Composition for preventing or treating inflammatory diseases containing poly(organic phosphazene) polymer

Abstract

The present invention relates to a temperature-sensitive poly(organic phosphazene) polymer loaded with a drug and a pharmaceutical composition for preventing or treating inflammatory diseases containing the same. The injectable polymer nanoparticle hydrogel system containing the hydrogel has a long-term anti-inflammatory effect and an effect of sustained release of a therapeutically effective concentration of the drug, and thus can be used for the prevention and treatment of various inflammatory diseases such as osteoarthritis.

Description

Technical Field

[0001] The present invention relates to a composition for preventing or treating inflammatory diseases containing poly(organic phosphazene) polymers, etc.

Background Art

[0002] Osteoarthritis (OA) is the most common type of degenerative joint disease. Degeneration and inflammation of joints lead to chronic pain, stiffness, and progressive loss of articular cartilage that cannot regenerate. Degenerated joints can be replaced with prostheses at the end stage of osteoarthritis, but prostheses are also limited and additional surgeries for joint replacement may be required. Intra-articular (IA) administration of autologous chondrocytes, other treatment strategies such as platelet-rich plasma, or replenishment of viscous substances such as hyaluronic acid (HA) to eroded cartilage can be other solutions for the regeneration of damaged articular cartilage. However, the anti-OA effect as a result of such methods is not satisfactory because of the poor target-specific action and in vivo retention time after IA administration. As drug therapies, analgesics, non-steroidal anti-inflammatory drugs (NSAIDs), specific cyclooxygenase (COX)-2 inhibitors, and opioids can be administered orally, but this treatment method is limited to the treatment of symptomatic pain and has little effect on local treatment of inflammation [Buchman A.L. Side effects of corticosteroid therapy. J.Clin.Gastroenterol. 2001;33(4):289-294]. Also, NSAIDs, COX-2 inhibitors, and opioids are known to cause serious side effects in the gastrointestinal (GI) system, heart, and brain. Therefore, such oral medications must be prescribed carefully for the elderly with underlying diseases in the gastrointestinal tract, heart, and brain. To enhance the bioavailability of the administered drug at the target site and reduce systemic side effects as much as possible, direct IA administration of corticosteroids such as methotrexate, diclofenac, and triamcinolone acetonide (TCA) is possible.However, the half-lives of these drugs are known to be 0.56 - 2.9, 5.2, and 1.47 hours, respectively, and they have been reported to disappear rapidly in the body [Larsen C., Ostergaard J., Larsen S.W., Jensen H., Jacobsen S., Lindegaard C., Andersen P.H. Intra-articular depot formulation principles: role in the management of postoperative pain and arthritic disorders. J. Pharmacol. Sci. 2008;97(11):4622 - 4654., etc.]. Such short residence times are not sufficient for reducing the persistent inflammatory response.

[0003] Therefore, for such reasons, research is underway on new methods for the ultimate treatment and early prevention of osteoarthritis. Potential new OA treatment methods using anti-OA drug delivery systems with long-term activity in the body have been applied as liposomes, nanoparticles, microparticles, hydrogels, etc., and it has been found that the duration of anti-OA drug release has been significantly extended over several weeks to several months [Pradal J., Zuluaga M.F., Maudens P., Waldburger J.M., Seemayer C.A., Doelker E., Gabay C., Jordan O., Allemann E. Intra-articular bioactivity of a p38 MAPK inhibitor and development of an extended-release system. Eur. J. Pharm. Biopharm. 2015;93:110 - 117., etc.]. However, such systems have been shown to be unsuitable for accurate drug delivery to the target site and to have low release efficiency [Kopecek J. Hydrogel biomaterials: a smart future? Biomaterials. 2007;28(34):5185 - 5192.].

[0004] On the one hand, a hydrogel-based drug delivery system can extend the drug release period and provide the advantages of a local drug depot that can provide a three-dimensional (3D) space that can be physically supported, such as the synovial fluid region. Such properties can further reduce synovial joint damage by reducing physical friction in inflammatory arthritis. Furthermore, the temperature-sensitive sol-gel transition property can provide the advantages of non-invasive administration. However, a hydrogel-based drug delivery system for anti-deformity arthritis has been reported to have a limitation in that it exhibits drug release characteristics only for several days [Park C.W., Ma K.W., Jang S.W., Son M., Kang M.J. Comparison of piroxicam pharmacokinetics and anti-inflammatory effect in rats after intra-articular and intramuscular administration. Biomol. Ther. (Seoul) 2014;22(3):260-266.].

[0005] The hybrid system of a nano drug delivery system and a 3D hydrogel drug delivery system can be a powerful tool for successful OA treatment. For this purpose, poly(organic phosphazene) substituted with hydrophobic and hydrophilic side chains can function as a biodegradable drug delivery system with injectable in situ hydrogel formation, drug loading process, and long-term drug release characteristics [Hong K.H., Kim Y.M., Song S.C. Fine-tunable and injectable 3D hydrogel for on-demand stem cell niche. Adv. Sci. 2019;6(17):1900597. doi:10.1002 / advs.201900597.]. Such polymers in aqueous solution exist in the form of chains that spread or nano-sized spherical particles in response to the change in hydrophobicity accompanying the change in temperature. Based on such characteristics, various drugs having hydrophobic moieties can be encapsulated into polymeric nanoparticles (PN) by participating in the self-assembly process of polymeric nanoparticles. Furthermore, polymeric nanoparticles can form a 3D hydrogel network at a specific concentration or higher.

[0006] The inventors have studied to enable local and sustained TCA delivery using such a poly(organic phosphazene)-based PN hydrogel system and completed the present invention.

Summary of the Invention

Problems to be Solved by the Invention

[0007] The technical problem to be achieved by the present invention is to provide a composition for preventing or treating inflammatory diseases containing a poly(organic phosphazene) polymer.

[0008] However, the technical problem to be achieved by the present invention is not limited to the problems described above, and other problems not mentioned will be clearly understood by those having ordinary knowledge in the technical field from the following description. Means for Solving the Problems

[0009] To solve the above problems,

[0010] on the phosphorus atom of the polyphosphazene skeleton represented by the following Chemical Formula 1,

[0011] the first moiety of the amino acid ester represented by the following Chemical Formula 2,

[0012] the second moiety of the polyethylene glycol represented by the following Chemical Formula 3,

[0013] a polyphosphazene-based polymer containing a third moiety containing aminoethanol and an organic acid in a molar ratio of a:b:c, and

[0014] a drug for controlling and treating inflammation in a therapeutically effective amount

[0015] A composition for preventing or treating inflammatory diseases containing the same is provided.

[0016]

Chem.

[0017]

Chem.

[0018]

Chem.

[0019] In the above chemical formula,

[0020] R1 is alkyl of C 1-6 alkenyl of C 1-6 or aryl-C 6-10 alkyl of C 1-6 and

[0021] R2 is hydrogen, methyl, isopropyl, 1-methylpropyl, 2-methylpropyl, thiomethyl, methylthioethyl, benzyl, hydroxybenzyl, or 2-indolylmethyl,

[0022] R3 is an alkyl of C 1-6 and

[0023] n is an integer from 3 to 100,000,

[0024] p is an integer from 1 to 20,

[0025] The molar ratio of a:b is from 5.5:1 to 7.5:1,

[0026] a may be 70 to 80 mol%.

[0027] According to one aspect, the organic acid in the third moiety may include glutaric acid.

[0028] According to one aspect, R1 may be methyl, ethyl, propyl, butyl, benzyl, or 2-prophenyl.

[0029] According to one aspect, R3 may be methyl.

[0030] According to one aspect, the drug for treating the inflammatory disease may be hydrophobic.

[0031] According to one aspect, the hydrophobic drug for treating the inflammatory disease may be any one or more selected from the group consisting of triamcinolone acetonide, methylprednisolone, dexamethasone, celecoxib, ibuprofen, naproxen, indomethacin, ketoprofen, etodolac, meloxicam, rofecoxib, etoricoxib, valdecoxib, lumiracoxib, and diclofenac.

[0032] According to one aspect, the polymer may sustainably release the drug for treating the inflammatory disease.

[0033] According to one aspect, the polyphosphazene polymer may be contained by being dissolved in a solvent at a concentration of 1 to 50% by weight. The solvent may be one or more selected from the group consisting of water, buffer solution, acidic solution, basic solution, salt solution, physiological saline, water for injection, cell culture solution, and glucose saline solution, but is not limited thereto.

[0034] According to one aspect, the polymer may exhibit sol-gel transition behavior in the range of 5 to 70°C and form a hydrogel at a predetermined temperature.

[0035] According to one aspect, when applied to a living body or in an in vitro environment, the polymer may gelate, lose its temperature sensitivity at a predetermined temperature, and maintain the gel state regardless of temperature changes.

[0036] According to one aspect, the inflammatory disease may be any one selected from the group consisting of osteoarthritis, rheumatoid arthritis, rheumatoid arthritis, osteoporosis, and Achilles tendinitis.

Advantages of the Invention

[0037] The present invention relates to a temperature-sensitive poly(organic phosphazene) polymer loaded with a drug, a hydrogel containing the same, and a composition for preventing or treating an inflammatory disease containing the same. The injectable polymer nanoparticle hydrogel system containing the hydrogel has an anti-inflammatory effect maintained in the body for a long time and has an effect of regenerating tendons (such as the Achilles tendon), ligaments, muscles, cells, etc., and thus can be used for the prevention and treatment of various inflammatory diseases such as osteoarthritis.

