone, Glycyrrhetinic Acid, Fluzacrocin, Fluclofenex, Flumethasone, Fluhuanol, Fluocinolone Acetate, Fluocinolone, Fluocinolone Butylester, Flucolone, Flucolone Hexanoate, Flucolone Neopentylester, Flumilone, Flunisolone Acetate, Fluprednisolone Acetate, Fluprednisolone, Fluticasone, Fluticasone Propionate, Formococcus, Halcinonide, Halobetasol Propionate, Halometasone, Halobetasol Acetate, Hydrocortisone, Hydrocortisone Acetate, Hydrocortisone-17-Propylene, Hydrocortisone-17-Butylpropionate, Hydrocortisone-17-Butylene, Hydrocortisone-17-Valerate, Clotiprine, Maprednisolone, Methylprednisolone, Methylprednisolone, Mometasone, Mometasone Furoate, Peramisone, Peramisone The anti-inflammatory agent may be one or more selected from the group consisting of pine acetate, prednicarbamate, prednisolone, prednisolone 25-diethylamino-acetate, prednisolone sodium phosphate, prednisone, prednisolone valerate, prednisolone, limexolone, tecortisone, tecortisone neovalerate, triamcinolone, triamcinolone acetonide, alcohol triamcinolone, benztriamcinolone, and hexamcinolone. In embodiments of the present invention, the anti-inflammatory agent may be dexamethasone, but is not limited thereto.

[0063] Furthermore, in embodiments of the present invention, the hydrophobic drug may be contained in 3 to 7% by weight of the microparticle structure, but is not limited thereto.

[0064] The hydrogel of the present invention may also contain hydrophobic drugs and hydrophilic drugs.

[0065] In this invention, the hydrophobic drug and the hydrophilic drug can be released simultaneously at the target site. In this case, the release of the hydrophobic drug can be based on erosion, while the release of the hydrophilic drug can be based on Fickian diffusion.

[0066] In the experimental examples described later, it was confirmed that the hydrogel of the present invention can load hydrophobic drugs into the hydrophobic regions of the hydrogel, i.e., the interior of the microparticles, and hydrophilic drugs into the hydrophilic regions of the hydrogel, and can simultaneously release both drugs to the target site requiring treatment. Furthermore, the degree of drug release from the hydrogel of the present invention can vary according to the concentration of nitric oxide, which means that drug release can be adjusted according to the severity of the disease, and also indicates that personalized treatment can be provided according to the state of the disease requiring treatment.

[0067] In this invention, compounds of chemical formulas 1 to 3 used to manufacture hydrogels can be co-injected onto the target site.

[0068] In this invention, "co-injection" refers to the injection of compounds of formulas 1 to 3 together in an injectable dosage form, such as a liquid, into the target site of the patient. In embodiments of this invention, the liquid composition comprising compounds 1 to 3 can be uniformly mixed after exiting the needle and before entering the target site in the patient, and then injected into the target site as a mixture, gelling in the body after removal from the syringe. The compounds of formulas 1 to 3 of this invention can spontaneously combine and form a hydrogel by means of a click chemistry reaction after co-injection into the target site.

[0069] In this invention, the simultaneous injection can be achieved using a dual syringe system or any other suitable syringe system, preferably a dual syringe system. In embodiments of this invention, compounds represented by chemical formulas 1 to 3 can be physically separated prior to simultaneous injection, for example, before a series of processes such as simultaneous extrusion, mixing, and injection into the patient via a needle occur. In embodiments of this invention, compositions comprising compounds represented by chemical formulas 1 and 3 can be physically separated from compositions comprising compounds represented by chemical formula 2. In embodiments of this invention, compositions comprising compounds represented by chemical formulas 1 and 2 can be physically separated from compounds represented by chemical formula 3. In embodiments of this invention, compositions comprising compounds represented by chemical formula 1 can be physically separated from compounds represented by chemical formulas 2 and 3.

[0070] The hydrogel of the present invention can form within 0.5 to 10 minutes, preferably within 0.5 to 1 minute, after the compounds of formulas 1 to 3 are injected into the target site. If the hydrogel formation time (gelation time) is less than this range, initial cross-linking after mixing of the compounds of formulas 1 to 3 may lead to difficulties in injection into the target site. If it exceeds this range, the injected substance may diffuse into surrounding tissues, making it unsuitable for localized hydrogel formation at the target site. Existing in-situ injectable hydrogels, developed conventionally through methods such as temperature or ultraviolet (UV) irradiation, suffer from problems such as slow phase transition times. The hydrogel of the present invention can gel in a very short time via click chemistry, thereby solving the problems existing in the prior art.

[0071] The hydrogel of the present invention can be used for the prevention, relief or treatment of inflammatory diseases.

[0072] In this invention, "inflammatory disease" refers to a disease with inflammation as the main pathological feature. In this invention, the inflammatory disease can be an inflammatory disease that presents with symptoms of excessive nitric oxide production in the body.

[0073] The hydrogel of this invention can capture and remove excessive nitric oxide. In cases where the immune system is paralyzed due to stress or other factors, leading to excessive nitric oxide production in the body, autoimmune diseases or inflammatory diseases may result. Therefore, the hydrogel of this invention, which can capture and remove excessive nitric oxide, can effectively treat diseases induced by excessive nitric oxide production.

[0074] Furthermore, the inflammatory disease can be a disease caused by joint inflammation, such as undifferentiated spondyloarthritis, undifferentiated arthropathy, arthritis, chronic inflammatory diseases caused by viral or bacterial infections, rheumatoid arthritis, reactive arthritis, osteoarthritis, and osteoporosis, but is not limited to these. In the implementation of this invention, the inflammatory disease can be rheumatoid arthritis, but is not limited to this.

[0075] Rheumatoid arthritis is an autoimmune disease caused by high concentrations of nitric oxide. Therefore, the hydrogel of this invention can inhibit cytokine-induced osteoclast-mediated bone resorption by capturing nitric oxide in a high-concentration nitric oxide environment, and can also alleviate or treat rheumatoid arthritis by preventing cell death in the joints.

[0076] In this invention, "relief or treatment" means reducing the severity of disease symptoms, prolonging the frequency and duration of periods without disease symptoms, or preventing damage or disability caused by disease-related suffering.

[0077] The hydrogel of this invention can reduce the levels of pro-inflammatory cytokines, namely tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6).

[0078] In this invention, "pro-inflammatory cytokines" mainly refer to cytokines generated by activated macrophages and promoting systemic inflammation. Pro-inflammatory cytokines include tumor necrosis factor-α (TNF-α), interleukin-1 (IL-1), interleukin-2 (IL-2), interleukin-6 (IL-6), and interleukin-8 (IL-8), which appear simultaneously during inflammatory responses and serve as indicators of these responses.

[0079] In the experimental examples described later, it was confirmed that treatment with the hydrogel of the present invention can reduce the levels of pro-inflammatory cytokines, namely tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), thereby confirming that the hydrogel of the present invention is effective for the treatment of inflammatory diseases.

[0080] The hydrogel of the present invention can be used as a visco-supplement.

[0081] In this invention, "visco-supplement" refers to a substance that replenishes viscous lubricating fluid by injecting gel-state hyaluronic acid into the joint area.

[0082] In the experimental examples described later, it was confirmed that the hydrogel of the present invention can exhibit almost complete recovery after rupture, accompanied by stress-induced flow, thus effectively serving as a viscosity supplement. This is not limited to a specific theory, but is believed to be caused by intermolecular hydrogen bonds between hyaluronic acid (HA) chains and entropy-driven reassociation of the microparticle structure.

[0083] In another aspect of the invention, an in situ hybridization hydrogel precursor composition comprising compounds represented by the following chemical formulas 1 to 3 is provided.

