Composition for preventing or treating inflammatory disease comprising micelle nanoparticles

Abstract

The present invention relates to a composition for preventing or treating inflammatory diseases, the composition comprising micelle nanoparticles. Specifically, the present invention has a micelle structure in which hydrophobic manganese oxide is supported on a core, wherein the micelle structure comprises nanoparticles that are formed from amphiphilic molecules in which hyaluronic acid and hydrophobic aliphatic hydrocarbons are linked by a reactive oxygen-decomposable linker, and thus may inhibit reactive oxygen species overexpressed in an inflammation-inducing environment and target inflammation-inducing M1 macrophages to facilitate repolarization into M2 macrophages, thereby providing an effect of intensive treatment of a site of inflammation.

Description

Composition for the prevention or treatment of inflammatory diseases containing micelle nanoparticles

[0001] The present invention relates to a composition for the prevention or treatment of inflammatory diseases comprising micelle nanoparticles.

[0002] Inflammatory response refers to a localized immune reaction that occurs to minimize damage and restore damaged areas when cells or tissues are damaged or destroyed by external biological causes (bacteria, viruses, parasites), physical causes (mechanical stimuli, heat, radiation, electricity), or chemical causes. As a useful defense mechanism for protecting the body and removing byproducts generated by tissue damage, the inflammatory response involves various inflammatory mediators and immune cells present in local blood vessels and body fluids. It entails enzyme activation, secretion of inflammatory mediators, fluid infiltration, cell migration, tissue destruction, erythema, edema, fever, or pain, or may induce dysfunction due to such symptoms. This inflammatory response involves various biochemical phenomena, and in particular, the reaction is initiated or regulated by various enzymes related to inflammation produced by immune cells. Among immune cells, macrophages include monocytes replenished from the blood and macrophages residing in tissues; they polarize into M1 and M2 types in response to specific stimuli and perform their respective functions. At the same time, macrophages play a crucial role in innate and adaptive immunity in response to stimuli. M1-type macrophages possess antimicrobial activity and function to induce inflammation, while M2-type macrophages function to suppress inflammation and promote wound healing.

[0003] Meanwhile, gouty arthritis is an inflammatory disease caused by the accumulation of uric acid crystals, primarily in joints such as the toes, due to the inability of uric acid to be excreted for various reasons. The uric acid that triggers gout is produced by a protein called "purine," which is consumed through food, metabolized into uric acid in the body, and excreted through urine. However, if uric acid is not excreted and accumulates in the body at high concentrations (blood uric acid levels exceeding 6.8 mg / dL), causing inflammation, it leads to gouty arthritis resulting from hyperuricemia. Typical gout treatments utilize drugs such as corticosteroids, xanthine oxidase inhibitors, and uric acid-lowering agents; however, these drugs have limited bioavailability and short half-lives, posing problems such as cardiovascular risks and nephrotoxicity. Therefore, research is needed on agents that target the site of inflammation and efficiently deliver active drugs to aid in the normalization of the inflammatory response.

[0004] The present invention aims to provide a pharmaceutical composition for the prevention or treatment of inflammatory diseases comprising micelle nanoparticles.

[0005] 1. A pharmaceutical composition for the prevention or treatment of inflammatory diseases comprising nanoparticles having a micelle structure in which hydrophobic manganese oxide is supported on a core, wherein the micelle structure is formed by an amphiphilic molecule in which hyaluronic acid and a hydrophobic aliphatic hydrocarbon are bonded by an active oxygen-degrading linker.

[0006] 2. The above pharmaceutical composition for the prevention or treatment of inflammatory diseases, wherein the hyaluronic acid has a molecular weight of 10 to 500 kDa.

[0007] 3. A pharmaceutical composition for the prevention or treatment of inflammatory diseases, wherein the hydrophobic aliphatic hydrocarbon is at least one selected from the group consisting of hexadecylamine, heptadecylamine, octadecylamine, stearamine, nonadecylamine, dodecylamine, bis(2-hydroxyethyl)laurylamine, linoleic acid, linolenic acid, palmitic acid, oleylamine, hexadecanoic acid, stearic acid, oleic acid, eicosencic acid, and erucic acid.

[0008] 4. In the above 1, the active oxygen-degrading linker is a thioketal linker, a TSPBA linker (N 1 -(4-boronobenzyl)-N 3 -(4-boronophenyl)-N 1 ,N 1 ,N 3 ,N 3 A pharmaceutical composition for the prevention or treatment of inflammatory diseases, comprising -tetramethylpropane-1,3-diaminium) or an AA linker (aminoacrylate).

[0009] 5. A pharmaceutical composition for the prevention or treatment of inflammatory diseases, wherein the hydrophobic manganese oxide is bonded to the surface of the manganese oxide with at least one hydrophobic substituent selected from the group consisting of a substituted or unsubstituted C1 to C30 alkyl group, a substituted or unsubstituted C3 to C30 cycloalkyl group, a substituted or unsubstituted C6 to C30 aryl group, a substituted or unsubstituted C2 to C30 heteroaryl group, a halogen group, a C1 to C30 ester group, and a halogen-containing group.

[0010] 6. A pharmaceutical composition for the prevention or treatment of inflammatory diseases, wherein, in accordance with 1 above, the amphiphilic molecule has the structure of the following chemical formula 1 or 2:

[0011] [Chemical Formula 1]

[0012]

[0013] [Chemical Formula 2]

[0014] .

[0015] 7. A pharmaceutical composition for the prevention or treatment of an inflammatory disease, wherein the inflammatory disease described in 1 above is at least one selected from the group consisting of gout, gouty nodules, gouty attack, gouty arthritis, gouty nephrolithiasis, gouty nephropathy, multiple sclerosis, ankylosing spondylitis, rheumatoid arthritis, lupus, fibromyalgia, psoriasis, asthma, atopy, Crohn's disease, ulcerative colitis, acute or chronic inflammatory disease, osteoarthritis, osteoporosis, endotoxemia, toxic shock syndrome, tendinitis, tenosynovitis, and syndrome.

[0016] The composition according to the present invention has an excellent effect of suppressing reactive oxygen species that are overexpressed in an inflammation-inducing environment.

[0017] The composition according to the present invention targets M1 macrophages that cause inflammation, so it has an excellent therapeutic effect at the site of inflammation.

[0018] The composition according to the present invention induces M0 repolarization of M1 macrophages and polarization of M2 macrophages, thereby having an excellent effect in alleviating inflammation.

[0019] Figure 1 is a chemical schematic of HTO micelle synthesis. TK is first modified with octadecylamine and then with ethylenediamine to produce the intermediate precursor NTO, and then hyaluronic acid is conjugated through a covalent EDC / NHS reaction.

[0020] Figure 2 is the result of an NMR spectrum showing a specific peak of HTO.

[0021] Figure 3 shows the FT-IR analysis results of HTO, HA, NTO, O, and TK.

[0022] Figure 4 shows the average hydrodynamic size and surface charge of HTO and HTO-MnO nanoparticles.

[0023] Figure 5 shows the results of HTO-MnO nanoparticles showing stability in three media: water, PBS, and 10% FBS.