[0038] In addition, the present inventors optimized the ratio of hydrophobic side chains and hydrophilic side chains forming the poly(organic phosphazene) polymer to exhibit ideal drug release characteristics in the body, and the drug can be administered into the body at a constant concentration for a long time.

[0039] The effects of the present invention are not limited to those described above, and other effects not mentioned will be clearly understood by those skilled in the art from the following description.

Brief Description of the Drawings

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Mode for Carrying Out the Invention

[0076] In order to solve the technical problems of the prior art, the present inventors provide the following poly(organic phosphazene) polymers and compositions for preventing or treating inflammatory diseases containing the same.

[0077] On the phosphorus atom of the polyphosphazene skeleton represented by the following chemical formula 1,

[0078] the first moiety of the amino acid ester represented by the following chemical formula 2,

[0079] the second moiety of the polyethylene glycol represented by the following chemical formula 3,

[0080] and a third moiety containing aminoethanol and an organic acid, are contained in a molar ratio of a:b:c, respectively, a polyphosphazene-based polymer, and

[0081] a therapeutically effective amount of an inflammation control and therapeutic drug,

[0082] is a composition for preventing or treating inflammatory diseases containing the same.

[0083]

Chem.

[0084]

Chem.

[0085]

Chem.

[0086] In the above chemical formula,

[0087] R1 is an alkyl of C 1-6 , an alkenyl of C 1-6 , or an aryl-C 6-10 alkyl of C 1-6 ,

[0088] R2 is hydrogen, methyl, isopropyl, 1-methylpropyl, 2-methylpropyl, thiomethyl, methylthioethyl, benzyl, hydroxybenzyl, or 2-indolylmethyl,

[0089] R3 is an alkyl of C 1-6 and

[0090] n is an integer from 3 to 100,000,

[0091] p is an integer from 1 to 20,

[0092] The molar ratio of a:b is from 5.5:1 to 7.5:1,

[0093] a may be 70 to 80 mol%.

[0094] Generally, a temperature-sensitive polymer is a polymer that exhibits a sharp change in solubility with a change in temperature. By raising the temperature, the hydrogen bond between the polymer and the solvent weakens and dehydration occurs, strengthening the hydrophobic attraction between the polymers and causing them to have a more hydrophobic structure. At the lower critical solution temperature (LCST), as the interaction between polymer-polymer and water-water becomes more favorable than the hydrogen bond between the polymer and water, rapid dehydration occurs from the polymer, resulting in a more hydrophobic structure. The lower critical temperature of the temperature-sensitive polymer changes depending on the balance between the hydrophobic groups and hydrophilic groups bonded to the polymer backbone. Generally, as the content of the hydrophilic groups increases, the phase transition temperature rises, and conversely, as the hydrophobic groups increase, the phase transition temperature decreases. Such characteristics can also be confirmed by changes in T max such as those due to changes in the ratio of hydrophobic side chains and hydrophilic side chains disclosed in Example 9 below.

[0095] When the essential constituent moieties constituting the poly(organic phosphazene) polymer have a predetermined ratio, a sol-gel transition of the polymer solution occurs reversibly due to a change in temperature below a certain temperature. However, after being exposed to a specific temperature or higher, such a sharp reversible transition characteristic is lost, and the gel state can be maintained for a long time. In particular, such a change in characteristics can be achieved by the ratio of the substituents introduced during the initial synthesis of the polymer.

[0096] In particular, in the present invention, when the first moiety and the second moiety are within a specific molar ratio range, it was confirmed that the optimal drug concentration required in vivo can be maintained and sustained release can be achieved (see Example 9 below). This is based on the discovery of the present inventors that the hydrophobic properties and the phase transition temperature of the polymer change according to the composition ratio of the hydrophobic first moiety and the hydrophilic second moiety. The higher the molar ratio of the hydrophobic side chain, the more hydrophobic the polymer, the stronger the interaction with the hydrophobic drug, and the more difficult it is for the drug to be released into the body. Therefore, by adjusting the molar ratio of the hydrophobic side chain to the hydrophilic side chain, the rate at which the drug is released into the body can be regulated.

[0097] Drugs for treating steroid inflammatory diseases such as TCA, or non-steroidal drugs containing celecoxib, all have a concentration range that is most suitable for their action in the body. In the case of TCA, as shown in FIG. 20, when outside a specific concentration range, it can be confirmed that the expression of inflammation-related factors rather increases.

[0098] As shown by the present inventors in Example 9 and FIG. 21 below, preferably, the molar ratio of the hydrophobic side chain may be in the range of 5.5:1 to 7.5:1 with respect to the hydrophilic side chain, more preferably 6:1 to 7:1, and most preferably 6.13:1. In the above-mentioned molar ratio range, a drug, preferably TCA, can be sustained released within a suitable concentration range before surgery. If outside the above-mentioned molar ratio range, the pharmacological effect of TCA may decrease.

[0099] According to one aspect, the organic acid in the third moiety may include glutaric acid. The glutaric acid means (Acid) disclosed in Example 10-2 below. This is produced and generated by the production method of Example 10-2, and may be glutaric acid bonded to a hydroxy functional group by an ester bond in the final product.

[0100] One or more functional moieties selected from the group consisting of a substance capable of regulating the degradation rate of a polymer, a substituent containing an ion group whose degradation rate can be regulated, a substituent capable of cross-linking, an additional compound capable of inducing tissue binding, a physiologically active substance, and a composite material formed by linearly connecting two or more of these functional materials may be further included as a fourth moiety directly or via a linker.

[0101] In this case, the fourth moiety may include at least one selected from the group consisting of folic acid, hyaluronic acid, cyclodextrin, imidazole-based compounds, anticancer agents, histidine, lysine, arginine, cysteine, thiol arylamine, spermine, spermidine, polyethyleneimine, polyhistidine, polylysine, polyarginine, protamine, heparin, chitosan, and peptides consisting of 1 to 20 amino acids.

[0102] According to one aspect, R1 may be methyl, ethyl, propyl, butyl, benzyl, or 2-prophenyl.

[0103] According to one aspect, R3 may be methyl.

[0104] According to one aspect, the drug for treating the inflammatory disease may be hydrophobic and may further contain a functional substance.

[0105] The functional substance may be a neovascular inhibitor such as preosteoblast, chondrocyte, osteoblast, adult stem cell, Schwann cell, oligodendrocyte progenitor cell, insulin, oxytocin, vasopressin, adrenocorticotropic hormone, fibroblast growth factor, epidermal growth factor, platelet-derived growth factor, insulin-like growth factor, vascular endothelial growth factor, transforming growth factor, brain-derived neurotrophic factor, antibiotic, and integrin α-5-β-1 antagonist, or a combination thereof.

[0106] The hydrophobic drug for treating osteoarthritis may be any one or more selected from the group consisting of triamcinolone, methylprednisolone, dexamethasone, ibuprofen, naproxen, and diclofenac, and most preferably triamcinolone.

[0107] According to one aspect, the polymer may sustainably release the drug for treating osteoarthritis. As used herein, "sustained release" means being maintained in the body for at least 21 days or more and releasing the drug, preferably maintaining the release concentration of the drug at the therapeutically effective concentration of the drug. Further, the sustained release is releasing the drug at a drug concentration within the range of the therapeutically effective amount, and the drug concentration within the range of the therapeutically effective amount is preferably 9.375 - 75 μg / ml, more preferably 10 - 50 μg / ml.

[0108] According to one aspect, the polyphosphazene-based polymer may be dissolved in a solvent at a concentration of 1 - 50% by weight. The solvent may be one or more selected from the group consisting of water, buffer solution, acidic solution, basic solution, salt solution, physiological saline, water for injection, cell culture solution, and glucose saline solution, but is not limited thereto. In the presence of the solvent, the polyphosphazene-based polymer of the present invention can be hydrogelated and exhibits the behavior of sol-gel transition as described below.

[0109] According to one aspect, the polymer may exhibit the behavior of sol-gel transition in the range of 5 - 70°C and form a hydrogel at a predetermined temperature.

[0110] According to one aspect, the polymer may gelate when applied to a living body or in an in vitro environment, lose its temperature sensitivity at a predetermined temperature, and maintain the gel state regardless of temperature changes.

[0111] The inflammatory disease may preferably be any one selected from the group consisting of osteoarthritis, rheumatoid arthritis, rheumatoid arthritis, osteoporosis, and Achilles tendinitis, and most preferably may be osteoarthritis.

[0112] The composition of the present invention has the use of "prevention" and / or "treatment" of inflammatory diseases, preferably osteoarthritis. In the preventive use, the composition of the present invention can be administered to an individual who has the disease or symptom described in the present invention or is suspected of being at risk of developing the disease. In the treatment use, the pharmaceutical composition of the present invention is administered to an individual such as a patient who already suffers from the disease described in the present invention in an amount sufficient to treat or at least partially arrest the disease or symptom described in the present invention. The effective amount for such use depends on the severity and course of the disease or symptom, previous treatment, the individual's health status and responsiveness to the drug, and the judgment of a physician or veterinarian.