[0084]

Chemical Formula 1

[0085]

[0086] [Chemical Formula 2]

[0087]

[0088]

Chemical Formula 3

[0089]

[0090] in,

[0091] R1 and R2 are each independently a C1-C10 alkylene group.

[0092] n and m are integers from 1 to 1000.

[0093] In this invention, "precursor" refers to a compound that participates in a chemical reaction that generates other compounds.

[0094] The precursor composition of the present invention may further comprise a hydrophobic drug that may be supported in a microparticle structure formed by the self-assembly of a compound represented by the chemical formula 2.

[0095] The precursor composition of the present invention may also contain hydrophobic and hydrophilic drugs.

[0096] In another aspect of the invention, a method for manufacturing an in situ hybridization hydrogel is provided, comprising the steps described below.

[0097] a) The step of manufacturing a first precursor composition comprising a compound represented by chemical formula 1 and a compound represented by chemical formula 3;

[0098] b) The step of manufacturing a second precursor composition comprising microparticle structures formed from a compound represented by the following chemical formula 2; and,

[0099] c) The step of simultaneously injecting the first precursor composition and the second precursor composition.

[0100]

Chemical Formula 1

[0101]

[0102] [Chemical Formula 2]

[0103]

[0104]

Chemical Formula 3

[0105]

[0106] in,

[0107] R1 and R2 are each independently a C1 to C10 alkylene group.

[0108] n and m are integers from 1 to 1000.

[0109] In embodiments of the present invention, the first precursor composition may contain 0.02 to 0.06 wt% of the compound represented by the chemical formula 1 compared to the total mass of the first precursor composition; 1.00 to 3.00 wt% of the compound represented by the chemical formula 2 compared to the total mass of the second precursor composition; and 1.00 to 1.50 wt% of the compound represented by the chemical formula 3 compared to the total weight of the first precursor composition; however, it is not limited thereto.

[0110] In the manufacturing method of the present invention, the second precursor composition may further contain a catalyst. In the embodiments of the present invention, the catalyst may be Cu(I), but is not limited thereto.

[0111] In the manufacturing method of the present invention, the second precursor composition may further comprise a hydrophobic drug that may be carried in a microparticle structure formed by the self-assembly of a compound represented by the chemical formula 2.

[0112] In the manufacturing method of the present invention, the first and second precursor compositions may further comprise a hydrophobic drug and a hydrophilic drug. In embodiments of the present invention, the hydrophobic drug may be contained in the second precursor composition, the hydrophilic drug may be contained in the first precursor composition, or both the hydrophobic drug and the hydrophilic drug may be contained in the first precursor composition or the second precursor composition, but this is not a limitation.

[0113] In the manufacturing method of the present invention, the simultaneous injection can be performed using a dual syringe system.

[0114] 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 non-oral administration, may be manufactured in a solid or semi-solid form, and may contain formulation agents such as suspending agents, stabilizers, solubilizers and / or dispersants.

[0115] In embodiments of the present invention, the first and second precursor compositions may be in liquid form or sterilized, but are not limited thereto. Furthermore, they may be stored in a form that prevents contamination by microorganisms such as bacteria or mold. Additionally, the first and second precursor compositions may be in powder form or sterilized. In the cases described above, they may be formulated into a liquid form using a preparation such as water for injection before use.

[0116] The present invention provides a method for the prevention, relief or treatment of inflammatory diseases, including the step of administering medication to an individual suffering from an inflammatory disease using the hydrogel of the present invention.

[0117] In this invention, "administration" refers to introducing the hydrogel of this invention into the patient's body through a suitable method. As the route of administration for the hydrogel of this invention, any general route can be used as long as the target tissue can be reached; preferably, it can be non-oral administration. The non-oral administration can be intramuscular, subcutaneous, intra-articular, or intraperitoneal administration, but is not limited to these. In the implementation of this invention, the administration can be intra-articular, but is not limited to this.

[0118] In this invention, an individual includes any human and non-human animal. The term "non-human animal" can refer to vertebrates such as non-human primates, sheep, dogs, and rodents such as mice, rats, and guinea pigs. Preferably, the individual can be a human, specifically a human suffering from a specific disease. In this specification, the term "individual" may be used interchangeably with "object" or "patient."

[0119] The matters mentioned in the in situ hybridization hydrogel, precursor composition, manufacturing method and treatment method of the present invention are equally applicable without contradiction, and repeated descriptions have been omitted to avoid making the specification too complicated.

[0120] The hydrogel of this invention can locally and selectively capture and remove excessive nitric oxide generated in vivo and in vitro, thus exhibiting excellent preventive, alleviating or therapeutic effects on inflammatory diseases caused by excessive nitric oxide generation.

[0121] Furthermore, the hydrogel of the present invention can carry hydrophobic and / or hydrophilic drugs, thus serving as a drug delivery system to deliver drugs to target sites, thereby achieving a combined therapeutic effect through nitric oxide capture and drug delivery.

[0122] Furthermore, the hydrogel of the present invention can be decomposed by means of nitric oxide sensitivity, so that the crosslinking points can be dissociated by means of nitric oxide sensitivity in the presence of nitric oxide, thereby delivering the drug loaded in the hydrogel locally and selectively to the target site.

[0123] Furthermore, the hydrogel of the present invention does not require a surgical shell, but can be formed locally at the target site through a minimally invasive method, thus providing excellent convenience for patients.

[0124] Furthermore, the hydrogel of the present invention can prevent rapid removal from the target site and diffusion to other organs, thus avoiding problems such as systemic toxicity present in the prior art.

[0125] Furthermore, the hydrogel of the present invention can effectively restore its mechanical properties by means of its self-healing ability, and therefore can be effectively used as a visco-supplement. Attached Figure Description

[0126] Figure 1 The formation process of the hydrogel of the present invention and the process by which the crosslinking points of the formed hydrogel dissociate by means of the reactivity of nitric oxide are illustrated.

[0127] Figure 2 The synthesis process of the compound represented by chemical formula 1 is illustrated.

[0128] Figure 3 It is the 1H NMR spectrum of the compound obtained in each step of the synthesis of the compound represented by chemical formula 1. 1 H NMR data.

[0129] Figure 4 The diagram illustrates whether the compound represented by chemical formula 1 can react with and decompose with nitric oxide.

[0130] Specifically, Figure 4 The mechanism by which the compound represented by chemical formula 1 reacts with nitric oxide and decomposes is illustrated.

[0131] Figure 4 b. A proton nuclear magnetic resonance spectrum used to confirm whether a compound represented by formula 1 reacts with and decomposes with nitric oxide. 1 The H NMR data were illustrated.

[0132] Figure 5 The Fourier transform infrared (FT-IR) (a) and ultraviolet-visible absorption spectrometry (UV-Vis) absorbance data (b) used to confirm whether the compound represented by chemical formula 1 reacts with and decomposes with nitric oxide are illustrated.

[0133] Figure 6 The synthesis process of the compound represented by chemical formula 2 is illustrated.

[0134] Figure 7 1H NMR spectra of compounds obtained in each step of the synthesis of the compound represented by chemical formula 2 ( 1 The 1H NMR spectral data are illustrated.

[0135] Figure 8 The data are obtained from Fourier transform infrared spectroscopy (FT-IR), gel permeation chromatography (GPC), transmission electron microscopy (TEM), and dynamic light scattering spectrophotometry (DLS) for compounds represented by chemical formula 2.

[0136] Specifically, Figure 8 The Fourier transform infrared (FT-IR) spectra of PLA-b-PEG-N3 are illustrated in figure a.

[0137] Figure 8 b. The gel permeation chromatography (GPC) data of PLA-b-PEG-N3 are illustrated.

[0138] Figure 8 c. Transmission electron microscope (TEM) images of PLA-b-PEG-N3 are illustrated.