[0024] Figure 6 shows the NTA size analysis results of HTO (top) and HTO-MnO (bottom).

[0025] Figure 7 is a schematic diagram of the ROS reaction behavior and cleavage of the TK linker, and the release of MnO loaded on HTO-MnO as a result.

[0026] Figure 8 is a TEM image of HTO (left) and HTO-MnO (right).

[0027] Figure 9 shows TEM images of HTO (left) and HTO-MnO (right) after exposure to H2O2 (hydrogen peroxide). The original nanomicelle structure collapses and larger aggregates are formed, exhibiting behavior in response to the presence of hydrogen peroxide.

[0028] Figure 10a shows the result of elemental mapping of metallic MnO in HTO-MnO before H2O2 treatment.

[0029] Figure 10b shows the result of elemental mapping of metallic MnO in HTO-MnO after H2O2 treatment. MnO is released from HTO due to the decomposition of TK linkers.

[0030] Figures 11a and 11b show the results of analyzing the catalytic activity of HTO and HTO-MnO for H2O2 decomposition. Compared to HTO, HTO-MnO showed a significantly reduced fluorescence intensity, indicating active catalysis of H2O2 decomposition and increased oxygen production.

[0031] Figure 12 shows the results of analyzing the H2O2 decomposition activity of HTO and HTO-MnO. HTO-MnO was confirmed to have H2O2 removal ability as its fluorescence intensity was significantly reduced compared to HTO.

[0032] Figure 13a shows the results of the cytotoxicity evaluation of HTO-MnO.

[0033] Figures 13b–13c show the results of the cytotoxicity evaluation of HTO and MnO, respectively.

[0034] Figures 14a–14c show the results of nitric oxide (NO) emission from MnO, HTO, and HTO-MnO, respectively. High NO emission was observed in the HTO-MnO nanoparticle treatment group, confirming the synergy between the HTO micelles and MnO according to the present invention (*p<0.5, **p<0.01, ***p<0.001, and p<0.0001).

[0035] Figures 15a and 15b show the results of quantifying the H2O2 decomposition ability (ROS values) of MnO, HTO, and HTO-MnO using the DCF-DA assay method, and the DCF-DA results as inversion microscope imaging. In the treatment groups containing manganese oxide (MnO and HTO-MnO), the fluorescence intensity was significantly reduced, confirming the catalase-mimicking ability to reduce H2O2 (*p<0.5, **p<0.01, ***p<0.001, and p<0.0001).

[0036] Figures 16a and 16b show the results of cellular uptake of MnO, HTO, and HTO-MnO and the quantification of the number of M1 macrophages. The number of M1 macrophages decreased significantly with HTO and HTO-MnO (*p<0.5, **p<0.01, ***p<0.001, and p<0.0001).

[0037] Figures 16c and 16d show the results of comparing cell uptake and quantifying the number of M1 macrophages after treating macrophages cultured with MSU with HA of various molecular weights. When treated with 37 kDa HA, the number of M1 macrophages decreased significantly (*p<0.5, **p<0.01, ***p<0.001, and p<0.0001).

[0038] Figures 17a and 17b show the effects of MnO, HTO, and HTO-MnO on the gene expression levels of the inflammatory cytokines interleukin-1β and iNOS in macrophages. A significant decrease in the mRNA expression of iNOS and IL-1β is confirmed by the synergy of HTO-MnO (ns: No significance; *p<0.05, **p<0.01, **p<0.001, and ****p<0.0001).

[0039] Figures 18a and 18b show the effects of MnO, HTO, and HTO-MnO on the protein expression levels of the inflammatory cytokines interleukin-1β and iNOS in macrophages. A significant reduction in the expression of iNOS and IL-1β is confirmed by the synergy of HTO-MnO (ns: No significance; *p<0.05, **p<0.01, **p<0.001, and ****p<0.0001).

[0040] Figures 19a and 19b show fluorescence images and quantification results regarding the effects of MnO, HTO, and HTO-MnO on the inflammatory cytokine iNOS in macrophages. Blue represents DAPI staining the nucleus, red represents the phalloidin cytoskeleton, and green represents the expression level of iNOS. A significant decrease in iNOS expression is confirmed by the synergy of HTO-MnO (*p<0.05, **p<0.01, **p<0.001, and ****p<0.0001).

[0041] Figures 19c and 19d show the effects of MnO, HTO, and HTO-MnO on the inflammatory cytokine interleukin-1β in macrophages, observed by fluorescence microscopy. Blue represents the expression level of DAPI staining the nucleus, red represents the phalloidin cytoskeleton, and green represents the expression level of IL-1β. A significant decrease in IL-1β expression is confirmed by the synergy of HTO-MnO (ns: No significance; *p<0.05, **p<0.01, **p<0.001, and ****p<0.0001).

[0042] Figure 20a shows the effects of MnO, HTO, and HTO-MnO on IκBα expression, which inhibits the NF-κB transcription factor in macrophages. It is confirmed that IκBα levels were significantly increased due to the synergy of HTO-MnO (*p<0.05, **p<0.01, **p<0.001, and ****p<0.0001).

[0043] Figure 20b shows the effects of MnO, HTO, and HTO-MnO on the expression of p65, a constituent protein of NF-κB, in macrophages. It is confirmed that p65 levels were significantly reduced due to the synergy of HTO-MnO (*p<0.05, **p<0.01, **p<0.001, and ****p<0.0001).

[0044] Figure 20c shows the results of confocal microscopy analysis confirming that the effect of p65 migration to the nucleus and increased expression due to IκBα reduction in macrophages was high in the HTO-MnO treatment group.

[0045] Figure 20d shows the quantification results of the upper confocal microscopy analysis (*p<0.05, **p<0.01, **p<0.001, and ****p<0.0001).

[0046] Figure 21a is the IVIS experimental plan for an MSU-induced gouty arthritis mouse model.

[0047] Figure 21b is an in vivo imaging system image showing the luminescence levels of intrinsic H2O2 after treating a mouse model of gouty arthritis with MnO, HTO, and HTO-MnO. The luminescence levels were significantly reduced in the HTO-MnO treatment group compared to the individual treatments of MnO and HTO.

[0048] Figure 22a is an experimental plan for the alleviation of ankle edema in a mouse model of gouty arthritis by treatment with MnO, HTO, and HTO-MnO.

[0049] Figure 22b shows ankle images of a mouse model of gouty arthritis before and after treatment with MnO, HTO, and HTO-MnO.

[0050] Figure 22c shows the results of a quantitative analysis of the difference in ankle edema calculated using a vernier caliper after treatment with MnO, HTO, and HTO-MnO. The HTO-MnO treatment group showed a significantly higher rate of ankle edema relief.

[0051] Figure 23 shows the results of analyzing immune cell infiltration after treating a mouse model of gouty arthritis with MnO, HTO, and HTO-MnO. Immune cell infiltration was significantly reduced in the HTO-MnO treatment group.

[0052] Figure 24a shows images (fluorescent probe 20 μM) and quantification results of iNOS expression analysis after treatment with MnO, HTO, and HTO-MnO in a mouse model of gouty arthritis (*p<0.05, **p<0.01, **p<0.001, and ****p<0.0001).