[0113] The composition may be any one dosage form selected from the group consisting of a sterilized aqueous solution, a non-aqueous solvent, a suspending agent, tablets, pills, powders, granules, capsules, internal liquid preparations, emulsions, syrups, emulsions, lyophilized agents, and suppositories, and may also be various dosage forms for parenteral or oral administration. When formulating, it is prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants that are commonly used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc. Such solid preparations are prepared by mixing at least one excipient, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc., with one or more compounds. In addition to mere excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral administration include suspending agents, internal liquid preparations, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients, such as wetting agents, sweeteners, flavoring agents, and preservatives, are included. Preparations for parenteral administration include sterilized aqueous solutions, non-aqueous solvents, suspending agents, emulsions, lyophilized agents, and suppositories. As non-aqueous solvents and suspending agents, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate are used. As the base of suppositories, witepsol, macrogol, tween 61, cocoa butter, lauric acid, glycerogelatin, etc. are used.

[0114] In the present invention, the composition of the present invention is formulated into a sterilized aqueous solution, a non-aqueous solvent, or a suspending agent for parenteral administration. Specifically, it may be formulated into an injection. The pharmaceutical composition of the present invention may further contain a suitable carrier, excipient, or diluent that is commonly used. In this case, the content of the injection composition of the present invention, which is the active ingredient contained in the pharmaceutical composition, is not particularly limited.

[0115] The composition of the present invention can be administered to an individual in a pharmaceutically effective amount.

[0116] The "pharmaceutically effective amount" in the present invention means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment. The level of the effective dose is determined by factors including the type and severity of the individual, age, gender, type of disease, activity of the drug, sensitivity to the drug, administration time, administration route and excretion rate, treatment period, factors including co-administered drugs, and factors known in other medical fields.

[0117] The composition of the present invention can be administered as an individual therapeutic agent or mixed with other therapeutic agents, and can be administered sequentially or simultaneously with conventional therapeutic agents, and can be administered once or repeatedly. Considering all the above factors, it is important to administer an amount that can obtain the maximum effect with the minimum amount without side effects, which may be easily determined by those skilled in the art. The preferred dosage of the composition of the present invention varies depending on the patient's condition and body weight, severity of the disease, drug form, administration route and period, and the administration can be once a day or divided into several times.

[0118] In the present invention, the composition of the present invention can be administered in admixture with the above-mentioned functional substances including stem cells, as described above.

[0119] The composition of the present invention can be administered without particular limitation to an individual for the purpose of preventing or treating osteoarthritis. The mode of administration includes without limitation any conventional method in the art. The composition can be administered into the joint cavity (for example, within cartilage), subcutaneously, intraperitoneally, intratracheally and intranasally, and for local treatment, it can be administered by an appropriate method including, if necessary, administration into the lesion, that is, into the joint cavity (for example, within cartilage). As an example, it can be administered (injected) by intra-articular injection, but is not limited thereto.

[0120] As used herein, the term "individual" means all animals in which osteoarthritis has occurred or may occur, and by administering the composition of the present invention to an individual suspected of having osteoarthritis, the individual can be efficiently treated. The individual is not particularly limited, and may be, for example, an animal such as a monkey, dog, cat, rabbit, guinea pig, rat, mouse, cow, sheep, pig, goat, or a bird, but is not limited thereto.

[0121] As used herein, the term "administration" means introducing the composition of the present invention into an individual suspected of having osteoarthritis by a suitable method, and the administration route can be through various parenteral routes as long as it can reach the target tissue. The composition of the present invention can be administered in a pharmaceutically effective amount, and the pharmaceutically effective amount is as described above.

[0122]

[0123] The terms used in the examples are for illustrative purposes only and should not be construed as limiting. Singular expressions include plural expressions unless otherwise clearly indicated in the context. Terms such as "comprising" or "having" in this specification are used to specify the presence of the features, numbers, steps, operations, components, parts, or combinations thereof described in the specification, and it should be understood that the presence or addition possibility of one or more other features, numbers, steps, operations, components, parts, or combinations thereof, etc. are not precluded in advance.

[0124] Unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by those of ordinary skill in the art to which the examples belong. Terms defined as in commonly used dictionaries should be construed to have a meaning consistent with the meaning in the context of the related art, and should not be construed in an ideal or overly formal sense unless clearly defined in the present application.

[0125] The present invention can be subjected to various transformations, can have various embodiments, and the following specific embodiments are illustrated in the drawings and will be described in detail in the detailed description. However, this is not intended to limit the present invention to specific embodiments, and it should be understood that the present invention includes all transformations, equivalents or alternatives included in the spirit and technical scope of the present invention. In the description of the present invention, if the specific description of related known technologies is determined to obscure the gist of the present invention, the detailed description thereof will be omitted. Mode for Carrying Out the Invention

[0126] In the following Examples 1 to 9, poly(organic phosphazene) loaded with TCA is disclosed. A schematic diagram of the mechanism of action of TCA-loaded poly(organic phosphazene) is shown in FIG. 1.

[0127]

[0128] Example 1. Experimental Method A

[0129] Example 1-1. Materials

[0130] Hexachlorocyclotriphosphazene was obtained from Sigma-Aldrich (USA) and purified by sublimation at 55 °C under vacuum (about 0.1 mmHg). Poly(dichlorophosphazene) was prepared according to the description by Bromberg L. et al. [Bromberg L., Temchenko M. Self-assembly in aqueous solutions of poly(ethylene oxide)-b-poly(propylene oxide)-b-poly(ethylene oxide)-b-poly(vinyl alcohol) Langmuir. 1999;15:8633-8639.]. Methoxypoly(ethylene glycol) with a molecular weight of 750 Da (Sigma-Aldrich, USA) was replaced by α-amino-ω-methoxypoly(ethylene glycol) (AMPEG) and substituted onto the poly(organic phosphazene) backbone. l-Isoleucine ethyl ester hydrochloride (IleOEt·HCl) (A&Z food additives, HangZhou, china, aminoethanol (Sigma-Aldrich, USA), glutaric anhydride (Alfa Aesar, USA), 4-(dimethylamino) pyridine (DMAP) (Alfa Aesar, USA) were obtained. THF (Tetrahydrofuran) and TEA (Trimethylamine) were refluxed and dried with metallic sodium and barium oxide respectively under dry nitrogen. Triamcinolone acetonide (TCA) was obtained from TCI (Japan). All other reagents were obtained commercially.

[0131]

[0132] Example 1-2. Synthesis of poly(organic phosphazene)

[0133] All reactions were carried out under a dry nitrogen atmosphere using standard Schlenk-line techniques.

[0134] 1-2-1. Synthesis of Precursor Polymer Composite (PP, Aminoethanol Poly(Organophosphazene))

[0135] The precursor polymer composite (PP) was prepared as follows. First, IleOEt·HCl (10.8 g, 55.22 mmol) was suspended in anhydrous THF (200 mL), and the reaction flask was cooled. Subsequently, poly(dichlorophosphazene) (10.00 g, 8.63 mmol) was dissolved in anhydrous THF (200 mL) and slowly added to the reaction flask containing IleOEt·HCl (23.64 g, 12.08 mmol) dissolved in anhydrous THA and TEA. The reaction mixture was stirred in a dry ice bath for 12 h and then at 45 °C for 24 h. Next, AEtOH (1.42 g, 2.33 mmol) and AMPEG750 (13.59 g, 18.12 mmol) were dissolved in anhydrous THF and added to the reaction mixture. The reaction mixture was stirred at room temperature for 24 h and then at 45 °C for an additional 24 h. The reaction mixture was filtered, poured into n-hexane to obtain a precipitate, and then reprecipitated twice with the same solvent system. The polymer product was further purified by dialysis against methanol at room temperature for 4 days and against distilled water at 4 °C for 4 days using a dialysis membrane (Spectra / Por, MWCO: 10 - 12 kDa). The dialysate was lyophilized to obtain PP. The yield was 73.7%, and the NMR data were as follows.

[0136] 1 H NMR (CDCl3), δ (ppm): 0.8 - 1.0 (s, 6H), 1.1 - 1.3 (b, 3H), 1.3 - 1.6 (b, 2H), 1.6 - 1.9 (b, 1H), 2.8 - 3.3 (b, 2H), 3.4 - 3.8 (b, 73H), 3.9 (s, 1H), 4.0 - 4.3 (b, 3H).

[0137] 1-2-2. Synthesis of Carboxylic Acid-Terminated Functional Poly(Organophosphazene) (CP)

[0138] To replace the hydroxyl terminus with a carboxylic acid terminus, PP (10 g, 12.25 mmol) was dissolved in dry THF. The reaction mixture was stirred at room temperature for 12 h and then further stirred at 40 - 45 °C for 24 h. The final product CP was dialyzed against methanol at room temperature for 3 days and against water at 4 °C for 3 days using a dialysis membrane. The purified CP was obtained by lyophilization. The newly appeared peak at 2.1 - 2.32 ppm (b, 4H, CH2) in the CP 1 1H NMR indicates the generated glutaric acid terminus that does not exist in the starting material. The yield was 96% and the NMR data were as follows.

[0139] 1 1H NMR (CDCl3), δ (ppm): 1 1H NMR (300 MHz, CDCl3, δ), d (ppm): 0.8 - 1.0 (s, 6H, CH3), 1.1 - 1.3 (b, 3H, CH3), 1.3 - 1.6 (b, 2H, CH2), 1.6 - 1.9 (b, 1H, CH), 2.1 - 2.32 (b, 4H, CH2), 2.8 - 3.3 (b, 2H, CH2), 3.4 - 3.8 (b, 62H, CH2), 3.9 (s, 1H, CH), 4.0 - 4.3 (b, 3H, CH3).