[0139] Figure 8 d illustrates the average hydrodynamic magnitude of PLA-b-PEG-N3 as determined by a dynamic light scattering spectrophotometer (DLS).

[0140] Figure 9 The synthesis process of the compound represented by chemical formula 3 is illustrated.

[0141] Figure 10 The proton nuclear magnetic resonance spectrum of the compound obtained during the synthesis of the compound represented by chemical formula 3 ( 1 The H NMR data were illustrated.

[0142] Figures 11 to 15 The properties of the nitric oxide-sensitive hydrogel (M-NO gel) of the present invention were analyzed.

[0143] Specifically, Figure 11 The nitric oxide-sensitive hydrogel (M-NO gel) was imaged using cryogenic scanning electron microscopy.

[0144] Figure 11b illustrates the self-healing vibrational rheological properties of a nitric oxide-sensitive hydrogel (M-NO gel) obtained through a dynamic transient test.

[0145] Figure 12 A schematic diagram of the self-healing process of a nitric oxide-sensitive hydrogel (M-NO gel) is illustrated.

[0146] Figure 12 b illustrates the frequency-dependent vibrational rheological properties of the nitric oxide-sensitive hydrogel (M-NO gel).

[0147] Figure 12 c. The NO concentration-dependent vibrational rheological properties of the nitric oxide-sensitive hydrogel (M-NO gel) and the control gel (NO non-reactive) are illustrated.

[0148] Figure 13 The NO removal capacity of the nitric oxide-sensitive hydrogel (M-NO gel) was confirmed. Data are presented as mean ± standard deviation (SD) (n = 4).

[0149] Figure 13 b. The relative swelling ratios of nitric oxide-sensitive hydrogels (MO gels) at different concentrations over time are illustrated. Data are expressed as mean ± standard deviation (SD) (n = 4).

[0150] Figure 13 c. The relative swelling ratios of the control group gel (NO non-reactive) at different concentrations over time are illustrated. Data are expressed as mean ± standard deviation (SD) (n = 3).

[0151] Figure 13 Images of nitric oxide-sensitive hydrogels (M-NO gels) and control gels (NO-nonreactive) after 2 hours of incubation in NO solutions of different concentrations are presented. Scale bar: 1 cm.

[0152] Figure 14 A schematic diagram of simultaneous dual drug release in a nitric oxide-sensitive hydrogel (M-NO gel) is illustrated.

[0153] Figure 14 b. The release curve of fluorescein isothiocyanate-labeled bovine serum albumin (BSA-FITC) (hydrophilic cargo model) on nitric oxide-sensitive hydrogel (M-NO gel) is illustrated.

[0154] Figure 14c. The release curve of fluorescein isothiocyanate-labeled bovine serum albumin (BSA-FITC) from the control group gel is illustrated.

[0155] Figure 14 The release curves of Nile Red (hydrophobic cargo model) loaded onto the microparticle structure of the nitric oxide-sensitive hydrogel (M-NO gel) are illustrated.

[0156] Figure 14 The Nile Red release curve of the control group gel is illustrated in the figure.

[0157] Figure 15 Figure a illustrates the average hydrodynamic size of PLA-b-PEG-N3 microparticle structures before hydrogel formation and the average hydrodynamic size of PLA-b-PEG-N3 microparticle structures confirmed in the supernatant after 24 hours of incubation of nitric oxide-sensitive hydrogel (M-NO gel) with NO solution (250 μM).

[0158] Figure 15 b illustrates a transmission electron microscope (TEM) image of the supernatant after erosion of a nitric oxide-sensitive hydrogel (M-NO gel). The white boxes within the images represent the morphology of the microparticle structures before gel formation.

[0159] Figure 16 as well as Figure 17 The reactivity and NO removal capacity of the nitric oxide-sensitive hydrogel (M-NO gel) after lipopolysaccharide (LPS) treatment in RAW 264.7 cells are illustrated.

[0160] Specifically, Figure 16 The NO concentrations before and after 24 hours of treatment with 5 μg / ml LPS in RAW 264.7 and NIH / 3T3 cell lines are illustrated.

[0161] Figure 16 b. The cell viability of RAW 264.7 cell lines treated with different concentrations of nitric oxide-sensitive hydrogels (M-NO gels) and the control gel is illustrated. Data are expressed as mean ± standard deviation (SD) (n = 5).

[0162] Figure 16c illustrates the NO removal capacity of the nitric oxide-sensitive hydrogel (M-NO gel). The results were obtained by treating RAW 264.7 cell lines with different concentrations of nitric oxide-sensitive hydrogel (M-NO gel) and culturing for 24 hours, and then confirming the NO level in the cell culture supernatant using the Griess assay.

[0163] Figure 16 Confocal microscopy images of RAW 264.7 cell lines activated with lipopolysaccharide (LPS) after treatment with nitric oxide-sensitive hydrogel (M-NO gel) and control gel are shown. Intracellular NO and nuclei were stained with 4,5-diaminofluorescein diacetate (DAF-2DA, green) and 4',6-diamidinyl-2-phenylindole (DAPI, blue), respectively. Scale bar: 100 μm.

[0164] Figure 17 The results of quantifying tumor necrosis factor-α (TNF-α) levels after treatment with nitric oxide-sensitive hydrogel (M-NO gel) and control gel are illustrated.

[0165] Figure 17 b. The results of quantifying interleukin-6 (IL-6) levels after treatment with nitric oxide-sensitive hydrogel (M-NO gel) and control gel are illustrated.

[0166] Figure 17 c. Images of nitric oxide-sensitive hydrogels (M-NO gels) and control gels after treating cultured RAW 264.7 or NIH / 3T3 cell lines (stimulated or unstimulated with lipopolysaccharide (LPS)) with nitric oxide-sensitive hydrogels (M-NO gels) and control gels overnight are illustrated.

[0167] Figure 17 The relative swelling ratios of cultured RAW 264.7 or NIH / 3T3 cell lines (stimulated or unstimulated with lipopolysaccharide (LPS)) after overnight culture using nitric oxide-sensitive hydrogel (M-NO gel) and control gel are illustrated.

[0168] Figure 17 e is a schematic diagram illustrating the in vitro NO- reactivity and removal capacity of a nitric oxide-sensitive hydrogel (M-NO gel) used to reduce levels of pro-inflammatory cytokines.

[0169] Figures 18 to 22The results confirming the therapeutic effect of the nitric oxide-sensitive hydrogel (M-NO gel) of the present invention on rheumatoid arthritis in a rheumatoid arthritis animal model, namely collagen-induced arthritis (CIA) mice, are illustrated.

[0170] Specifically, Figure 18 The overall experimental timeline is illustrated in diagram a.

[0171] Figure 18 Figure b illustrates the changes in relative foot volume in a mouse model of rheumatoid arthritis (RA) after sample treatment. Regarding relative foot volume, the group treated with saline (B) was compared with other experimental groups, and statistical analysis was performed using one-way ANOVA (**p<0.01, ***p<0.001).

[0172] Figure 19 Representative images of mouse hind feet at weeks 4, 6, and 8 are illustrated.

[0173] Figure 20 The time-dependent mean clinical scores of rheumatoid arthritis (RA) mice after sample treatment are illustrated. Data are presented as mean ± standard deviation (SD) (n = 8).

[0174] Figure 20 b. The mean clinical scores of rheumatoid arthritis (RA) mice at week 8 after sample treatment are illustrated. The group of RA model mice treated with saline (B) was compared with other experimental groups, and statistical analysis was performed using one-way ANOVA (**p<0.01, ***p<0.001).