[0053] Figure 24b shows images (fluorescent probe 20 μM) and quantification results of analyzing the number of CD86 M1 macrophages after treatment with MnO, HTO, and HTO-MnO in a mouse model of gouty arthritis (*p<0.05, **p<0.01, **p<0.001, and ****p<0.0001).

[0054] Figure 24c shows images (fluorescent probe 20 μM) and quantification results analyzing the number of CD206 M2 macrophages after treatment with MnO, HTO, and HTO-MnO in a mouse model of gouty arthritis (ns: No significance; *p<0.05, **p<0.01, **p<0.001, and ****p<0.0001).

[0055] Figure 25 shows the results of MSU, MnO, HTO, and HTO-MnO treatment in a mouse model of gouty arthritis, showing no damage to other major organs.

[0056] Figures 26a and 26b are schematic diagrams of the synthesis of micelle nanoparticles, ROS-based MnO release, and the alleviation of gouty arthritis severity by HTO-MnO nanoparticles.

[0057] The present invention relates to a pharmaceutical composition for the prevention or treatment of inflammatory diseases comprising micelle nanoparticles.

[0058] In the present invention, 'micelle nanoparticles' have a micelle structure in which hydrophobic manganese oxide is supported on a core, and the micelle structure may be a nanoparticle formed by an amphiphilic molecule in which hyaluronic acid and a hydrophobic aliphatic hydrocarbon are combined by an active oxygen decomposition linker.

[0059] The present invention provides a pharmaceutical composition for the prevention or treatment of inflammatory diseases comprising nanoparticles having a micelle structure in which hydrophobic manganese oxide is supported on a core, wherein the micelle structure is formed by an amphiphilic molecule in which hyaluronic acid and a hydrophobic aliphatic hydrocarbon are bonded by an active oxygen-degrading linker.

[0060] In the present invention, the hydrophobic manganese oxide is treated such that hydrophobic substituents are present on the surface of the manganese oxide so that the manganese oxide can be stably supported inside the micelles.

[0061] The hydrophobic substituent may be at least one selected from the group consisting of, for example, a substituted or unsubstituted C1 to C30 alkyl group, a substituted or unsubstituted C3 to C30 cycloalkyl group, a substituted or unsubstituted C6 to C30 aryl group or a substituted or unsubstituted C2 to C30 heteroaryl group, a halogen group, a C1 to C30 ester group and a halogen-containing group.

[0062] Hydrophobic substituents can be substituted with, for example, halogen atoms, nitro, hydroxy, cyano, amino, thiol, carboxyl, amide, nitrile, sulfide, disulfide, sulfphenyl, formyl, formyloxy, formylamino, formylamino, and aryl.

[0063] Manganese oxide may have hydrophobic substituents on its surface by treating, for example, 1-dodecanethiol, 1-octadecanethiol, or 1-hexanediol.

[0064] In one embodiment, the surface of the manganese oxide may be modified with a hydrophobic substituent by 1-dodecanethiol treatment.

[0065] Manganese oxide may be any one of MnO, Mn2O3, MnO2, Mn3O4, and Mn2O5, but is not limited thereto.

[0066] Manganese oxide particles can be stably supported inside micelles by surface-modifying them with hydrophobic substituents as described above. The manganese oxide particles may be distributed throughout the entire hydrophobic region inside the micelle or may be supported on the core. It is more preferable for the manganese oxide to be supported on the core so that it can circulate stably within the body and be released at the site of inflammation.

[0067] Hydrophobic manganese oxide particles may have a particle size of 10 to 30 nm. When the hydrophobic manganese oxide falls within the above particle range, it can be evenly distributed and stably supported inside the micelles, and its reactivity with active oxygen at the target site can be increased, thereby increasing oxygen generation.

[0068] Hydrophobic manganese oxide may be included in an amount of 5 to 15, more preferably 7 to 12, parts by weight per 100 parts by weight of the amphiphilic molecule. When hydrophobic manganese oxide is included in micelles within the above range, the micelles can stably capture hydrophobic manganese oxide and effectively release hydrophobic manganese oxide at the site of inflammation. In addition, the inclusion of hydrophobic manganese oxide in an appropriate amount can increase the amount of oxygen generated by the hydrophobic manganese oxide released at the target site, thereby changing the microenvironment of the target site and enhancing the effect of the bioactive substance.

[0069] In the present invention, hyaluronic acid (HA) includes what is referred to as hyaluronan, hyaluronate, or sodium hyaluronate. In the present invention, hyaluronic acid may be referred to interchangeably as 'HA'.

[0070] Hyaluronic acid is a type of complex polysaccharide composed of amino acids and uronic acid, and is a high-molecular-weight compound consisting of N-acetylglucosamine and glucuronic acid. It is a hydrophilic substance due to its high hydroxyl group (-OH) content and is known to play a role in moisturizing the skin of animals and preventing bacterial invasion or the penetration of toxins. It is found in the extracellular matrix and cell surfaces of most human tissues and is therefore used as a biomaterial, such as a drug delivery system or a scaffold for tissue engineering, because it has excellent biocompatibility and is biodegradable by hyaluronidase, an enzyme found in the blood.

[0071] The hyaluronic acid used to form the micelle structure of the nanoparticles according to the present invention may have a molecular weight in the range of 10 to 500 kDa. Preferably, it may be 10 to 400 kDa, or 10 to 300 kDa, or 10 to 200 kDa, or 10 to 100 kDa. More preferably, it may have a molecular weight range of 10 to 50 kDa. In the above range, the binding ability with CD44 receptors may be increased.

[0072] In the present invention, the hydrophobic aliphatic hydrocarbon is a hydrocarbon group derived from a hydrophobic hydrocarbon compound, and may have an aliphatic hydrocarbon group having 12 to 22 carbon atoms, or may have a straight-chain aliphatic hydrocarbon group.

[0073] Hydrophobic aliphatic hydrocarbons may be at least one selected from the group consisting of hexadecylamine, heptadecylamine, octadecylamine, stearamine, nonadecylamine, dodecylamine, bis(2-hydroxyethyl)laurylamine, linoleic acid, linolenic acid, palmitic acid, oleylamine, hexadecanoic acid, stearic acid, oleic acid, eicosencic acid, and erucic acid, but not limited thereto.

[0074] The hydrophobic aliphatic hydrocarbon compound may have a weight average molecular weight in the range of 100 to 500 g / mol, preferably 150 to 350 g / mol, and more preferably 180 to 300 g / mol, but is not limited thereto.

[0075] As hydrophobic aliphatic hydrocarbon groups form a hydrophobic core, the hydrophobic core can possess a certain degree of fluidity. This fluidity offers an advantageous characteristic compared to hydrophobic cores formed by hydrophobic polymers, particularly in that it allows for the rapid release of hydrophobic manganese oxide located within the core in response to reactive oxygen species (ROS). By forming a hydrophobic core with long-chain aliphatic hydrocarbon groups, sufficient hydrophobicity is provided to the core to stably support hydrophobic manganese oxide while maintaining a certain degree of fluidity. Consequently, the ROS-degradable linker becomes sensitive to ROS surrounding the target tissue, reacting with them to easily degrade.