[0140]

[0141] Examples 1 - 3. Characterization of CP

[0142] The structure of the synthesized CP was 1Estimated by measurement using ¹H NMR (Varian Gemini-300 spectrometer operating at 300 MHz in Fourier transform mode using CDCl₃). The surface charge of CP in aqueous solution was measured with a Zetasizer Nano ZS (Malvern Instruments Ltd., Malvern, UK). The molecular weight (MW) of CP was calculated by gel permeation chromatography system (Tosoh, EcoSEC HLC-8320 GPC) equipped with a refractive index detector and two Styragel columns (TSKgel Supermultipore HZ-M and TSKgel Super HZ-2500) connected in series at a flow rate of 0.35 mL / min at 25 °C. THF containing 0.1 wt% tetrabutylammonium bromide was used as the mobile phase. Polystyrene (MW: 162; 580; 1920; 3090; 9590; 27,810; 70,500; 133,500; 290,300; 729,500; 1,074,000; 2,703,000) was used as a standard.

[0143]

[0144] Examples 1-4. Biocompatibility experiments

[0145] 1-4-1. Experiment on cytotoxicity of CP

[0146] The test for cytotoxicity of CP was performed using a mouse fibroblast cell line (NIH3T3). The cells were seeded at 2×10 4Dispensed at the density of / well and adhered overnight in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin in a humidified atmosphere of 5% CO2 at 37°C. The culture medium was replaced with fresh DMEM after 24 hours, and treated with various concentrations of CP (0 mg / mL - 10 mg / mL in DMEM, n = 6). After 24 hours / 48 hours / 72 hours, MTT solution (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) was added, and after aspirating the medium, cultured for 3 hours. The precipitate was dissolved in 100 μl of DMSO, and the absorbance of the resulting purple solution was measured at an excitation wavelength of 570 nm using Spectra MAX 340 (BIO-RAD, Hercules, CA). The cell viability (%) was calculated as [ab]test / [ab]control×100%.

[0147] 1-4-2. Confirmation of the mutagenicity of CP

[0148] The mutagenicity of CP was evaluated in various auxotrophic strains of the bacterium Salmonella typhimurium. Mutagenicity was determined by evaluating the number of revertant colonies formed (Ames test). It was performed using a sterilized liquid medium (2.5% Oxoid Nutrient Broth No.2) for the tests of T98 and T100. After culturing for 10 hours in a shaking water bath (37 °C, 200 rpm), 0.1 mL of the cultured bacteria was mixed with 2 mL of top agar, 0.1 mL of the CP solution, and the S9 fraction. Then, this mixture was gently poured into a previously prepared petri dish containing 20 mL of a minimal glucose agar plate and cultured until it solidified. After culturing at 37 °C for 48 hours, the number of colonies was counted. Phosphate buffered saline (PBS) was used as the negative control group, and 2-nitrofluorene, benzo(a)pyrene, and sodium azide were used as the positive control groups specific for each bacterium T98 and T100. All negative and positive control groups were performed in the same manner. The minimal glucose agar plate was prepared using Bacto agar (Difco), Vogel-Bonner medium E, and 2% glucose.

[0149]

[0150] Example 1-5. Production of self-assembled polymer nanoparticles (PNs) and TCA-encapsulated polymer nanoparticles (TePN)

[0151] The synthesized CP was dissolved in PBS solution at 4 °C using a magnetic stirrer (a 12 wt% CP solution was prepared and diluted 10-fold). The prepared CP solution was observed by dynamic light scattering (DLS) using a Zetasizer Nano ZS (Malvern Instruments Ltd., Malvern, UK) and transmission electron microscopy (TEM, CM30 electron microscope, Philips, CA). The observed size and morphology of the self-assembled PN were confirmed. TCA-encapsulated polymer nanoparticles (TePN) were prepared with three concentrations of TCA (0.3, 1.0, and 1.5 mg / mL). TCA was directly added to the PNs solution and gently mixed. The mixed solution was incubated at room temperature for 30 minutes to induce TCA encapsulation. The properties of TePN were verified by DLS and TEM.

[0152]

[0153] Example 1-6. Temperature-responsive sol-gel transition behavior of TePN solution

[0154] The viscosity of the PN aqueous solution (12 wt%) was measured in the range of 5 - 75 °C at a fixed shear rate of 0.1 s-1 using a Brookfield RVDV-III+ viscometer. The measurement was processed at a set spindle speed of 0.2 rpm and a heating rate of 0.33 °C / min. Rheological measurements (MSC 102, Anton Paar, DE) were performed on the PN solution (PN 12 wt% in PBS) and the TePN solution (added to the 12 wt% PN solution prepared with 0.3, 1.0, 1.5 mg / mL of TCA). The measurements of storage modulus (G’) and loss modulus (G”) were carried out at a vibration frequency of 1 Hz, a vibration deformation rate of 5%, temperatures of 4 °C and 60 °C, and an interval length of 0.3 mm.

[0155]

[0156] Example 1-7. In vivo degradation study of TePN hydrogel

[0157] A TePN solution (1.5 mg / mL of TCA, 200 μL) was subcutaneously injected into the back of mice (Balb / c nude mice, 6 weeks old, male, Orient Bio, Korea) using a 31-gauge needle. The locally generated TePN hydrogel was confirmed immediately after injection. The remaining TePN hydrogel was weighed on a pre-determined day.

[0158]

[0159] Example 1-8. In vitro release of TCA from TePN

[0160] The TePN solutions were prepared with 12 wt% PN solutions containing 0.3, 1.0, and 1.5 mg / mL of TCA, respectively. 0.3 mL of the prepared TePN solution was placed in a tube and heated to 37 °C to confirm the formation of the hydrogel. 6 mL of PBS solution was gently added to the hydrogel, and the tube was cultured in a 37 °C water bath (KMC-12055W1, Vision, Korea) with gentle shaking (50 rpm). The PBS solution was replaced with fresh PBS solution at each time point. The amount of released TCA was measured by high-performance liquid chromatography (HPLC, Agilent) using water / acetonitrile (50 / 50, v / v%) as the eluent and calculated with a standard sample established using UV detection at 240 nm.

[0161]

[0162] Example 1-9. In vivo anti-OA effect of the TePN hydrogel system

[0163] 1-9-1. Intra-articular injection of TePN in rats with osteoarthritis (OA)

[0164] Monosodium iodoacetate (MIA, Sigma-Aldrich, USA) (0.5 mg / 50 μl) was injected into the intra-articular site of Sprague Dawley (SD) rats (6 weeks old, male, Orient Bio, Korea) (n = 6 per group) to induce OA. One week later, the OA-induced rats were treated with 0.3 mL of TePN solution by intra-articular injection. Untreated MIA-induced OA rat models were used as the control group. For additional analysis, the rats were sacrificed 8 weeks after TePN injection.

[0165]

[0166] 1-9-2. Intra-articular structure and X-ray, micro-computed tomography (μ-CT) scan

[0167] After sacrificing the rats 8 weeks after injection, the isolated knee joints were cut from the femur and tibia. Samples of the isolated knee joints were scanned using X-ray (In-Vivo Series, DXS PRO, Carestream, USA) and μ-CT (Aibira CT system, Carestream, USA). The distance of the damaged cartilage was measured using μ-CT images and ImageJ software.

[0168]

[0169] 1-9-3. Histological analysis

[0170] All collected tissues were embedded in paraffin and sectioned with a microtome (thickness 8 μm). Tissue sections were deparaffinized, rehydrated, and stained for histological evaluation. After staining with H&E (hematoxylin and eosin) and safranin-O, the histological appearance of individual knee joints was observed with an optical microscope (Nikkon; E400, Japan).

[0171]

[0172] 1-9-4. RNA extraction and real-time polymerase chain reaction

[0173] Blood samples were collected at the following time points: OA induction, 1 week, 4 weeks, and 8 weeks after TePN hydrogel treatment. RNA was extracted using an RNA blood kit (QIAamp, QIAGEN) according to the manufacturer's instructions. cDNA was synthesized using Accupower CycleScriptRT Pre Mix (dT20) (BIO-RAD) according to the manufacturer's protocol. Real-time polymerase chain reaction (PCR) amplification and detection were performed using an ABI7300 Real-Time Thermal cycler (Applied Biosystems, Foster City, CA, USA). Gene expression of systemic inflammatory cytokines, such as metalloproteinase-3 (MMP-3), MMP-13, interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α), and anti-inflammatory cytokines, such as IL-4, IL-10, and IL-13, was investigated. The sequences of each gene marker are listed in Table 1 below.

[0174] [Table 1]

[0175]

[0176]

[0177] Example 2. Synthesis and properties of poly(organophosphazenes)

[0178] Poly(organophosphazene) mainly containing hydrophobic l-isoleucine ethyl ester (IleOEt) and hydrophilic α-amino-ω-methoxy poly(ethylene glycol) (AMPEG) in the side chain was synthesized and used as a temperature-responsive and in situ gelling drug storage system. Also, a carboxylic acid terminal was introduced to mimic the extracellular matrix of natural cartilage mainly composed of chondroitin sulfate and hyaluronic acid, and interact with various cytokines involved in cartilage homeostasis and regeneration, which is as shown in the following chemical formula.

[0179] [Chemical Formula]

[0180] The molar ratio of the substituted side chains was calculated using NMR spectra. The concentrations of IleOEt, AMPEG, and carboxylic acid were 70%, 10.5%, and 13.5% respectively in the final polymer. The substitution rate of each polymer composite was evaluated by the integration of specific peaks at 0.8 - 1.0 ppm for the six protons of IleOEt, 2.1 - 2.32 ppm for the four protons of carboxylic acid, and 3.4 - 3.8 ppm for the 62 protons of AMPEG as 1H NMR data (Figure 2). The charge of the final polymer was characterized by a negative charge of -4.0 ± 1.6 in zeta potential measurement, mimicking the negative charge of the extracellular matrix of cartilage, and GPC analysis using a polystyrene standard showed a molecular weight of 50,085 Da.