[0175] Figure 20 c. The rotarod drop delay time of rheumatoid arthritis (RA) mice after sample treatment, analyzed by a fatigue rotarod apparatus, is illustrated. Data are expressed as mean ± standard error of mean (SEM) (n = 8).

[0176] Figure 20 d. The time to bar drop at week 8 in rheumatoid arthritis (RA) mice after sample treatment is illustrated. The group (B) of RA model mice treated with saline was compared with other experimental groups, and statistical analysis was performed using one-way ANOVA (**p<0.01, ***p<0.001).

[0177] Figure 21The results confirming the therapeutic effect of the nitric oxide-sensitive hydrogel (M-NO gel) of the present invention on rheumatoid arthritis in a rheumatoid arthritis animal model, namely collagen-induced arthritis (CIA) mice, are illustrated.

[0178] Specifically, Figure 21 The image illustrates a micro-CT scan of the forefoot of a mouse model of rheumatoid arthritis (RA). Red arrows represent bone destruction and damage.

[0179] Figure 21 Figure b illustrates the histological analysis results of the joint tissue after sample processing. Scale bar: 100 μm.

[0180] Figure 22 Figure a shows the mean histological scores of joint tissues determined by hematoxylin-eosin (H&E), masson's trichrome, and safranin-O staining. The group treated with saline in a mouse model of rheumatoid arthritis (RA) (B) was compared with other experimental groups, and statistical analysis was performed using one-way ANOVA (*p<0.05, ***p<0.001; no significant difference in ns).

[0181] Figure 22 b. The quantitative results of tumor necrosis factor-α (TNF-α) levels in serum samples from rheumatoid arthritis (RA) mice after sample processing are illustrated.

[0182] Figure 22 c. Interleukin-6 (IL-6) levels in serum samples from rheumatoid arthritis (RA) mice after sample processing are illustrated. Data are presented as mean ± standard deviation (SD) (n = 4). # was compared with other groups, and statistical analysis was performed using one-way ANOVA (*p < 0.05, ***p < 0.001; no significant difference in ns).

[0183] Figure 22 The quantitative results of tumor necrosis factor-α (TNF-α) levels in the foot tissue fluid of rheumatoid arthritis (RA) mice after sample processing are illustrated in Figure d.

[0184] Figure 22The quantitative results of interleukin-6 (IL-6) levels in the paw tissue fluid of rheumatoid arthritis (RA) mice after sample treatment are illustrated. Data are expressed as mean ± standard deviation (SD) (n=4). The group of RA model mice treated with saline (B) was compared with other experimental groups, and statistical analysis was performed using one-way ANOVA (*p<0.05, ***p<0.001; no significant difference in ns).

[0185] Figure 22 The quantitative results of NO levels in the paw tissue fluid of treated rheumatoid arthritis (RA) mice at week 8 are illustrated. Data are expressed as mean ± standard deviation (SD) (n = 4). The group treated with saline in RA mice (B) was compared with other experimental groups, and statistical analysis was performed using one-way ANOVA (*p < 0.05, ***p < 0.001; no significant difference in ns). Detailed Implementation

[0186] The present invention will now be described in more detail with reference to embodiments. However, these embodiments are merely illustrative and the scope of the invention is not limited by these embodiments. Furthermore, terms not specifically defined in this specification should be understood to have the meaning commonly used in the technical field to which this invention pertains.

[0187] Example 1. Synthesis of a compound of chemical formula 1

[0188] To synthesize the click crosslinking agent DA-NOCCL that reacts with nitric oxide, the amino group at the C2 position of 4-nitro-o-phenylenediamine was first selectively protected with a tert-butyloxycarbonyl (Boc) group using guanidine hydrochloride as a catalyst. Next, 4-NO2 was reduced to 4-NH2, and 4-pentynoic acid was bonded to each amino group via an amide bond. The synthesis was then completed by deprotecting the tert-butyloxycarbonyl (Boc) group under acidic conditions. The synthesis process of the crosslinking agent DA-NOCCL is as follows: Figure 2 As shown, by nuclear magnetic resonance hydrogen spectrum (NMR) 1 The results of H NMR (hydrogen spectroscopy) confirming the successful synthesis of each compound obtained during the synthesis process and the crosslinking agent DA-NOCCL are as follows: Figure 3 As shown.

[0189] The specific synthesis steps are described below.

[0190] Step 1: After dissolving 4-nitro-o-phenylenediamine (1 g, 6.53 mmol) and guanidine hydrochloride (95 mg, 0.91 mmol) in 20 mL of ethanol and droppingwise adding di-tert-butyl dicarbonate (2.85 g, 13.06 mmol), the reaction mixture was vigorously stirred at 25 °C for 24 hours. The organic solvent in the reaction solution was evaporated under reduced pressure and resuspended in distilled water. The aqueous solution was extracted three times with ethyl acetate (EA), and the organic fraction was washed with Na₂SO₄ to obtain an orange-yellow crude solid. Compound 1 (1.22 g, yield: 73.8%) was then purified by silica gel column chromatography (eluent: ethyl acetate (EA)-hexane mixture) to obtain compound 1.

[0191]

Compound 1

[0192]

[0193] 1 H NMR (500MHz, DMSO-d6): δ = 8.56 (brs, 1H, D2O exchangeable),8.30(d,J=1.6Hz,1H),7.78(dd,J=9.0,2.7Hz,1H),6.72(d,J=9.0Hz,1H),6.46(brs,2H,D2Oexchangeable),1.48(s,9H).

[0194] Step 2: Activated carbon (10% Pd / C) supporting 100 mg of palladium was added to 10 mL of anhydrous tetrahydrofuran (THF) solution of Compound 1 (400 mg, 1.58 mmol) under nitrogen. Hydrogen was then pumped in until the pressure reached a maximum of 40 psi, and the reaction mixture was stirred at 25 °C for 36 hours. After the reaction was complete, Pd / C was removed by filtration of the reaction mixture through a diatomaceous earth 545 pad. The resulting solid, Compound 2 (322 mg, yield: 91.2%), was then evaporated under reduced pressure to obtain a purple solid.

[0195]

Compound 2

[0196]

[0197] 1H 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,D2Oexchangeable),4.01(brs,2H,D2O exchangeable),1.45(s,9H)

[0198] Step 3: Compound 2 (200 mg, 0.90 mmol), 4-pentyneic acid (200 mg, 2.04 mmol), 1-hydroxybenzotriazole (HOBt, 280 mg, 2.07 mmol), and triethylamine (TEA, 190 mg, 1.88 mmol) were completely dissolved in 10 mL of anhydrous N,N-dimethylformamide (DMF). After adding N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC, 400 mg, 2.09 mmol) to the solution, the reaction mixture was vigorously stirred at 25 °C for 24 hours. After the reaction was complete, the reaction mixture was evaporated under reduced pressure and suspended in CH2Cl2. The organic solvent was extracted three times with distilled water, and the organic fraction was washed with Na2SO4 to obtain a light brown crude solid. Compound 3 (285 mg, 0.74 mmol, yield: 82.7%) was then purified by silica gel column chromatography (elution: ethyl acetate (EA)-hexane mixture).

[0199]

Compound 3

[0200]

[0201] 1 H NMR (500MHz, DMSO-d6): δ=9.99(brs,1H,D2Oexchangeable),9.41(brs,1H,D2Oexchangeable),8.26(brs,1H,D2O exchangeable),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)

[0202] Step 4: 5 ml of trifluoroacetic acid (TFA) was added dropwise to compound 3 (200 mg, 0.52 mmol) suspended in 20 ml of CH₂Cl₂ at 0 °C. The reaction mixture was then vigorously stirred at 25 °C for 12 hours. After the reaction was complete, the reaction mixture was evaporated under reduced pressure and neutralized with a saturated sodium bicarbonate (NaHCO₃) solution. The neutralized solution was extracted three times with 5% (v / v) methanol in CH₂Cl₂, and the dark brown solid crosslinking agent DA-NOCCL (138 mg, 0.49 mmol) was obtained by washing the organic fraction with Na₂SO₄. 93.5%.