[0076] In addition, compared to the case where a large hydrophobic polymer is used, even if the size of the micelles formed by self-assembly in the aqueous phase is relatively small, a sufficient amount of hydrophobic manganese oxide can be stably captured in the hydrophobic core due to a certain degree of fluidity of the aliphatic hydrocarbon group.

[0077] In the present invention, the reactive oxygen species-degradable (ROS-labile) linker is a linker that connects hydrophilic hyaluronic acid with hydrophobic aliphatic hydrocarbons, and has high sensitivity to reactive oxygen species, so that the bond is broken upon reaction with reactive oxygen species.

[0078] Reactive oxygen species degrading linkers are, for example, thioketal linkers and TSPBA linkers (N 1 -(4-boronobenzyl)-N 3 -(4-boronophenyl)-N 1 ,N 1 ,N 3 ,N 3 It may be -tetramethylpropane-1,3-diaminium) or AA linker (aminoacrylate).

[0079] In one embodiment, the nanoparticle may be an amphiphilic polymer compound in which hyaluronic acid and a hydrophobic aliphatic hydrocarbon group are covalently bonded to a thioketal linker (TL). Specifically, one end of the thioketal linker may be modified to ethylenediamine or 1,3-propylenediamine and then covalently bonded to hyaluronic acid, while the other end is covalently bonded to the amide group of the hydrophobic aliphatic hydrocarbon group, and self-assembles in the aqueous phase to form micelles. The thioketal linker is a linker that connects a water-soluble polymer and a hydrophobic hydrocarbon group, and has very high sensitivity to reactive oxygen species and excellent binding stability under normal physiological conditions, which can prevent the early leakage of hydrophobic manganese oxide particles from the micelles during circulation in the blood.

[0080] In the present invention, the amphiphilic molecule may have a structure of the following chemical formula 1 or 2:

[0081] [Chemical Formula 1]

[0082]

[0083] [Chemical Formula 2]

[0084] .

[0085] The amphiphilic molecule having the structure of Chemical Formula 1 above is a molecule in which one end of the thioketal linker is modified into ethylenediamine and then covalently bonded to hyaluronic acid.

[0086] The amphiphilic molecule having the structure of Chemical Formula 2 above is a molecule in which one end of the thioketal linker is modified to 1,3-propylenediamine and then covalently bonded to hyaluronic acid.

[0087] The micelle nanoparticles according to the present invention may have a diameter of 50 to 400 nm. Preferably, they may have a diameter of 100 to 300 nm or 150 to 250 nm. Although not limited thereto, within the above ranges, circulation in vivo is facilitated, and the decomposition of thioketal links by reactive oxygen species and the release of hydrophobic manganese oxide can be effectively achieved.

[0088] In the present invention, 'inflammatory disease' refers to any disease mediated by inflammation. For example, it may be at least one selected from the group consisting of gout, gouty nodules, gouty attacks, gouty arthritis, gouty nephrolithiasis, gouty nephropathy, multiple sclerosis, ankylosing spondylitis, rheumatoid arthritis, lupus, fibromyalgia, psoriasis, asthma, atopic dermatitis, edema, dermatitis, allergy, atopy, Crohn's disease, ulcerative colitis, acute or chronic inflammatory diseases, renal failure, conjunctivitis, periodontitis, rhinitis, otitis media, pharyngitis, tonsillitis, pneumonia, gastritis, cystitis, hepatitis, osteoarthritis, osteoporosis, endotoxemia, toxic shock syndrome, tendinitis, tenosynovitis, and polydactyly. It is not limited thereto. It is known that CD44 receptors are overexpressed in inflammatory diseases, and recently, there have been reports that the progression of inflammation can be inhibited by injecting CD44 antibodies that can specifically bind to these CD44 receptors into the body. Therefore, by using the micelle nanoparticles of the present invention to specifically bind to CD44 receptors, it is possible to effectively prevent or treat diseases mediated by inflammatory responses.

[0089] The pharmaceutical composition of the present invention may further include various bioactive substances within the micelle. The bioactive substances may be released together with hydrophobic manganese oxide at the hypoxic site of the target area, and may exhibit a higher effect due to the tissue microenvironment altered by oxygen evolution induced by hydrophobic manganese oxide.

[0090] The bioactive substance is a bioactive substance / biological function regulator that can be loaded inside micelles and delivered to an organism to exhibit activity, and may be at least one selected from the group consisting of low molecular weight drugs, gene drugs, protein drugs, extracts, nucleic acids, nucleotides, proteins, peptides, antibodies, antigens, RNA, DNA, PNA, aptamers, chemicals, enzymes, amino acids, sugars, lipids, compounds (natural compounds and / or synthetic compounds) and components constituting these.

[0091] The bioactive substance may be a therapeutic agent capable of providing direct or indirect therapeutic, physiological, and / or pharmacological effects to a human or animal organism. The therapeutic agent may be, for example, a general medicine, a drug, a prodrug or target group, or a drug or prodrug containing a target group.

[0092] The pharmaceutical composition of the present invention may be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy, and biological response modifiers for the prevention or treatment of inflammatory diseases.

[0093] When formulating the pharmaceutical composition of the present invention, it may be prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Solid dosage forms for oral administration include tablets, pills, powders, granules, and capsules, and these solid dosage forms may be prepared by mixing at least one excipient with the above compounds, for example, starch, calcium carbonate, sucrose or lactose, gelatin, etc. In addition, lubricants such as magnesium, stearate, and talc may be used in addition to simple excipients. Liquid dosage forms for oral administration include suspensions, liquid formulations, emulsions, and syrups, and may include various excipients, for example, wetting agents, sweeteners, flavoring agents, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used as non-aqueous solvents and suspensions. Witepsol, macrogol, Tween 60, cacao oil, laurin oil, glycerozelatin, etc. may be used as bases for suppositories.

[0094] The pharmaceutical composition of the present invention may be administered orally or parenterally (e.g., intravenously, subcutaneously, intraperitoneally, or topically) depending on the intended method, and the dosage may vary depending on the patient's health condition, body weight, age, gender, diet, excretion rate, severity of disease, drug form, time of administration, route of administration, and duration of administration, but may be appropriately selected by those skilled in the art. However, for a desirable effect, the hyaluronic acid nanoparticles of the present invention may be administered at a dose of 0.1 mg / kg (body weight) to 30 mg / kg (body weight), 0.1 mg / kg (body weight) to 20 mg / kg (body weight), or 1 mg / kg (body weight) to 10 mg / kg (body weight) based on the daily active ingredient, and the administration may be performed once a day or divided into several doses. The above dosage does not limit the scope of the present invention in any way.

[0095] The present invention will be described in detail below through examples. However, these are presented as preferred examples of the present invention and should not be interpreted as limiting the present invention.

[0096] Details not listed here can be sufficiently technically inferred by a person skilled in this field, so their explanation will be omitted.

[0097]

[0098] Preparation Example

[0099]

[0100] 1. HTO Nanomicelle Fabrication

[0101] The chemical scheme of HTO nanomicelle synthesis is shown in Figure 1.