[0181] In addition, the biocompatibility of poly(organophosphazene) was evaluated as follows. The Ames test is a test for evaluating the potential mutagenicity of test chemicals generally related to carcinogenicity. For the mutagenicity test, Salmonella typhimurium TA98 or TA100 was treated with 200 μg of the polymer. The polymer-treated group showed almost the same revertant colonies as the negative control group ((a) in Figure 3). Such non-toxicity and mutation reaction imply the safety of synthetic polymers, and such results support the possibility of using the polymer as a bioavailable substance. Especially in the case of anti-OA treatment, since the TCA carrier should not promote an immune reaction at the lesion site, highly biocompatible properties are required. Therefore, the examined biocompatibility of the synthesized poly(organophosphazene) was confirmed to be appropriate as an anti-OA drug delivery system.

[0182] Cytotoxicity and mutagenicity tests were conducted as they are necessary for generating toxicity data on new drugs or biologically available substances. The toxicity of the synthesized poly(organophosphazene) was examined by MTT assay and Ames test. The cytotoxicity of the polymer was treated with NIH 3T3 cell line (polymer concentration in the medium 0 mg / mL - 30 mg / mL). 90 - 95% cell viability was observed at all polymer concentrations 24 hours after treatment ((b) in Figure 3). There was no cytotoxic effect even after treating the polymer of the same composition with CP for 48 and 72 hours, which is shown in Figure 4. This indicates that CP is a cell-compatible substance.

[0183]

[0184] Example 3. Characteristics of Self-Assembled PN and TCA Encapsulation

[0185] The side chains of the polymer consist of a hydrophobic part and a hydrophilic part, and have balanced amphiphilicity. The polymer exists as spherical nanoparticles in an aqueous environment to minimize the exposed area of the hydrophobic IleOEt units of the polymer to water molecules. Also, hydrophobic drugs such as TCA interact with the hydrophobic core part of the PN (Figure 5(a)). Self-assembled PN and TePN were investigated by TEM and size distribution studies. Spherical PN was identified in the TEM image (Figure 5(b)), and the size was ~140 nm at 25 °C (120 mg / mL of PN in phosphate buffered saline (PBS) solution), with an error range of ≦4 nm. TCA, a drug with low water solubility, showed irregular nano- and micro-sized crystallin phases in the range of 232.9 nm to 3343 nm (error range ≧1450 nm in DLS and TEM data) in a PBS solution (1.5 mg / mL of TCA in PBS) (Figure 5(c) and Figure 6). However, fine-sized TCA aggregates disappeared from the size distribution after mixing and culturing with the PN-dispersed PBS solution. Only particles with a size of ~140 nm were measured with an error range of ≦4 nm in the mixed solution of TCA and PN (120 mg / mL of PN and 1.5 mg / mL of TCA in PBS solution) (Figure 5(d)). This result means the successful encapsulation of TCA into the PN nanoparticles. The values of the average size and polydispersity index (PDI) of these particles are shown in Figure 5(e) and Figure 7. The change and significant decrease in size prove that TCA can be successfully encapsulated into the hydrophobic core of PN by hydrophobic interaction.

[0186]

[0187] Example 4. Confirmation of the behavior of the temperature-sensitive sol-gel transition of the TePN solution

[0188] In an aqueous solution of PN with a certain concentration or higher, an obvious viscosity change and phase transition occur with the change in temperature. Such a phase transition occurs as the hydrogen bonds between the hydrophilic PEG chains of PN and water molecules weaken with increasing temperature, while the hydrophobic interaction between PNs increases. In this study, a 12 wt% PN solution was prepared to have the maximum storage modulus so that it has good injectability, sufficient viscosity, and becomes gel-like at body temperature. As shown in Fig. 8(a), the PN solution showed a viscosity close to 0 Pa·s up to 32 °C. The viscosity value increased continuously at 32 °C and showed 518.75 Pa·s at 37 °C. Similarly, the values of both the storage and loss moduli increased at 32 °C, and the value of the storage modulus (G’) was larger than the loss modulus value (G”) between 32 °C and 44 °C in the rheology study. The values of G’ and G” at 37 °C were 1284.3 and 765 Pa, respectively, indicating a gel state (Fig. 8(b)).

[0189] Also, as shown in Fig. 8(c), since the physical properties of the drug delivery hydrogel may have a significant impact on the drug release pattern or duration, the behavior of the sol-gel transition after TCA loading was monitored. The storage and loss coefficients of the TePN solution were measured after loading TCA at three different doses. In all groups, similar changes were shown in the values of the storage and loss moduli with the change in temperature. This result can prove that the loaded TCA is homogeneously encapsulated inside the PN and has almost no effect on the behavior of the sol-gel transition, and such characteristics can predict that the hydrogel shows similar behavior even at different drug doses.

[0190]

[0191] Example 5. In vivo degradation of TePN hydrogel

[0192] Since biodegradation is an essential requirement for biomaterials and drug delivery systems, the hydrogel degradation rate and general symptoms around the tissue where the PN hydrogel was locally placed were monitored. The prepared PN solution was injected subcutaneously into mice with a 31-gauge needle. After injection, the formation of the PN hydrogel was directly confirmed, and the remaining PN hydrogel was measured through a simple separation process on the skin at the intended time point (Figure 9(a)). The amount of the remaining PN hydrogel decreased over 42 days (Figure 9(b) and Figure 10). Such biodegradation characteristics, mild conditions for PN hydrogel formation, and long-term retention at the injected site indicate that the developed PN hydrogel system is a long-term drug depot and suitable for use as a drug delivery carrier.

[0193]

[0194] Example 6. Confirmation of the sustained release profile of TCA from TePN in vitro

[0195] Steroidal anti-inflammatory drugs such as TCA have a short residence time after intra-articular administration and are rapidly removed from the body. Therefore, the long-term anti-inflammatory effect during general administration is limited. In the TePN hydrogel system, strongly interacting drugs tend to strongly depend on the degradation characteristics of the material, and by controlling the dissociation and degradation rates of the PN hydrogel, the TCA release pattern and duration can be controlled. The TCA release behavior was experimentally studied using freshly prepared TePN solutions containing three different concentrations of TCA. After forming the TePN hydrogel at 37 °C, each TePN hydrogel was immersed in PBS buffer. In contrast to the PN group showing rapid swelling within less than 1 day, most hydrogels with TCA showed swelling after 1 week, which is thought to be because the hydrophobic interaction between TCA and the hydrogel improves the stability of the polymer structure in the aqueous solution (Figure 11). Also, all gels started to show mass loss after 4 weeks (Figure 12(a)). This was closely related to the TCA release pattern of the hydrogel for 42 days (Figure 12(b)). Suppressed initial release of TCA was confirmed during the first 24 hours in the three TePN hydrogel groups with TCA concentrations of 0.3, 1.0, and 1.5 mg / mL, and the observed values were read as 14.45%, 9.55%, and 9.72% respectively for the amount of all loaded TCA. Such results were in contrast to the fact that the half-life of TCA without a delivery carrier was only 1.27 hours in the human body in the case of direct administration. Approximately half of the amount of TCA loaded at 7 days, 14 days, and 16 days in each TePN hydrogel group was released at values of 0.3, 1.0, and 1.5 mg / mL respectively. The sustained release process continued until the TePN hydrogel was completely degraded. On the other hand, the TePN hydrogel group with the lowest TCA concentration showed a faster release pattern than the other two groups. The low TCA concentration is affected by the loss of the hydrophobic network, rapid swelling, and rapid dissolution. In the TePN hydrogel groups containing 1.0 mg / mL and 1.5 mg / mL of TCA, the TCA release pattern over time was linear. The degradation of the TePN hydrogel and the TCA release time were almost similar.The suppressed initial release and continuous TCA release indicate that TCA interacts strongly with the polymer and is better encapsulated by PN.

[0196]

[0197] Example 7.Confirmation of the long-term anti-inflammatory and cartilage degeneration preventive effects by a single injection of TePN hydrogel in an OA rat model

[0198] The long-term anti-inflammatory and cartilage degeneration inhibitory effects were verified by a single injection of the TePN hydrogel storage system. For the treatment of the TePN hydrogel system, a rat model with MIA (monosodium iodoacetate)-induced OA was prepared. The injection of TePN was performed 1 week after OA induction to mimic the initial stage of OA. In addition to the TePN hydrogel group, a direct injection group of the TCA solution and a saline group were added as control groups. 300 μl solutions of physiological saline, TePN, and TCA were prepared respectively and injected into the injury site of the rats.