[0203] [DA-NOCCL]

[0204]

[0205] 1 H NMR (500MHz, DMSO-d6): δ = 9.70 (brs, 1H, D2Oexchangeable), 9.06 (brs, 1H, D2Oexchangeable ),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).

[0206] exist Figure 4 as well as Figure 5 The mechanism by which the synthesized crosslinking agent DA-NOCCL reacts with nitric oxide and decomposes, and the 1H NMR spectrum used to confirm this mechanism (…) 1 Absorption data from H NMR, Fourier transform infrared (FT-IR), and ultraviolet-visible absorption spectroscopy (UV-Vis) were illustrated.

[0207] pass Figure 4 as well as Figure 5 It can be confirmed that after culturing in a saturated NO solution, the characteristic peak of the primary amine disappears and a benzotriazole group is formed, indicating that the crosslinking agent DA-NOCCL can react with and decompose NO.

[0208] Example 2. Synthesis of compounds with chemical formula 2

[0209] To synthesize a compound of formula 2 capable of click reaction with a crosslinking agent represented by formula 1, polyethylene glycol (PEG) was used as the starting material. One of the hydroxyl groups of PEG was selectively tosylated under silver oxide (Ag₂O) catalysis, followed by substitution of the methanesulfonyl group with bromide. The resulting PEG-Br was then cyclized with D,L-lactide to form a PLA-b-PEG-Br block copolymer. Finally, the synthesis was completed by substituting the bromide group with azide. After dissolving 10 mg of the synthesized PLA-b-PEG-N3 in 1 ml of tetrahydrofuran (THF), it was dropwise added to 9 ml of water to form a microparticle structure. The remaining THF was removed by dialysis. The specific synthetic steps are described below, and the synthetic process is as follows. Figure 6 As shown. By nuclear magnetic resonance hydrogen spectroscopy (NMR 1H) 1 The successful synthesis of PLA-b-PEG-N3 was confirmed by 1H NMR, Fourier transform infrared spectroscopy (FT-IR), and gel permeation chromatography (GPC), as shown in the figures below. Figure 7 , 8 As shown in a and 8b.

[0210] The specific synthesis steps are described below.

[0211] Step 1: Silver oxide (I) (1.74 g, 7.51 mmol) and potassium iodide (KI, 0.33 g, 1.99 mmol) were added to a vacuum-dried 2 kDa polyethylene glycol (PEG) solution (10 g, 5.00 mmol) dissolved in 160 mL of anhydrous dichloromethane (CH2Cl2). Next, 20 mL of p-toluenesulfonyl chloride (TsCl, 1 g, 5.25 mmol) in anhydrous CH2Cl2 was added, and the mixture was vigorously stirred overnight at 0 °C. After the reaction was complete, 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 recrystallization from multiple CH2Cl2 / diethyl ether ((C2H5)2O) to obtain a white solid precipitate (monoTos-PEG, 9.72 g, yield: 90.9%).

[0212] Step 2: MonoTos-PEG (4.0 g, 1.86 mmol) and potassium bromide (KBr, 1.1 g, 9.24 mmol) were completely dissolved in 40 mL of anhydrous dimethylformamide (DMF), and the reaction was carried out at 65 °C for 18 hours with stirring. After the reaction was complete, the reaction mixture was evaporated under reduced pressure and redispersed in CH2Cl2. The product was then purified by passing it through short silica gel and by recrystallization from multiple CH2Cl2 / (C2H5)2O to obtain a white solid precipitate (monoBr-PEG, 3.59 g, yield: 93.5%).

[0213] 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 performing multiple freeze-thaw cycles on the solution, polymerization was initiated by adding tin(II)2-ethylhexyl ester catalyst (Sn(Oct)2, 0.01 g, 0.02 mmol). The reaction mixture was vigorously stirred at 120 °C for 36 hours. After the reaction was complete, the mixture was evaporated under reduced pressure and redispersed in CH2Cl2. The product was then purified by multiple recrystallizations of CH2Cl2 / (C2H5)2O to obtain a discolored amorphous solid precipitate. The product was further purified (PLA-b-PEG-Br) by dialyzing with distilled water for 2 days followed by freeze-drying. The molecular weight was determined by gel permeation chromatography (GPC) under tetrahydrofuran (THF) (Mn = 5540, PDI = 1.50).

[0214] 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 dimethylformamide (DMF), and the reaction mixture was vigorously stirred at 50 °C for 3 days. After the reaction was completed, the product was purified by dialyzing with distilled water for 2 days and then freeze-drying. The molecular weight was determined by gel permeation chromatography (GPC) at tetrahydrofuran (THF) (Mn = 5864, PDI = 1.55).

[0215] Furthermore, transmission electron microscopy (TEM) and dynamic light scattering spectrophotometry (DLS) data of the microparticle structures formed by the self-assembly of the fabricated PLA-b-PEG-N3 are as follows: Figure 8 As shown in c and 8d, through Figure 8 c and 8d confirm that PLA-b-PEG-N3 can form a uniform microparticle structure of approximately 284.1 ± 7.8 nm in size through self-assembly under aqueous conditions.

[0216] Example 3. Synthesis of compounds with chemical formula 3

[0217] After replacing the chloride group of 2-chloroethylamine with an azide group, the substituted 2-azidoethylamine was bonded to hyaluronic acid via an amide bond, thereby synthesizing a hyaluronic acid-based polymer chain (HA-N3) that can obtain a hydrogel backbone through a click reaction with a compound represented by Formula 1. The synthesis process of the hyaluronic acid-based polymer chain is as follows: Figure 9 As shown, the proton NMR spectra of the compounds obtained in each step of the synthesis are as follows: 1 H NMR spectrum as follows Figure 10 As shown.

[0218] The specific synthesis steps are described below.

[0219] Step 1: Dissolve 1.16 g of 2-chloroethylamine hydrochloride (10.00 mmol) and 1.3 g of NaN3 (20.00 mmol) in 30 ml of distilled water, and vigorously stir the reaction mixture at 80 °C for 12 hours. After the reaction is complete, add 1 M sodium hydroxide (NaOH) to adjust the pH of the solution to 10-11. Extract the alkaline solution three times with CH2Cl2, and wash the organic fraction with anhydrous sodium sulfate (Na2SO4) to obtain a pale yellow oily product (2-azidoethylamine, 0.62 g, yield: 72.0%).

[0220] 1 H NMR (500MHz, D2O): δ = 3.41 (t, J = 5.7Hz, 1H), 2.80 (t, J = 5.7Hz, 1H).

[0221] Step 2: 100 kDa hyaluronic acid (HA, 200 mg, 0.50 mmol (repeat unit)) was completely dissolved in 40 ml of 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 in the dark. After the reaction was complete, the product was purified by continuous dialysis with 0.1 M sodium chloride (NaCl) and distilled water, and HA-N3 was obtained by freeze-drying.

[0222] Manufacturing Example 4. Manufacturing of Hydrogels

[0223] The nitric oxide-sensitive hydrogel of the present invention (hereinafter referred to as M-NO gel) was prepared by the following method.

[0224] Specifically, M-NO gel was prepared into a dual-syringe system. One syringe of the dual-syringe system contained compounds represented by chemical formulas 1 and 3, while the other syringe contained a compound represented by chemical formula 2 and a Cu(I) catalyst. The syringes containing chemical formulas 1 and 3 were prepared in 1% (v / v) dimethyl sulfoxide (DMSO) at concentrations of 0.04 wt% and 2.5 wt%, respectively. The syringe containing the compound represented by chemical formula 2 and the Cu(I) catalyst was prepared in distilled water at concentrations of 2 wt% and 0.02 wt%, respectively. The Cu(I) catalyst was prepared by adding 15.5 mg of CuSO... 4· It is prepared by mixing 5H2O and 22.0 mg of sodium ascorbate into 0.2 mL of distilled water.