[0102]

[0103] STEP 1: Production of Thioketal Linker (TK)

[0104] 49 mmol of 3-mercaptopropionic acid was dissolved in anhydrous acetone (98 mmol) in a 1:2 ratio and stirred with dry hydrochloric acid at room temperature for about 6 hours. The product was cooled with ice and salt until crystallized, then filtered, washed with n-hexane, washed once more with cold distilled water (DW) using a paper filter, and freeze-dried to obtain thioketal linker (hereinafter referred to as TK).

[0105]

[0106] STEP 2: Preparation of Aminethioketal Octadecylamine (NTO)

[0107] A mixed solution was prepared by mixing EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide), NHS (N-hydroxysuccinimide), and octadecylamine in a molal ratio of 2:2:1. 100 mg of thioketal linker obtained in STEP 1 was dissolved in an aqueous solution of DMF (N,N-Dimethylformamide) and reacted at 100°C for 3 hours. Subsequently, the mixed solution was added and the reaction was carried out for an additional 3 hours. The reaction product was diluted 10-fold in distilled water and dialyzed for 24 hours under a molecular weight cutoff (MWCO) of 12-14 kDa to remove reaction byproducts, and then freeze-dried to obtain amine thioketal octadecylamine (hereinafter referred to as NTO).

[0108]

[0109] STEP 3: Preparation of Hyaluronic Acid Thioketal Linker Octadecylamine (HTO)

[0110] The NTO obtained in STEP 2 above was synthesized through a covalent EDC / NHS reaction with hyaluronic acid (HA). A warm aqueous formamide solution containing 100 mg of hyaluronic acid (weight average MW: 37 kDa) dissolved in EDC and NHS at a 1:1 molal ratio was reacted with NTO dissolved in 5 mL of DMF for 3 hours. The product was lyophilized after dialyzing with PBS (pH 7.4) for 24 hours and then dialyzing with distilled water for an additional 48 hours to obtain hyaluronic acid thioketal linker octadecylamine (hereinafter referred to as HTO).

[0111]

[0112] 2. Preparation of Hydrophobic Manganese Oxide (MnO)

[0113] A distilled water solution containing 4 mmol of dissolved sodium hydroxide (NaOH) was added dropwise to 2 mmol of manganese chloride (MnCl2) while stirring. Then, 1-dodecanethiol was added in a half-molar ratio, the precipitate was collected using a centrifuge, and the precipitate was washed several times with ethanol and water to obtain hydrophobic manganese oxide (hereinafter referred to as MnO).

[0114]

[0115] 3. Preparation of Hydrophobic Manganese Oxide-Hyaluronic Acid Nanoparticles (HTO-MnO)

[0116] HTO-MnO was synthesized using an oil-in-water (O / W) emulsion method. MnO and HTO were dissolved in dimethyl sulfoxide (DMSO) at a ratio of 1:10 and slowly added dropwise to 10 mL of distilled water using continuous probe sonication. The resulting mixture was continuously stirred for 24 hours, then dialyzed with distilled water using an MWCO 10-12 kDa membrane, and finally hydrophobic manganese oxide-hyaluronic acid nanoparticles (hereinafter referred to as HTO-MnO) were obtained by freeze-drying.

[0117]

[0118] 4. Synthesis of Needle-shaped Monosophium Ilorate (MSU) Crystals

[0119] MSU crystals were synthesized by alkaline titration. 4 g of uric acid was dissolved in 800 mL of distilled water at a temperature of 60°C. The pH was adjusted to 8.9 using a 0.5 M sodium hydroxide solution, and the temperature was maintained at 60°C until the uric acid was completely dissolved. After obtaining a clear solution, the reaction was stopped and incubated at 40°C for 24 hours. Subsequently, the white precipitate was transferred and dried at 100°C for 6 hours. The obtained crystalline monosophium yorate (hereinafter referred to as MSU) was purified at 180°C before use.

[0120]

[0121] Experimental method

[0122]

[0123] 1. Cell viability

[0124] In vitro cell viability was used to evaluate the toxicity of HTO, MnO, and HTO-MnO using RAW264.7 cells. Cells were seeded into 96-well plates at a concentration of 1×10⁴ cells / well in 100 μL of DMEM medium supplemented with 10% FBS and 1% antibiotics and cultured for 12 hours. Cells were treated with various concentrations of HTO, MnO, and HTO-MnO in 100 μL of DMEM medium, and cytotoxicity profiles were examined using the WST-1 assay after 24 hours.

[0125]

[0126] 2. Cell uptake study

[0127] Cell uptake was investigated by examining intracellular uptake using the IR780 fluorescent probe. IR780 was loaded onto HTO nanomicels instead of MnO. Cell uptake was evaluated by inoculating cells into 8-well chambers and 6-well plates. After cell inoculation, the cells were exposed to MSU (1 μg / mL) for 12 hours to induce the development of M1 phenotype macrophages.

[0128] PEG-based PTO-IR780 was used as a control to confirm HA-induced cellular uptake. After washing the cells, they were exposed to IR780, HTO-IR780, and PTO-IR780 for 2 hours. Cells were pre-exposed for 4 hours to demonstrate HA-promoted CD44-mediated cellular uptake. After 4 hours, the cells were rinsed three times with DPBS and then treated with 4% paraformaldehyde for 15 minutes. Subsequently, the samples were fixed with ProLong Gold Antifade reagent containing DAPI, and cellular uptake was observed using an inverted fluorescence microscope. To quantify cellular uptake, cells were seeded into a 6-well chamber, treated with 4% paraformaldehyde, and analyzed using a flow cytometer. This demonstrated the cellular internalization of the stains by an indirect means. To demonstrate direct cellular uptake, M1 macrophages were exposed to PTO-MnO and HTO-MnO for 2 and 4 hours, respectively. Subsequently, cell samples were sampled after centrifugation and acid digestion with aqua regia for ICP-MS analysis.

[0129]

[0130] 3. Intracellular ROS Analysis

[0131] Intracellular ROS measurements were performed using 2,7-dichlorofluorescein diacetate (DCFH-DA). RAW264.7 cells treated with 100 μg / mL of MSU were cultured in 24-well plates for 12 hours, followed by treatment with various concentrations of HTO, MnO, and HTO-MnO for 24 hours. The cells were then exposed to 15 μM of DCFH-DA for 40 minutes and washed with a wash solution. The DCFH-DA signal at an emission wavelength of 530 nm was quantified using a multi-plate reader.

[0132]

[0133] 4. Flow cytometry

[0134] Flow cytometry was performed using antibodies purchased from Biolegend. M1 macrophages repolarized to return to a non-inflammatory M0 macrophage state were analyzed using flow cytometry, and CD11b (APC) and CD86 (PE-A) antibodies were used. RAW cells were cultured in 90 mm dishes and treated with 100 μg / mL MSU for 12 hours. Subsequently, they were exposed to HTO, MnO, and HTO-MnO for 24 hours. Cells were treated with antibodies for 45 minutes after trypsinization, fixed with 2% paraformaldehyde, and subjected to flow cytometry. Data were analyzed using FlowJo software.