[0199] Eight weeks after the start of treatment, the knee joints of the dead rats were dissected, and as a result, only the group treated with TePN showed a significantly rare inflammatory reaction compared to the other groups (Figure 13). For morphological studies, X-ray and μ-CT images were also taken and shown in Figure 14(a). The saline-treated group showed advanced osteoarthritis with severe cartilage defects and cartilage destruction. However, each TePN-treated group showed well-maintained morphological characteristics, indicating that the inflammation at the initial stage of OA was well suppressed by sustained-release TCA. Furthermore, all TePN hydrogel groups showed a significant improvement in the anti-OA effect compared to the TCA solution group. The destroyed cartilage state in the direct solution injection group of TCA reflects the failure of long-term inflammation treatment and prevention of cartilage degeneration. The pathological state of cartilage in the TCA solution group was similar to or worse than that in the saline-treated group. This was expected because the possible side effects of direct IA injection of TCA on chondrocyte toxicity and local treatment with high-dose TCA can reduce hydroxyproline production, inhibit fibroblast growth and collagen synthesis, thereby reducing the structural stability of the connective tissue around the knee. For statistical evaluation, the distance of the articular cartilage (DIC) was measured (Figure 14(b)). Many DICs increased similarly in both the left and right sides in the saline group and the TCA solution-treated group, but only a statistically significant difference was confirmed between the saline group and the high-concentration TCA solution (1.5 mg / mL) group in the left part of the cartilage. Severe destruction of cartilage may occur for two reasons: 1) the possibility of toxicity of direct high-dose TCA exposure in a short time and 2) the inappropriate sustained anti-inflammatory effect due to the rapid removal of TCA.

[0200] In addition, histological staining demonstrated a contrasting result where cartilage degeneration was well-prevented in the TePN hydrogel group, while OA treatment failed in the direct injection group of the TCA solution (Figure 15). Hematoxylin and eosin (H&E) and safranin O staining showed the morphological characteristics of each experimental group. In the saline and TCA solution treatment groups, the cartilage became thinner, the tissue with chondrocytes was lost, and the characteristic red staining of glycosaminoglycans, which was confirmed in healthy cartilage, disappeared. Such results indicate that the treatments with saline and TCA solution have little effect on the progression of OA after the initial onset. However, the TePN hydrogel group showed morphological similarity to normal cartilage, and the cellular characteristics of chondrocytes were well-maintained. In this experiment, it was confirmed that the sustained release of TCA by a single injection of the TePN hydrogel system could effectively prevent further inflammation and the progression of OA.

[0201]

[0202] Example 8. Confirmation of the effect of sustained TCA release using the TePN hydrogel system on the suppression of genes for systemic inflammation and the activation of anti-inflammatory genes

[0203] The changes in the gene expression of inflammation during the experimental period were evaluated. The expression levels of genes for systemic inflammation and anti-inflammation were evaluated from calibrated blood samples. One week after the onset of MIA-induced osteoarthritis, the expression levels of genes for MMP-3, MMP-13, IL-6, and TNF-α, which are systemic inflammatory cytokines, increased 5.60, 4.25, 4.80, and 5.35 times, respectively, compared to the normal group (Fig. 16(a)). The expression patterns of genes to be controlled in each experimental group were observed at 1 week, 4 weeks, and 8 weeks after the injection of TePN or TCA solution. The increased expression levels of genes for systemic inflammatory cytokines decreased over time in the TePN hydrogel group, while they continuously increased in the TCA solution group. Since the expression of systemic inflammatory cytokines and MMPs causes cartilage degeneration, the degradation of cartilage could be successfully prevented by effectively reducing inflammatory cytokines and MMPs cytokines. After 8 weeks, in the TePN hydrogel with the highest TCA concentration, the gene levels of MMP-3, MMP-13, IL-6, and TNF-α cytokines were confirmed to have decreased 1.62 times, 1.67 times, 1.49 times, and 1.62 times, respectively, compared to the saline-treated group. In contrast, the gene levels of each systemic inflammatory cytokine in the TCA solution group continuously increased over time. Since directly administered TCA is rapidly removed, the anti-inflammatory effect was insufficient to prevent extracellular matrix degradation and cartilage collagen degradation by a single IA injection of the TCA solution.

[0204] Genes of anti-inflammatory cytokines such as IL-4, IL-10, and IL-13 were simultaneously monitored ((b) in Fig. 16). The expression of such genes is known to be related to the stimulation of the survival, proliferation, and differentiation of immune cells such as macrophages and specific T cells (Th2). In addition, the degree of expression of such cytokines can serve as a criterion for inhibiting the synthesis of systemic inflammatory cytokines such as interferon-γ, interleukin, and TNF-α. One week after the onset of MIA-induced osteoarthritis, the expression levels of IL-4, IL-10, and IL-13 genes were almost similar to the steady state in all treatment groups. There was no significant difference among the treatment groups until 4 weeks after inflammation induction. However, after 8 weeks, the expression levels of each gene significantly increased in the TePN hydrogel-treated group. In addition, the margin of increase in gene expression was expanded depending on the concentration of loaded TCA. After 8 weeks, in the TePN hydrogel with the highest TCA concentration, the expression levels of IL-4, IL-10, and IL-13 genes were 1.91-fold, 2.25-fold, and 1.92-fold higher than those in the saline-treated group, respectively. Such results support the efficiency of sustained TCA release and the long-term anti-inflammatory effect by a single injection. There were no abnormal symptoms with respect to body weight (Fig. 17). Treatment with PN hydrogel without TCA did not show a change in the expression levels of systemic inflammatory and anti-inflammatory genes. That is, it shows that the carrier PN hydrogel system does not affect the systemic inflammatory response.

[0205]

[0206] Example 9. Optimization of Release Characteristics by the Molar Ratio of IleOEt / AMPEG

[0207] Since the hydrophobic properties of PN, the related drug loading capacity, and the sustained release capacity vary depending on the molar ratio of IleOEt / AMPEG, an attempt was made to adjust the molar ratio of IleOEt / AMPEG to confirm a polymer composition having optimal release characteristics.

[0208] Poly[(isoleucine ethyl ester) 1.47 (aminomethoxypolyethylene glycol 750) 0.24 (ethanol)0.21 (acid) 0.08 phosphazene n (hereinafter, PPZ 43), poly[(isoleucine ethyl ester) 1.37 (aminomethoxypolyethylene glycol 750) 0.25 (ethanol) 0.27 (acid) 0.11 phosphazene n (hereinafter, PPZ 46), and poly[(isoleucine ethyl ester) 1.44 (aminomethoxypolyethylene glycol 750) 0.30 (ethanol) 0.08 (acid) 0.18 phosphazene n (hereinafter, PPZ 24) was prepared as follows, and the NMR data of the prepared PPZ are shown in Fig. 18.

[0209] PPZ 43 was prepared as follows. The precursor polymer (PP) was prepared as follows. First, IleOEt·HCl (24.65 g, 112.98 mmol) was suspended in anhydrous THF (200 mL), and the reaction flask was cooled. Subsequently, poly(dichlorophosphazene) (10.00 g, 8.63 mmol) was dissolved in anhydrous THF (200 mL), and then slowly added to the reaction flask containing IleOEt·HCl (24.65 g, 112.98 mmol) dissolved in anhydrous THA and TEA. The reaction mixture was stirred in a dry ice bath for 12 hours and then at 45 °C for 24 hours. Subsequently, AEtOH (1.05 g, 17.26 mmol) and AMPEG750 (22 g, 29.34 mmol) were dissolved in anhydrous THF and added to the reaction mixture. The reaction mixture was stirred at room temperature for 24 hours and then at 45 °C for an additional 24 hours. The reaction mixture was filtered, poured into n-hexane to obtain a precipitate, and then reprecipitated twice in the same solvent system. The polymer product was further purified by dialysis against methanol at room temperature for 4 days and against distilled water at 4 °C for 4 days using a dialysis membrane (Spectra / Por, MWCO: 10 - 12 kDa). The dialysate was lyophilized to obtain PP.

[0210] To replace the hydroxyl terminus with a carboxylic acid terminus, PP (10 g, 12.25 mmol) was dissolved in dry THF. To this was added a reaction mixture of glutaric anhydride (2.80 g, 24.5 mmol) and dimethylaminopyridine (2.99 g, 24.5 mmol), and the mixture was stirred at room temperature for 12 hours and then further heated to 40 - 45 °C for 24 hours. The CP of the final product was dialyzed against methanol at room temperature for 3 days and against water at 4 °C for 3 days using a dialysis membrane. The purified CP was obtained by lyophilization. The newly appeared peak at 2.1 - 2.32 ppm (b, 4H, CH2) indicates the generated glutaric acid terminus that does not exist in the CP 1 in the 1H NMR.

[0211] PPZ 46 was prepared as follows. The precursor polymer (PP) was prepared as follows. First, IleOEt·HCl (23.98 g, 122.53 mmol) was suspended in anhydrous THF (200 mL), and the reaction flask was cooled. Subsequently, poly(dichlorophosphazene) (10.00 g, 8.63 mmol) was dissolved in anhydrous THF (200 mL) and then slowly added to the reaction flask containing IleOEt·HCl (23.98 g, 122.53 mmol) dissolved in anhydrous THA and TEA. The reaction mixture was stirred in a dry ice bath for 12 hours and then stirred at 45 °C for 24 hours. Subsequently, AEtOH (1.32 g, 21.57 mmol) and AMPEG750 (21.36 g, 28.48 mmol) were dissolved in anhydrous THF and added to the reaction mixture. The reaction mixture was stirred at room temperature for 24 hours and then further stirred at 45 °C for 24 hours. The reaction mixture was filtered, poured into n - hexane to obtain a precipitate, and then reprecipitated twice in the same solvent system. The polymer product was further purified by dialysis against methanol at room temperature for 4 days and against distilled water at 4 °C for 4 days using a dialysis membrane (Spectra / Por, MWCO: 10 - 12 kDa). The dialysate was lyophilized to obtain PP.