[0225] In addition, the nitric oxide non-reactive hydrogel used as a control group was manufactured by replacing DA-NOCCL with DA-NONCL (dialkyne-functionalized non-cleavable cross-linker) in the manufacturing method of the M-NO gel.

[0226] Experimental Example 1. Morphological Analysis of M-NO Gel

[0227] To perform morphological analysis on the M-NO gel of this invention, the M-NO gel prepared as a dual-syringe system was imaged using cryogenic scanning electron microscopy, and the results are as follows. Figure 11 As shown in a.

[0228] Through the above Figure 11 It can be confirmed that the microparticle structures are uniformly distributed and fixed within the network hydrogel.

[0229] Experimental Example 2. Mechanical Properties Analysis of M-NO Gel

[0230] To confirm the use of the M-NO gel of the present invention as a visco-supplements for joints, the self-healing properties of the M-NO gel after rupture were analyzed by a dynamic transient test.

[0231] Specifically, step strain (ε = 0.5 to 200%, ωrad / s, 25℃) was measured over several cycles to confirm whether the mechanical properties of the viscous supplement, a crucial variable in the viscous additive, exhibit self-healing characteristics after the network structure fractures under high strain. The results are as follows: Figure 11 As shown in b and 12a.

[0232] The results confirm that the M-NO gel can almost completely restore the flow induced by stress, thus the M-NO gel of the present invention can be effectively used as a viscosity supplement.

[0233] Experimental Example 3. Analysis of NO concentration-dependent mechanical property changes in M-NO gel

[0234] To confirm the changes in the mechanical properties of M-NO under different NO concentrations, frequency-dependent rheological parameters were analyzed.

[0235] Specifically, the frequency-dependent vibrational rheological properties (ε%, 25°C) and concentration-dependent rheological properties (ωrad / s, ε%, 25°C in ωrad / s) of the NO-non-reactive gel and the NO-reactive M-NO gel of the present invention were measured and compared after quantification, and the results are as follows: Figure 12As shown in b and 12c.

[0236] pass Figure 12 As confirmed by b and 12c, the storage modulus of the NO-reactive M-NO gel gradually decreases with increasing NO concentration, thus exhibiting a tendency for decreased mechanical strength when the crosslinking agent decomposes under NO-mediated conditions. Conversely, in the NO-non-reactive gel, which serves as a control group, no significant change in storage modulus was observed even in high-concentration NO solutions. That is, it can be confirmed that the M-NO gel of the present invention exhibits a decomposition pattern that increases with increasing NO concentration.

[0237] Experimental Example 4. Confirmation of the NO removal capacity of M-NO gel

[0238] Regarding the NO removal capacity of the M-NO gel of the present invention, after preparing a 25 μM Py-NO solution using Py-NO (a synthetic NO donor that releases NO under aqueous conditions, consisting of N-diazenium diolates-incorporated pyrrolidine), the M-NO gel and a NO-non-reactive gel were treated with the prepared 25 μM Py-NO solution and incubated for 1 hour. The residual NO level in the supernatant was then quantified by Grise analysis (NO concentration was determined by comparing the absorbance at 548 nm with a standard curve plotted using NaNO2). Furthermore, the swelling level of the hydrogel was measured and confirmed, and the relative swelling ratio was calculated using the following formula, the results of which are as follows: Figure 13 As shown in a, 13b, 13c and 13d.

[0239] <Calculation Formula>

[0240] -Expansion ratio = (Mf - Mi) / Mi

[0241] (Mf: weight of the hydrogel that swells in NO solution, Mi: weight of the dried hydrogel)

[0242] - Relative expansion ratio = Expansion ratio / Expansion ratio in water at 0h

[0243] pass Figure 13 a, 13b, 13c, and 13d confirm that the M-NO gel of the present invention can significantly reduce the amount of NO solution along with the concentration of NO, and expand significantly compared with the NO non-reactive gel used as a control group.

[0244] That is, it can be confirmed that the M-NO gel of the present invention can effectively remove nitric oxide in a concentration-dependent manner, and has the expansion characteristics caused by the increase in pore size when the network structure of the hydrogel breaks based on the NO concentration, thus enabling on-demand controlled delivery based on NO concentration.

[0245] Experimental Example 5. Confirmation of Simultaneous Dual Drug Release from M-NO Gel

[0246] The simultaneous dual-stage release of NO-reactive drugs by M-NO gel was confirmed through the following experiments.

[0247] Specifically, a hydrophilic drug model, namely fluorescein isothiocyanate-labeled bovine serum albumin (BSA-FITC), is dissolved at a final concentration of 1 wt% in a solution containing HA-N3 and encapsulated in the hydrophilic region of the hydrogel, while a hydrophobic drug model, namely Nile Red, is encapsulated in the hydrophobic region of PLA-b-PEG-N3 microparticles. Both the hydrophilic and hydrophobic drug models are co-encapsulated within the M-NO gel during hydrogel formation.

[0248] Next, 200 μL of M-NO gel was incubated at 25 °C with 5 mL of NO solution (0, 2.5, 25, and 250 μM). The supernatant was replaced with NO solution at predetermined time intervals. After concentrating the obtained supernatant by freeze-drying, the fluorescence (excitation / emission peaks at 485 / 510 nm) and absorbance at 585 nm were compared with standard curves to investigate the time-dependent NO-responsive simultaneous release curves of fluorescein isothiocyanate-labeled bovine serum albumin (BSA-FITC) and Nile red.

[0249] A schematic diagram of dual-drug release is shown below. Figure 14 As shown in a, the results are as follows Figure 14 As shown in b, 14c, 14d, and 14e.

[0250] pass Figure 14b, 14c, 14d, and 14e confirm that the M-NO gel of the present invention can simultaneously release hydrophilic molecules with release characteristics due to Fickian diffusion and hydrophobic molecules with erosion-based release characteristics at high NO concentrations. Figure 14 b and 14d), but the NO non-reactive gel, as a control group, did not exhibit a special release pattern even with increasing NO concentration ( Figure 14 c and 14e).

[0251] Furthermore, the ability of the M-NO gel of the present invention to achieve erosion-based release of hydrophobic drugs from microparticle structures was confirmed by comparing the average hydrodynamic magnitude of the PLA-b-PEG-N3 microparticle structures before hydrogel formation with the average hydrodynamic magnitude of the PLA-b-PEG-N3 microparticle structures observed in the supernatant after 24 hours of incubation of the M-NO gel with NO solution (250 μM). The results are as follows: Figure 15 As shown in a and 15b.

[0252] Through the above Figure 15 a and 15b confirm that microparticle structures with similar size and morphology in hydrodynamics to the PLA-b-PEG-N3 microparticle structures before hydrogel formation were observed in the supernatant of M-NO gel cultured with NO solution, suggesting that hydrophobic drugs can be released through an erosion-based mechanism.

[0253] Experimental Example 6. Confirmation of the Cytotoxicity of M-NO

[0254] The volume-dependent cytotoxicity of M-NO of the present invention and the control group was confirmed by the MTT assay.

[0255] Specifically, RAW 264.7 cells were cultured at a rate of 1×10⁻⁶. 4 Cells were initially seeded at a density of 100 cells / well into 96-well plates and cultured at 37°C in Duchenne Modified Igor Medium (DMEM) containing 10% fetal bovine serum (FBS), 100 U / mL penicillin, and 100 μg / mL streptomycin.