[0135]

[0136] 5. Nitric Oxide (NO) Analysis

[0137] Nitric oxide analysis was performed by quantifying the levels of nitrite, a metabolite of nitric oxide, in the cell culture medium using the grease reagent method. RAW264.7 cells were seeded into 24-well plates at a density of 5 × 10⁴ cells per well and exposed to 100 μg / mL MSU for 12 hours. After 12 hours, cells were additionally treated with various concentrations of HTO, MnO, and HTO-MnO for 24 hours, followed by incubation of 100 μL of the cell supernatant with an equal amount of grease reagent for 30 minutes. Nitrite concentrations were measured at a wavelength of 540 nm using a BioTek visible light spectrometer (Agilent Technologies, USA). Nitric oxide levels within each treatment group were calculated using a sodium nitrite production standard curve.

[0138]

[0139] 6. PCR

[0140] 1 × 10⁶ RAW264.7 cells per well 5Cells were inoculated into 6-well plates at a certain density. Cell RNA was extracted using RNAiso Plus (TaKaRa, Japan), and complementary DNA (cDNA) was synthesized using RT premix (AccuPower, Biomeer) according to the manufacturer's protocol. Subsequently, PCR mastermix (AccuPower, Biomeer) was used as a template for PCR amplification. The PCR conditions consisted of an initial denaturation step at 95°C for 5 minutes, denaturation at 95°C for 1 minute, annealing for 30 seconds at a temperature determined by the specific primers used, extension at 72°C for 1 minute, and a final extension step at 72°C for 5 minutes, followed by storage at 4°C.

[0141]

[0142] 7. Western Blot

[0143] Proteins were extracted using RIPA lysis buffer supplemented with protease and phosphatase inhibitors, and the protein fractions were separated by centrifugation at 12,000 rpm for 20 minutes at 4°C. NE-PER nuclear and cytoplasmic extraction reagents were used to extract cytoplasmic and nuclear proteins, following the manufacturer's protocol. Subsequently, equal amounts of the extracted proteins were loaded onto an SDS-PAGE gel, separated by electrophoresis, and transferred to a PVDF membrane. The membrane was incubated in 5% skim milk at room temperature for 1 hour to block non-specific binding sites. It was then exposed overnight at 4°C with primary antibodies targeting inducible nitric oxide synthase (iNOS), interleukin-1β, p65, lamin B1, IKBα, and β-actin. After primary antibody culture, iNOS and β-actin were treated with goat anti-mouse IgG, and interleukin-1β was treated with mouse anti-Armenia hamster antibodies for 2 hours at room temperature. Protein bands were visualized using Immobilon Chemiluminescent substrate.

[0144]

[0145] 8. Immunofluorescence and Confocal Imaging

[0146] 5×10⁶ RAW264.7 cells in a 6-well plate 4 Cells were inoculated at a density of cells / mL. After treatment with MSU, HTO, Mn, and HTO-MnO, the cell culture medium was aspirated, washed with phosphate-buffered saline (PBS), and fixed in 4% paraformaldehyde for 20 minutes. Subsequently, the cells were washed again with PBS, permeated with 0.5% Triton X-100 for 15 minutes, and incubated in 5% bovine serum albumin (BSA) solution for 1 hour to block non-specific binding sites, followed by washing with PBS. Afterward, the cells were exposed to primary antibodies diluted 1:500 against iNOS, IL-1β, and p65 for 1 hour.

[0147] Next, the samples were washed with PBS, treated with a fluorescently labeled secondary antibody, and nuclear stained with DAPI for 5 minutes. Finally, the samples were attached to a 6-well plate with coverslips and examined at 20x magnification using an Agilent BioTek Lionheart FX fluorescence imaging system and at 40x magnification using a ZEISS LSM 700 confocal fluorescence microscope.

[0148]

[0149] 9. Treatment of gouty arthritis

[0150] In vivo animal experiments were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC; Project No.: IACUC-H-2022-28) of Chonnam National University College of Medicine, and all in vivo experiments were conducted in accordance with relevant regulations. Six-week-old male C57BL / 6 mice were purchased from the Korea Orient Research Institute. Gouty arthritis was induced in mice by directly administering 100 mg / kg of MSU crystals in 50 mL of PBS into the ankle joints. After 12 hours, the fabricated nanoparticles were administered, and joint edema was monitored for up to 96 hours. Joint edema was evaluated using vernier calipers, and after 12 hours of HTO-MnO treatment, L012 (100 mg / kg, 200 μL) was injected intraperitoneally into the mice, and observation was performed using the NightOWL II LB 983 IVIS system.

[0151]

[0152] Experimental Example

[0153]

[0154] 1. Characterization of HTO and HTO-MnO nanoparticles

[0155] Characteristic peaks appeared in the HTO molecule prepared by the method of Preparation Example 3, confirming the presence of specific parts. In 1H NMR, the chemical shift at δ 1.23 ppm corresponds to the methylene group (CH2CH2CH2), and the shift at δ 0.83 ppm corresponds to the terminal methyl group (CH3) of the C18 chain. Additionally, a characteristic peak of the acetamido portion (-NHCOCH3) in the N-acetyl-d-glucosamine residue of hyaluronic acid was observed at δ 1.95 ppm (Fig. 2). The NH proton of the amide group may appear in the range of 5-9 ppm, and as a result of comparing the intensity of specific signals in the NMR spectrum to confirm how much octadecylamine (long chain molecule) is attached to the polymer, approximately 80% of the attachment sites were identified as octadecylamine.

[0156] FT-IR analysis was performed to repeatedly confirm whether NTO and HA were successfully attached to both ends of the TK molecule, and SC and C=O bonds, which are part of the amide structure, were identified. When the FT-IR spectrum of the HTO polymer was compared with that of HA and NTO alone, a peak confirming CH bonds was found. The CMC of HTO was calculated to be 10.03 μg / mL (Fig. 3).

[0157] The average hydrodynamic size and surface charge were determined using DLS. HTO has an average size of approximately 193±12.3 nm and -23.47 mV, while HTO-MnO has an average size of approximately 212±31.3 nm and -22.76 mV (Fig. 4).

[0158] The stability of the nanoparticles was tested for 72 hours in three different media: water, PBS, and 10% FBS, and they were found to be stable in all three media without significant changes in size (Fig. 5).

[0159] The atomic percentage of Mn present in MnO was analyzed using EDAX and XPS, and according to ICP-MS analysis, the amount of MnO loaded in HTO-MnO was calculated to be ~5.8%.

[0160] NTA size analysis of HTO-MnO showed that the number of particles in 5 mg / mL HTO-MnO was 2.9E+10 particles / mL, in 2.5 mg / mL HTO-MnO was 1.5E+9 particles / mL, and in 1.25 mg / mL HTO-MnO was 9.2E+8 particles / mL (Fig. 6).

[0161]

[0162] 2. Analysis of ROS Reaction Behavior and Hydrogen Peroxide Decomposition of HTO-MnO Nanoparticles

[0163] Figure 7 is a schematic diagram showing that in an environment rich in H2O2 (hydrogen peroxide), the TK linker is cleaved by ROS reaction behavior, and as a result, MnO loaded on HTO-MnO is released.