[0212] To replace the hydroxyl terminus with a carboxylic acid terminus, PP (10 g, 12.25 mmol) was dissolved in dry THF. Anhydrous glutaric acid (2.80 g, 24.5 mmol) and dimethylaminopyridine (2.99 g, 24.5 mmol) were added to this, and the reaction mixture was stirred at room temperature for 12 hours and then further heated to 40 - 45 °C for 24 hours. The CP of the final product was dialyzed against methanol at room temperature for 3 days and against water at 4 °C for 3 days using a dialysis membrane. The purified CP was obtained by lyophilization. The newly appeared peak at 2.1 - 2.32 ppm (b, 4H, CH2) indicates the generated glutaric acid terminus that does not exist in the CP 1 in the 1H NMR.

[0213] PPZ 24 was prepared as follows, and the precursor polymer (PP) was prepared as follows. First, IleOEt·HCl (26.43 g, 134.61 mmol) was suspended in anhydrous THF (200 mL), and the reaction flask was cooled. Subsequently, poly(dichlorophosphazene) (10.00 g, 8.63 mmol) was dissolved in anhydrous THF (200 mL) and then slowly added to the reaction flask containing IleOEt·HCl (26.43 g, 134.61 mmol) dissolved in anhydrous THA and TEA. The reaction mixture was stirred in a dry ice bath for 12 hours and then stirred at 45 °C for 24 hours. Subsequently, AEtOH (1.05 g, 17.26 mmol) and AMPEG750 (15.53 g, 20.71 mmol) were dissolved in anhydrous THF and added to the reaction mixture. The reaction mixture was stirred at room temperature for 24 hours and then further stirred at 45 °C for 24 hours. The reaction mixture was filtered, poured into n - hexane to obtain a precipitate, and then reprecipitated twice in the same solvent system. The polymer product was further purified by dialysis against methanol at room temperature for 4 days and against distilled water at 4 °C for 4 days using a dialysis membrane (Spectra / Por, MWCO: 10 - 12 kDa). The dialysate was lyophilized to obtain PP.

[0214] To replace the hydroxyl terminus with a carboxylic acid terminus, PP (10 g, 12.25 mmol) was dissolved in dry THF. To this was added a reaction mixture of glutaric anhydride (2.80 g, 24.5 mmol) and dimethylaminopyridine (2.99 g, 24.5 mmol), and the mixture was stirred at room temperature for 12 hours and then further heated to 40 - 45 °C for 24 hours. The CP of the final product was dialyzed against methanol at room temperature for 3 days and against water at 4 °C for 3 days using a dialysis membrane. The purified CP was obtained by lyophilization. The newly appeared peak at 2.1 - 2.32 ppm (b, 4H, CH2) indicates the generated glutaric acid terminus that does not exist in the CP 1 in the 1H NMR.

[0215] The T max of the produced PPZ43, 46 and 24 was measured and shown in Fig. 19(a). As shown in the figure, the higher the molar ratio of IleOEt / AMPEG, the stronger the hydrophobicity and the lower the T max value. Fig. 19(b) shows the cumulative release amounts over time in mg and % respectively after loading TCA onto each compound. It was confirmed that the stronger the hydrophobic property, the more the sustained release of the hydrophobic drug TCA.

[0216] Referring to the release profiles of TCA shown in Example 6 and Fig. 12, the release rate of TCA in TePN1.5 (TCA-encapsulated PN loaded at a concentration of 1.5 mg / ml) was 0.011 - 0.018 mg / day (11 - 18 μg / day). TePN is applied in the body at about 200 - 300 μl and gradually decomposes over about 30 days to release TCA. Therefore, an attempt was made to calculate the amount of TCA that can be released and exposed in the body per day and apply it to the in vitro experiment. For this purpose, the 1.5 mg / ml TCA loaded on TePN1.5 was diluted 20 - 160 times respectively to be loaded at concentrations of 9.375, 18.75, 37.5, and 75 μg / ml.

[0217] The complex of TCA and PPZ carried was treated with chondrocytes in an induced inflammatory environment, and the expression of inflammation-related factors was measured by PCR. The induction of the inflammatory environment was achieved by treating human chondrocytes with IL-1β at 10 ng / ml to form an in vitro arthritis model. The relative expression levels of MMP3, MMP13, IL-6, and TNF-α when treated with the TCA-PPZ complex carried at each of the above concentrations in the model were measured and shown in Figure 20. As can be seen from the figure, MMP3, MMP13, and IL-6 (inflammatory cytokine) increased rapidly after treatment with IL-1β, confirming that the inflammatory environment was successfully induced. At this time, when treated with the TCA-PPZ complex at 9.375 - 37.5 μg / ml, it was confirmed that the expression of MMP3, MMP13, and IL-6 decreased rapidly. In contrast, at the highest dose of 75 μg / ml, the decrease in relative expression was less, indicating that the anti-inflammatory effect decreased more than that at the concentration of 9.375 - 37.5 μg / ml. That is, it is shown that high-dose TCA can have an adverse effect, suggesting that the property of the drug delivery system that gradually releases a certain concentration of the drug is more useful for OA treatment.

[0218] To confirm the optimal molar ratio of IleOEt / AMPEG, experiments were conducted as follows. In the in vitro release experiment, it was confirmed that 200 ul of the TCA-PPZ complex was released in the amounts shown in Table 2.

[0219]

Table 2

[0220]

[0221] Based on this, assuming the case of treatment with 5 ml of the TCA-PPZ solution, the predicted values of the amount of TCA released were calculated and shown in Table 3 below. The in vitro OA model mainly involves treating chondrocytes with IL-1β, and often uses 10 ml of the culture medium. Assuming the case of treating with 5 ml of the TCA-PPZ hydrogel, which is half of the volume of the culture medium.

[0222]

Table 3

[0223]

[0224] Based on the predicted values in Table 3 above, in order to confirm the optimal molar ratio of IleOEt / AMPEG, the following calculations were performed. First, since the volume of the total culture medium was 10 ml, the value obtained by dividing the predicted value of the TCA release amount in Table 3 by 10 was predicted to be the TCA release concentration in μg / ml units. The predicted TCA release concentrations were displayed for each date and are shown in Fig. 21. Referring to Fig. 21, the polymer composition in which the expression levels of MMP3, MMP13, and IL-6 are all included within the optimal range of 9.375 μg / ml to 37.5 μg / ml from the first day to the fourth day corresponds to 43GA, and the molar ratio of IleOEt / AMPEG was confirmed to be approximately 6.125, and it was found that the best sustained-release characteristics are exhibited within the range of this molar ratio.

[0225]

[0226] In Examples 10 and later described below, the preparation of poly(organic phosphazene) for loading celecoxib (CXB) and its effects are disclosed. A schematic diagram of the treatment of poly(organic phosphazene) loaded with celecoxib in Achilles tendonitis (AT) is shown in Fig. 22.

[0227]

[0228] Example 10. Experimental method B

[0229] Example 10-1. Materials

[0230] Hexachlorocyclotriphosphazene (Sigma-Aldrich, USA) was recrystallized at 60 °C under vacuum for purification. Poly(dichlorophosphazene) was synthesized as disclosed in Example 2 above. l-Isoleucine ethyl ester hydrochloride (IleOEt·HCl) (A&Z food additives, Hangzhou, China) was obtained and dried under vacuum conditions at 55 °C for 10 days. Methoxy Polyethylene glycol (750 Da) was obtained from Sigma-Aldrich (USA) and replaced with α-amino-ω-methoxy-poly(ethylene glycol) (AMPEG). Ethanolamine (Sigma-Aldrich, USA), glutaric anhydride (Alfa Aesar, USA), 4-(dimethylamino)pyridine (DMAP) (Alfa Aesar, USA) were prepared. Tetrahydrofuran (THF) and triethylamine (TEA) were dissolved in nitrogen gas, and THF was dried using sodium metal (Aldrich, USA) and benzophenone (Daejung, Korea). TEA was dried using barium oxide (Daejung, Korea). Celecoxib (CXB) was obtained from EDQA (Europe).

[0231]

[0232] Example 10-2. PNP Synthesis

[0233] The reaction was carried out under a dry nitrogen atmosphere using a standard Schlenk line. IleOEt·HCl (10.38 g, 53.06 mmol) was dissolved in anhydrous THF. The reactor was stirred in a dry ice water bath, and 50 mL of dry TEA was added. Poly(dichlorophosphazene) (5.00 g, 43.14 mmol) was dissolved in dried THF and added to the reactor. The reaction temperature was set at 50 °C for 24 h, and the polymer was further reacted for 48 h by adding ethanolamine (0.91 g, 15.1 mmol) and AMPEG (13.59 g, 18.12 mmol). The polymer solution was filtered, and the solvent was removed using a rotary evaporator. The polymer was precipitated by pouring it twice into n-hexane (Daejung, Korea), and further purified using a dialysis membrane (Spectra / Por, MWCO: 10 - 12 kDa) against methanol for 4 days and using distilled water for 4 days. The purified polymer solution was filtered using a syringe filter with 0.45 μm pores and freeze-dried to obtain aminoethanol-bonded poly(organophosphazene). Dry THF was added to aminoethanol-poly(organophosphazene) (10 g, 158.93 mmol) to bond with carboxylic acid groups. Anhydrous glutaric anhydride (5.98 g, 52.41 mmol) and DMAP (6.40 g, 52.41 mmol) were dissolved in anhydrous THF and added to the aminoethanol PPZ solution. Then, the solution was stirred and incubated at 45 °C for 24 h. Next, the polymer was dialyzed against methanol using a dialysis membrane for 4 days and using distilled water for 4 days before freeze-drying. Through such a manufacturing process, a polymer composed of (IleOEt), (AMPEG750), (EtOH), and (Acid) could be obtained.