[0256] After 24 hours of culture, the medium was replaced with fresh medium containing the sample, and then cultured for another 40 hours. Next, the cells were washed and cultured for 4 hours in medium containing 0.5 mg / mL tetrazolium blue (MTT) solution. Finally, the medium was replaced with 200 μL of dimethyl sulfoxide (DMSO), and cell viability was investigated by measuring absorbance at 570 nm. The results are as follows: Figure 16 As shown in b.

[0257] pass Figure 16 b. It can be confirmed that the M-NO gel of the present invention has almost no impact on cell survival rate, thereby confirming the safety of the M-NO of the present invention.

[0258] Experimental Example 7. Confirmation of NO Removal Capacity of M-NO

[0259] The NO-scavenging ability of the compositions of the present invention and the associated cytokine levels were confirmed by using lipopolysaccharide (LPS)-stimulated NO-releasing RAW 264.7 macrophage cell line.

[0260] (1) Confirmation of NO removal capacity at different concentrations

[0261] RAW 264.7 cell line was cultured at 5 × 10⁻⁶ cells / year. 4 Cells were seeded at a density of 10 cells / well in 12-well plates and cultured at 37°C in Duchenne Modified Igor Medium (DMEM) containing 10% fetal bovine serum (FBS), 100 U / mL penicillin, and 100 μg / mL streptomycin.

[0262] After culturing overnight, the medium was replaced with fresh medium containing the sample and 5 μg / mL of lipopolysaccharide (LPS), and then cultured for another 24 hours.

[0263] Finally, the culture medium was centrifuged (3,000 rpm, 10 min, 4 °C) to obtain the supernatant, which was then transferred to a 96-well plate. The residual NO level in the supernatant was then confirmed by Grise analysis, and the results are as follows: Figure 16 As shown in c.

[0264] pass Figure 16 c. It can be confirmed that the M-NO gel of the present invention can significantly reduce the NO level of RAW264.7 cell line stimulated with lipopolysaccharide (LPS) to 25%, which is similar to the observation in RAW 264.7 cells that have not been treated with lipopolysaccharide (LPS).

[0265] (2) Confirmation of NO removal capability using confocal microscopy

[0266] NO removal capacity was assessed by imaging intracellular NO levels using confocal microscopy.

[0267] Specifically, cells are divided into 1×10 5 Cells were seeded at a density of 10 cells / well into coverslips configured in 6-well plates and cultured at 37°C in Duchenne Modified Igor Medium (DMEM) containing 10% fetal bovine serum (FBS), 100 U / mL penicillin, and 100 μg / mL streptomycin.

[0268] After culturing overnight, the medium was replaced with fresh medium containing the sample (25 μg / mL crosslinking agent, 250 μg / mL gel) and 5 μg / mL lipopolysaccharide (LPS), and then cultured for another 24 hours.

[0269] Next, after replacing the sample medium with fresh medium containing 5 μm of 4,5-diaminofluorescein diacetate (DAF-2DA) and culturing for another 40 minutes, the samples were washed with Duchenne phosphate buffer (DPBS) and then incubated with fresh medium for 20 minutes.

[0270] Next, the sample was washed with Duchenne phosphate-buffered saline (DPBS) and treated with 10% neutral formalin (NBF) under dark conditions at room temperature. After 30 minutes, the sample was observed using a confocal microscope. The results are as follows: Figure 16 As shown in d.

[0271] pass Figure 16 It can be confirmed that high fluorescence intensity was observed in the RAW 264.7 cell line stimulated with lipopolysaccharide (LPS), but the signal was significantly reduced after treatment with M-NO gel, thereby confirming that the M-NO of the present invention can effectively remove NO.

[0272] (3) Quantification of cytokine levels

[0273] Although NO is an important pro-inflammatory mediator, excessive production can increase the levels of representative cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in vitro. Therefore, it was confirmed whether the M-NO gel of the present invention can reduce the levels of cytokines, namely tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6).

[0274] Specifically, after treating RAW 264.7 cell lines stimulated with lipopolysaccharide (LPS) (250 μg / mL) using M-NO gel and control gel, and culturing for 24 hours, the levels of pro-inflammatory cytokines, namely tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), in the supernatant of the cell culture medium were quantified by enzyme-linked immunosorbent assay (ELISA). The results are as follows: Figure 17 As shown in a and 17b.

[0275] pass Figure 17 As can be confirmed by a and 17b, after treatment with M-NO gel, the levels of NO-induced pro-inflammatory cytokines, namely tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), are significantly reduced. This confirms that the M-NO gel of the present invention can effectively remove excessive NO, thereby reducing inflammation and treating various inflammatory diseases.

[0276] (4) Confirmation of swelling of M-NO gel during NO capture

[0277] After treating RAW 264.7 or NIH / 3T3 cell lines with or without lipopolysaccharide (LPS) stimulation with M-NO gel and control gel, the cells were cultured overnight. The swelling of the M-NO gel during NO capture was then confirmed. The results are as follows: Figure 17 As shown in c and 17d.

[0278] Through the above Figure 17 c and 17d confirm that the M-NO gel can react with NO and swell significantly in RAW264.7 cell lines stimulated with lipopolysaccharide (LPS), thereby confirming that the M-NO gel of the present invention has conditions conducive to on-demand release.

[0279] Case 6. Confirmation of the therapeutic effect on rheumatoid arthritis (1)

[0280] The therapeutic effect of the M-NO gel of the present invention on rheumatoid arthritis was confirmed in mice, a mouse model of rheumatoid arthritis (RA) and collagen-induced arthritis (CIA). Figure 18 a).

[0281] (1) Preparation of a collagen-induced arthritis mouse model

[0282] The first immunization was performed by subcutaneously injecting 100 μL of an emulsion (1:1, v / v) of a mixture of type II collagen (CII, 2 mg / mL) dissolved in 10 mM acetic acid and complete Freund's adjuvant (CFA, 1 mg / mL) into the tail of 6-week-old DBA / 1j mice.

[0283] Two weeks later, 100 μL of an emulsion (1:1, v / v) of a mixture of type II collagen (CII, 2 mg / mL) dissolved in 10 mM acetic acid and incomplete Freud's adjuvant (IFA, 1 mg / mL) was subcutaneously injected into the tail of mice. Next, four weeks after the first immunization, rheumatoid arthritis (RA) model mice were randomly assigned to eight groups, and 20 μL of each sample was injected into the joints of collagen-induced arthritis (CIA) mice. In situ hybridization hydrogels were injected using a dual-injector system (one syringe containing HA-N3 (2.5 wt%) and a crosslinking agent (0.04 wt%), and the other syringe containing microparticles (2 wt%) and Cu(I) (0.02 wt%)). The M-NO gel loaded with dexamethasone was diluted 2-fold to make it consistent with the hydrogel system at 1 wt% (loading: 5.02%, capacity: 0.5 mg / kg).

[0284] The experimental groups are classified as follows.

[0285] (A) Healthy mice that have not been immunized

[0286] (B) Group of mice with rheumatoid arthritis (RA) model treated with saline solution

[0287] (C) Group of mice with rheumatoid arthritis (RA) model treated with dexamethasone-loaded microparticle structures.

[0288] (D) Group of mice treated with DA-NOCCL to treat a model of rheumatoid arthritis (RA).

[0289] (E) Group of mice treated with M-NO gel in a rheumatoid arthritis (RA) model

[0290] (F) Group of mice treated with control gel (NO non-reactive) to treat rheumatoid arthritis (RA) model mice

[0291] (G) Group of mice treated with M-NO gel loaded with dexamethasone to create a model of rheumatoid arthritis (RA).

[0292] (H) Group of mice treated with a control gel (NO non-reactive) loaded with dexamethasone

[0293] (2) Confirmation of foot volume changes and clinical scoring assessment related to arthritis.