[0164] When compared with TEM images of HTO alone and HTO-MnO (Fig. 8), both HTO and HTO-MnO nanoparticles after exposure to 1 mM H2O2 showed structural degradation in an ROS-rich environment induced by H2O2, clearly demonstrating the ROS reaction behavior of the TK linker. In particular, the size of HTO-MnO nanoparticles increased significantly to 400–900 nm upon exposure to H2O2; this size expansion suggests that the original nanomicelle structure collapsed and larger aggregates were formed, exhibiting behavior responsive to the presence of hydrogen peroxide (Fig. 9).

[0165] As a result of observing the elemental mapping of metallic MnO in HTO-MnO nanoparticles before and after H2O2 treatment, it was confirmed that MnO is released from HTO due to the decomposition of TK linkers (Figs. 10a-10b).

[0166] The catalytic activity of HTO-MnO for H2O2 decomposition was analyzed using the fluorescent probe terephthalic acid (TA). As a result, the fluorescence intensity of HTO-MnO nanoparticles decreased significantly over time compared to HTO (Fig. 11a), and the oxygen generation capacity increased (Fig. 11b).

[0167] To confirm the H2O2 decomposition activity of HTO-MnO, a concentration-dependent Ru(ddp) assay was performed in the presence of H2O2. The Ru(ddp) fluorescence intensity decreases in the presence of oxygen, and the high H2O2 removal ability of HTO-MnO nanoparticles was confirmed as the fluorescence intensity in the HTO-MnO-treated group decreased significantly compared to the HTO-alone treated group (Fig. 12).

[0168]

[0169] 3. Analysis of Anti-inflammatory Effects of MnO, HTO, and HTO-MnO Nanoparticles in In Vitro MSU-Activated Mouse Macrophages

[0170]

[0171] (1) Cytotoxicity evaluation

[0172] The antioxidant efficiency of the nanoparticles on RAW264.7 cells was evaluated. To establish the optimal dose for further therapeutic studies, the toxicity of HTO-MnO, HTO, and MnO was evaluated. HTO-MnO nanoparticles showed low cytotoxicity at concentrations below 50 μg / mL (Fig. 13a), while HTO and MnO were found to be safe with low cytotoxicity at concentrations below 150 μg / mL and below 6.25 μg / mL, respectively (Figs. 13b–13c).

[0173]

[0174] (2) Evaluation of nitric oxide (NO) reduction

[0175] NO release is a key marker for reducing inflammation and is directly proportional to the release of inflammatory cytokines. The NO reducing ability of HTO-MnO, HTO, and MnO was evaluated using the nitrate quantification method. RAW cells were treated with 100 μg / mL MSU and cultured for 12 hours, after which MnO, HTO, and HTO-MnO were treated at various concentrations for 24 hours. The supernatant was then separated and tested using Greiss reagent. The negative control (MSU) was treated with PBS. As a result, NO release was higher in the HTO-MnO nanoparticle-treated group compared to the MnO or HTO-treated groups, confirming the synergy between the HTO nanomicelles according to the present invention and MnO (Figs. 14a–14b).

[0176] Since NO can regulate ROS production by modulating enzymes such as NADPH oxidase, ROS levels were quantified using the DCF-DA assay. As a result, the group of RAW264.7 cells cultured with MSU treated with PBS showed a significant increase in ROS levels, while the MnO and HTO-MnO treatment groups showed a significant decrease in ROS levels, demonstrating their catalase-mimicking ability to reduce H2O2. The HTO treatment group showed a decrease in H2O2 levels compared to the PBS treatment group, suggesting that the TK linker within HTO reacted with H2O2, leading to a reduction in its concentration (Fig. 15a). Evaluating H2O2 reduction using average fluorescence intensity via inversion microscopy imaging of the DCF-DA results, the fluorescence intensity was significantly reduced, particularly in the treatment groups containing manganese oxide (MnO and HTO-MnO) (Fig. 15b).

[0177]

[0178] (3) Evaluation of M0 repolarization of M1 macrophages

[0179] To further analyze the cellular uptake of nanoparticles, the samples were loaded with the fluorescent dye IR780 and analyzed using a flow cytometer after 4 hours of treatment. The analysis results showed that HTO and HTO-MnO exhibited superior cellular uptake compared to the group treated with PBS (MSU) on RAW264.7 cells cultured with MSU (Fig. 16a). This indicates that increased cellular uptake is due to HA's ability to target CD44 receptors on the surface of M1 macrophages. Quantification results showed that compared to the PBS-treated group, the groups treated with HTO and HTO-MnO experienced a significant decrease in the number of M1 macrophages, with the reduction being even greater due to the synergy of HTO-MnO (Fig. 16b). HA showed differences in cellular uptake depending on its molecular weight, with a particularly significant decrease in the number of M1 macrophages observed in the experimental group treated with 37 kDa HA (Figs. 16c–16d).

[0180]

[0181] (4) Evaluation of IL-1β and iNOS inhibition

[0182] To investigate the effects of HTO, MnO, and HTO-MnO nanoparticles on MSU-induced inflammation, the levels of the inflammatory cytokines interleukin-1β and iNOS were evaluated in vitro using polymerase chain reaction (PCR) and Western blot. PCR results showed that the PBS-treated group significantly increased the mRNA expression of iNOS and IL-1β. Quantification graphs indicated that IL-1β mRNA expression decreased in all HTO, MnO, and HTO-MnO treatment groups, with a particularly synergistic effect observed in the HTO-MnO nanoparticle treatment group (Figs. 17a–17b). Furthermore, Western blot results confirmed a significant reduction in the expression of iNOS and IL-1β due to the synergy of HTO-MnO, verifying the anti-inflammatory ability of HTO-MnO nanoparticles at the protein expression level (Figs. 18a–18b).

[0183] The inhibition of iNOS and IL-1β by MnO, HTO, and HTO-MnO was also confirmed by immunofluorescence analysis, and HTO-MnO nanoparticles showed a significant anti-inflammatory effect compared to MnO and HTO (Figs. 19a-19d).

[0184]

[0185] 4. Effects of MnO, HTO, and HTO-MnO nanoparticles on the NF-κB pathway

[0186] Since the NF-κB pathway plays a crucial role in mediating the expression of inflammatory genes, we investigated the effects on the expression of IκBα and p65, which inhibit NF-κB transcription factors. RAW cells were treated with 100 μg / mL MSU and cultured for 12 hours, after which they were treated with various concentrations of MnO, HTO, and HTO-MnO for 24 hours, followed by observation of IκBα and p65 levels. PBS was used as a negative control. The results showed that while IκBα levels decreased significantly in the PBS-treated group, they increased in all MnO, HTO, and HTO-MnO-treated groups, with the increases in HTO and HTO-MnO being particularly high. This suggests that HA inhibits the degradation of IκBα. In particular, IκBα levels increased significantly in the HTO-MnO-treated group (Fig. 20a). A decrease in IκBα levels indicates an increase in the translocation of p65 to the nucleus. As p65 levels were measured via nuclear protein detection, the reduction in p65 expression was high in the HTO and HTO-MnO treatment groups, and was particularly significant in the HTO-MnO treatment group (Fig. 20b). Confocal microscopy analysis further confirmed that the reduction in IκBα in the HTO-MnO treatment group led to a high increase in p65 translocation to the nucleus and expression (Fig. 20c). These results demonstrate that the regulation of the NF-κB pathway is superior when treated with the combined form of HTO-MnO compared to individual treatments of HTO or MnO.