[0294] Regarding the changes in paw volume over time in a mouse model of rheumatoid arthritis (RA), statistical analysis was performed by comparing each experimental group of the RA mouse model with the group treated with saline (B). The results are as follows: Figure 18 As shown in b. Furthermore, in Figure 19 The images show representative hind paw images of mice at weeks 4, 6, and 8.

[0295] pass Figure 18 b and Figure 19 It can be confirmed that a significant reduction in foot volume was observed in the groups using the M-NO gel of the present invention and the M-NO gel loaded with dexamethasone.

[0296] In addition, the clinical score of arthritis over time was assessed in a blinded manner using a standard scoring method (scale 0-5) while monitoring clinical scores during weeks 4 to 8. The results are as follows: Figure 20 As shown in a and 20b. The clinical scoring and assessment criteria for arthritis evaluation are described below.

[0297] 0: No clinical symptoms

[0298] 1: Minimal diffuse erythema / edema (affecting one or two toes)

[0299] 2: Mild diffuse erythema / edema (affecting more than 3 toes)

[0300] 3: Moderate diffuse erythema / edema (entire foot)

[0301] 4: Obvious diffuse erythema / edema (the entire foot and ankle)

[0302] 5: Severe diffuse erythema / edema (entire foot), inability to bend toes (joint adhesions)

[0303] pass Figure 20 a and 19b confirm that significant reductions in clinical scores were observed in the groups using the M-NO gel of the present invention and the M-NO gel loaded with dexamethasone.

[0304] The results confirm that the M-NO gel of the present invention, and the M-NO gel loaded with dexamethasone, can not only slow the progression of the disease but also alleviate the symptoms of arthritis itself. In particular, the M-NO gel loaded with dexamethasone exhibits almost complete relief of arthritis symptoms.

[0305] (3) Performance assessment

[0306] A rotarod assay was performed to assess kinetic activity in a mouse model of rheumatoid arthritis (RA).

[0307] Specifically, before the first immunization, mice underwent 3 days of fatigue rotarod training to achieve a stable latency to fall. The latency to fall was then recorded at fixed speeds of 10 rpm and 120 s, and the results are as follows: Figure 20 As shown in c and 20d.

[0308] Through the above Figure 20 c and 20d confirmed that the stick drop delay time was increased in the groups treated with the M-NO gel of the present invention and the M-NO gel loaded with dexamethasone, especially in the group treated with the M-NO gel loaded with dexamethasone, which showed a significant increase in stick drop delay time, thereby confirming that the motility of the mice was gradually improved.

[0309] Case 7. Confirmation of the therapeutic effect on rheumatoid arthritis (2)

[0310] To conduct a focused investigation into the therapeutic effect of the M-NO gel of the present invention on rheumatoid arthritis, a series of additional trials were performed in week 8.

[0311] (1) Confirmation of bone and joint condition

[0312] The skeletal and joint morphology was confirmed using representative forefoot microcomputed tomography (micro-CT) images from each experimental group after sample processing. The results are as follows: Figure 21 As shown in a.

[0313] pass Figure 21 It can be confirmed that, compared with other experimental groups, in the groups treated with the M-NO gel of the present invention and the M-NO gel loaded with dexamethasone, obvious bone boundaries can be observed due to less bone erosion.

[0314] This indicates that the hydrogel of the present invention can prevent oxidative / nitrifying stress and upregulation of osteoclasts by removing excessive nitric oxide generated in rheumatoid arthritis (RA) lesions.

[0315] (2) Histological analysis

[0316] At week 8, histological analysis was performed on the joint tissues of euthanized mice by staining with hematoxylin and eosin (H&E), masson's trichrome, and safranin-O. The results, along with the degree of cartilage damage assessed by a blinded test using a standard scoring method (scale 0-4), were recorded. Figure 21 As shown in b and 22a (B: bone, C: cartilage). Clinical scoring and assessment criteria are as follows.

[0317] 0: No damage, normal activity is possible.

[0318] 1: Minimal erosion limited to a single location.

[0319] 2: Mild / moderate erosion within a limited area

[0320] 3: Obvious / extensive erosion

[0321] 4: Most of the damage

[0322] Through the above Figure 21 b and 22a confirm that the M-NO gel of the present invention and the M-NO gel loaded with dexamethasone can restore damaged cartilage and joint cavity.

[0323] (3) Confirmation of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) levels and NO concentration.

[0324] In week 8, in accordance with the manufacturer's protocol, the levels of pro-inflammatory cytokines, namely tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), in serum and foot tissue fluid were quantified and confirmed using enzyme-linked immunosorbent assay (ELISA). NO levels were determined using Grise analysis, and the results are as follows: Figure 22 As shown in b to 22e.

[0325] pass Figure 22From b to 22e, it can be confirmed that in the groups treated with the M-NO gel of the present invention and the M-NO gel loaded with dexamethasone, the levels of pro-inflammatory cytokines in both serum and foot tissue fluid showed a significant reduction. In particular, in the group treated with the M-NO gel loaded with dexamethasone, the levels were significantly reduced to normal.

[0326] In addition, data quantifying NO levels in the foot tissues of sample-treated rheumatoid arthritis (RA) mice at week 8 were obtained, such as... Figure 22 As shown in f. Figure 22 As shown in f, it can be confirmed that the NO concentration in the foot tissue fluid of the group treated with the M-NO gel of the present invention and the M-NO gel loaded with dexamethasone was significantly reduced.

Claims

1. An in situ hybridization hydrogel manufactured using compounds represented by the following chemical formulas 1 to 3: 【Chemical Formula 1】 [Chemical Formula 2] 【Chemical Formula 3】 in, R1 and R2 are each independently a C1-C10 alkylene group. n and m are integers from 1 to 1000.

2. The in situ hybridization hydrogel according to claim 1, R1 and R2 are C2 alkylene groups.

3. The in situ hybridization hydrogel according to claim 1, The compound represented by the chemical formula 2 forms microparticle structures by means of self-assembly, the microparticle structures having a uniform diameter of 10 to 1000 nm.

4. The in situ hybridization hydrogel according to claim 1, The hydrogel is manufactured by a click chemistry reaction between a compound represented by chemical formula 1 and compounds represented by chemical formulas 2 and 3.

5. The in situ hybridization hydrogel according to claim 1, The hydrogel further comprises a hydrophobic drug, a hydrophilic drug, or a combination thereof.

6. The in situ hybridization hydrogel according to claim 5, The hydrophobic drug is loaded into a microparticle structure formed by the self-assembly of the compound represented by the chemical formula 2.

7. The in situ hybridization hydrogel according to claim 5, The hydrophobic drug is an anti-inflammatory agent.

8. The in situ hybridization hydrogel according to claim 6, The hydrophobic drug is contained in 3 to 7% by weight of the microparticle structure compared to the total weight of the structure.

9. The in situ hybridization hydrogel according to claim 5, The hydrophobic and hydrophilic drugs are released simultaneously at the target site.

10. The in situ hybridization hydrogel according to claim 1, The compounds of chemical formulas 1 to 3 are simultaneously injected into the target site.

11. The in situ hybridization hydrogel according to claim 10, The simultaneous injection is performed using a dual-injector system.

12. The in situ hybridization hydrogel according to claim 1, The hydrogel is intended for the treatment of inflammatory diseases.

13. The in situ hybridization hydrogel according to claim 12, The inflammatory disease is characterized by symptoms of excessive nitric oxide production in the body.

14. The in situ hybridization hydrogel according to claim 1, The hydrogel is used to reduce tumor necrosis factor-α and interleukin-6 levels.

15. The in situ hybridization hydrogel according to claim 1, The hydrogel is intended for use as a viscous supplement.

Images