[0187]

[0188] 5. Analysis of Anti-inflammatory Effects of MnO, HTO, and HTO-MnO Nanoparticles in an In vivo MSU-induced Arthritis Mouse Model

[0189] Gouty arthritis was induced in a mouse model by injecting 2 mg of MSU (50 μL of PBS) into the ankle joint. Acute swelling and inflammation developed in the right sole of the foot within 12 hours of MSU injection, and this time point was established using the In-vivo Imaging System (IVIS) and the plantar swelling test. IVIS imaging was performed using Luminol solution L-012, and luminescence increased as H2O2 levels increased (Fig. 21a). Strong luminescence was observed in the PBS-treated groups, and a decrease in luminescence in the MnO, HTO, and HTO-MnO-treated groups was visualized as H2O2 levels decreased (Fig. 21b). This confirms that MnO alleviated ROS in the mouse ankle by decomposing H2O2 into water and oxygen. Additionally, luminescence was significantly reduced in the HTO-treated groups, suggesting that the TK linker reacted with H2O2, causing H2O2 levels to decrease over time in vivo. The significant reduction in luminescence and H2O2 levels in the HTO-MnO treatment group indicates a synergistic effect between MnO and HTO, thereby confirming the synergy of HTO-MnO nanoparticles.

[0190] To verify whether nanoparticle treatment reduces ankle edema in a mouse model of gouty arthritis, the same concentration of MSU was injected into the left foot. Ankle edema was measured 12 hours after MSU treatment, and then measured 96 hours after injecting MnO, HTO, and HTO-MnO, respectively (Fig. 22a). As a result, alleviation of ankle edema was confirmed in the MnO, HTO, and HTO-MnO treatment groups compared to the PBS treatment group (Fig. 22b), and quantitative data showed that nanoparticles containing HA had a high edema-reducing effect, with the HTO-MnO group showing particularly superior treatment efficacy (Fig. 22c).

[0191] H&E staining was performed to confirm immune cell infiltration in mouse models treated with MnO, HTO, and HTO-MnO. While cells were sparsely distributed in the normal control group, they were found to be densely distributed in the PBS-treated group. Given the sparse distribution of cells in the MnO, HTO, and HTO-MnO treatment groups, it can be confirmed that immune cell infiltration caused by nanoparticle treatment was significantly reduced (Fig. 23).

[0192] In mouse models treated with MnO, HTO, and HTO-MnO, the inflammatory factor iNOS, inflammation-inducing M1 macrophages, and M2 macrophages that reduce inflammation and promote tissue repair were analyzed using a confocal microscope. In the PBS-treated group, iNOS expression surged, while in the MnO, HTO, and HTO-MnO treatment groups, iNOS expression gradually decreased (Fig. 24a). This is consistent with the in vitro NO release results shown in Figs. 14a–14c. Evaluation of M1 macrophages by staining with CD86 antibody revealed that inflammatory M1 macrophages surged in the PBS-treated group, whereas the number of M1 macrophages decreased in the MnO, HTO, and HTO-MnO treatment groups (Fig. 24b). When M2 macrophages were evaluated using CD206 antibodies, the number of M2 macrophages decreased in the MSU-treated group (PBS), but increased in the MnO, HTO, and HTO-MnO treatment groups (Fig. 24c). When the effects of the substances used in the mouse model (MSU, MnO, HTO, and HTO-MnO) on major organs were confirmed by H&E staining, no damage to other organs was observed in any of them.

[0193]

[0194] The present invention is the product of research conducted with the support of the Korea Institute for Industrial Technology Promotion research project funded by the government (Ministry of Trade, Industry and Energy) in 2024 (No. P002794).

[0195] [Assignment No.] P002794

[0196] [Ministry Name] Ministry of Trade, Industry and Energy

[0197] [Specialized Research Management Agency] Korea Institute for Industrial Technology Promotion

[0198] [Research Project Name] Promotion of Open Ecosystems for Bio-based Industries

[0199] [Research Project Title] Support for Core Non-Clinical Demonstration for the Commercialization of Innovative New Drug Materials

[0200] [Organizing Organization] DR Cure Co., Ltd.

[0201] [Research Period] 2024.04.01~2024.12.31

Claims

1. A pharmaceutical composition for the prevention or treatment of inflammatory diseases comprising nanoparticles having a micelle structure in which hydrophobic manganese oxide is supported on a core, wherein the micelle structure is formed by an amphiphilic molecule in which hyaluronic acid and a hydrophobic aliphatic hydrocarbon are bonded by an active oxygen-degrading linker.

2. A pharmaceutical composition for the prevention or treatment of inflammatory diseases, wherein the hyaluronic acid has a molecular weight of 10 to 500 kDa.

3. A pharmaceutical composition for the prevention or treatment of inflammatory diseases according to claim 1, wherein the hydrophobic aliphatic hydrocarbon is at least one selected from the group consisting of hexadecylamine, heptadecylamine, octadecylamine, stearamine, nonadecylamine, dodecylamine, bis(2-hydroxyethyl)laurylamine, linoleic acid, linolenic acid, palmitic acid, oleylamine, hexadecanoic acid, stearic acid, oleic acid, eicosencic acid, and erucic acid.

4. In Claim 1, the active oxygen decomposing linker is a thioketal linker, a TSPBA linker (N 1 -(4-boronobenzyl)-N 3 -(4-boronophenyl)-N 1 ,N 1 ,N 3 ,N 3 A pharmaceutical composition for the prevention or treatment of inflammatory diseases, comprising -tetramethylpropane-1,3-diaminium) or an AA linker (aminoacrylate).

5. A pharmaceutical composition for the prevention or treatment of inflammatory diseases according to Claim 1, wherein the hydrophobic manganese oxide is bonded to the surface of the manganese oxide with any one hydrophobic substituent selected from the group consisting of a substituted or unsubstituted C1 to C30 alkyl group, a substituted or unsubstituted C3 to C30 cycloalkyl group, a substituted or unsubstituted C6 to C30 aryl group, a substituted or unsubstituted C2 to C30 heteroaryl group, a halogen group, a C1 to C30 ester group, and a halogen-containing group.

6. A pharmaceutical composition for the prevention or treatment of inflammatory diseases, wherein the amphiphilic molecule of claim 1 has a structure of the following chemical formula 1 or 2: [Chemical Formula 1] [Chemical Formula 2] .

7. A pharmaceutical composition for the prevention or treatment of an inflammatory disease according to claim 1, wherein the inflammatory disease is at least one selected from the group consisting of gout, gouty nodules, gouty attack, gouty arthritis, gouty nephrolithiasis, gouty nephropathy, multiple sclerosis, ankylosing spondylitis, rheumatoid arthritis, lupus, fibromyalgia, psoriasis, asthma, atopy, Crohn's disease, ulcerative colitis, acute or chronic inflammatory disease, osteoarthritis, osteoporosis, endotoxemia, toxic shock syndrome, tendinitis, tenosynovitis, and syndrome.

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