[0234]

[0235]

[0236] Example 10 - 3. Characterization of PNP

[0237] 1 The results of 1H NMR were as follows.

[0238] 11H NMR (CDCl3), δ (ppm): 0.8 - 1.0 (s, 6H), 1.1 - 1.3 (b, 3H), 1.3 - 1.6 (b, 2H), 1.6 - 1.9 (b, 1H), 2.1 - 2.3 (b, 4H), 2.8 - 3.3 (b, 2H), 3.4 - 3.8 (b, 62H), 3.9 (s, 1H), and 4.0 - 4.3 (b, 3H)

[0239] The molecular weight of the polymer was measured using a gel permeation chromatography system (EcoSEC HLC - 8320 GPC, Tosoh) and a refractive index detector. Two Styragel columns (TSKgel Supermultipore HZ - M and TSKgel SuperHZ - 2500) were connected and used at a flow rate of 0.35 mL min-1 at 40 °C. THF was used as the mobile phase together with 0.1% (w / v) tetrabutylammonium bromide. Polystyrene (MW: 162; 580; 1920; 3090; 9590; 27 810; 70 500; 133 500; 290 300; 729 500; 1 074 000; and 2 703 000 Da) was used as the standard. Fourier transform infrared spectroscopy (FTIR) was performed using an FTIR spectrometer (Nicolet i20 FTIR spectrometer, Thermo Fisher Scientific) to characterize the presence of CXB and PNP with PCNP. The sample was dissolved in chloroform at 1 wt% and coated on a KBr circular cell (Sigma - Aldrich, USA). After drying the chloroform, the coated sample was analyzed and scanned 32 times in the wavenumber range of 4000 - 500 cm-1 to obtain the FTIR spectrum.

[0240]

[0241] Example 10 - 4. Cell viability test

[0242] The NIH3T3 fibroblast cell line and PNP hydrogel serially diluted (0 - 10 mg / mL) were used. The cells were seeded at 2×10 4Cells were dispensed at a density of cells / well and cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin in a 37°C, CO₂ incubator for 24 hours. The culture medium was replaced with serum-free DMEM with different concentrations of PNP, CXB, and PCNP. After 24 hours, MTT solution (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) was added, and then incubated in a 37°C, CO₂ incubator for 2 hours. Dimethyl sulfoxide (200 μl; Daejung, Korea) was added, and the absorbance of the solution was measured at 570 nm using a Spectra MAX 340 spectrophotometer (BIO-RAD, Hercules, CA). The viability of NIH3T3 cells was calculated using the absorbance of the experimental group and the control group with the following formula.

[0243] Cell viability (%) = Absorbance (experimental group) / Absorbance (control group) × 100

[0244]

[0245] Example 10-5. Rheology Test

[0246] The temperature-sensitive sol-gel transition of PPZ was measured using a rheometer (Anton Parr, AT). PNP and PCNP hydrogel solutions were prepared at a concentration of 10 wt%. The modulus was measured at the exact temperature. The rheometer was used with a 25.0 mm parallel plate and a 0.5 mm zero gap. The modulus was measured under oscillatory stress at a frequency of 0.8 Hz, 10% strain, and various temperatures. A frequency sweep test was also performed at 37°C under 10% shear strain. All modulus data were analyzed using Anton Parr software of Rheocompass.

[0247]

[0248] Example 10-6. Acquisition of Cryo-Scanning Electron Microscope (cryo-SEM) Images

[0249] The Quanta 3D FEG (FEI, Netherlands) equipped with an Alto 2500 cryo-transfer system (Gatan, UK) was used for cryo-SEM experiments. The PNP and PCNP samples were quickly immersed in a liquid nitrogen slush over 0.5 seconds. The cryo chamber emptied after a few seconds, and the samples were transferred to a preparatory chamber that had been pre-evacuated to about -190 °C at 10 -5 mbar. Metal evaporation was performed by a plasma sputterer at a current of 3 mA for 60 seconds. The metal-coated samples were vacuum-treated at a pressure of 10 -5 mbar and transferred to a microscope chamber that had been pre-cooled to a temperature of -190 °C. The cryo-SEM images were acquired with a 5-keV beam at 11.8 pA.

[0250]

[0251] Example 10-7. Dynamic Light Scattering (DLS)

[0252] The particle sizes of PNP, CXB, and PCNP were examined using a Zetasizer Nano ZS (Malvern Instruments Ltd., Malvern, UK). The nanoparticles were dissolved in phosphate-buffered saline (Well Gene, KR) at a concentration of 0.1 wt%, and the particle sizes were measured at 5, 25, 37, and 45 °C over 2 cycles.

[0253] For zeta potential evaluation, 0.5 wt% of PNP and PCNP (4 mg of CXB) were prepared at room temperature. The measurements were performed using DLS (Malvern Instruments Ltd., Malvern, UK).

[0254]

[0255] Example 10-8. Hydrogel Degradation In Vitro and In Vivo

[0256] To confirm swelling and degradation in vitro, 200 μl of 10 wt% PNP hydrogel was weighed in an insert well and incubated in an oven at 37 °C for 5 min to induce complete gelation. Subsequently, the hydrogel block was immersed in PBS at 37 °C. The change in the mass of the hydrogel was measured at determined time points.

[0257] To measure the swelling and degradation of PNP, PNP was dissolved in PBS at 10 wt% and stirred at 4 °C. Next, 200 μl of the polymer solution was slowly subcutaneously injected into the back of each mouse (CrljOri:CD1(ICR), 6 weeks old, male, Orient Bio, Korea). The injected PNP was extracted and the remaining amount was measured at regular time points.

[0258]

[0259] Example 10 - 9. In vitro release of CXB from PCNP

[0260] The PCNP hydrogel was prepared by stirring 10 wt% of PNP with CXB. Subsequently, PNCP (200 μl) was placed in a Millicell (diameter: 12 mm, Millipore, US) and placed in an oven at 37 °C for 5 min. Subsequently, the PCNP was immersed in a 15 - mL conical tube containing 6 mL of PBS and cultured in a shaker bath (KMC - 12 - 55W1, Vision, KR) at 37 °C and 50 rpm. The PBS was changed at specific time points. The released CXB was measured using high - performance liquid chromatography (Agilent Technologies) with methanol (75%) and water (25%) as the mobile phase.

[0261] The kinetic model was used to fit the experimental data. This model includes

Equation

Equation

[0262]

[0263] Example 10 - 10. In Vivo Tendon Regeneration Test

[0264] All experiments using mice were conducted in accordance with the relevant laws and institutional guidelines of the Animal Experimentation Committee (IACUC) of Korea University Guro Hospital and received IACUC approval (approval number KOREA - 2018 - 0049). Collagenase (Thermo Fisher Inc., US, 50 μL) was injected into 9 - week - old mice (DooYeol Biotec, Seoul, KR), and after leaving them for 1 week, a CXB solution with a concentration of 1 or 4 mg / (100 μL) dissolved in DMSO was injected to treat the damaged tendon. PCNP (CXB 1 mg) and PCNP (CXB 4 mg) hydrogels were prepared by stirring 1 mg or 4 mg of CXB into 100 μL of 10 wt% PNP hydrogel at 4°C. Then, the PCNP hydrogel was injected into the damaged tendon model.

[0265]

[0266] Example 10 - 11. Real - Time Polymerase Chain Reaction (RT - PCR)

[0267] Blood samples were collected 4 weeks after injecting the CXB solution or PCNP into the damaged tendon. An RNA blood kit (QIAGEN, QIAamp) was used for RNA extraction. cDNA was synthesized using Accupower CycleScript RT Premix (dT20) (BIO - RAD). Real - time polymerase chain reaction (RT - PCR) was performed using an ABI7300 real - time thermal cycler (Applied Biosystems, USA) to examine inflammatory cytokines (COX - 2, IL - 1, IL - 6, MMP - 3, MMP - 13, and TNF - α), and anti - inflammatory cytokines (IL - 4 and IL - 10). The gene marker sequences are shown in Table 4 below.

[0268]

Table 4

[0269]

[0270] Example 10 - 12. Histological Processing and Analysis

[0271] The damaged tendons treated with PCNP or CXB solution were extracted and washed with DPBS. Each tendon was immersed in 4% paraformaldehyde for 24 hours to fix the tissue, then the tendon was rinsed and cut with paraffin (5 - μm sections). The paraffin sections were placed in an oven at 60°C and the paraffin was removed with xylene containing 100%, 90%, 80% and 70% ethanol. The sections were stained with H&E and MT. Each group was triplicated (n = 3).

[0272]

[0273] Example 10 - 13. Analysis of Hydroxyproline Content

[0274] To demonstrate the relaxation of AT by PCNP and CXB solution treatment, the collagen content of mouse tendon tissue was evaluated by hydroxyproline analysis. To hydrolyze the tendon tissue, 5 mg was immersed in 6N HCl solution and heated at 120°C for 3 hours. After hydrolysis, the tendon tissue was centrifuged at 3000 rpm for 3 minutes, and the supernatant was collected and transferred to a 96 - well plate. Then, the 96 - well plate was incubated in a drying oven at 60°C for 12 hours. Chloramine T reagent (6 μL) was mixed with oxidation buffer (94 μL), and the mixture was added to each sample or standard solution. The resulting solution was slowly transferred to the 96 - well plate, and p - dimethylaminobenzaldehyde reagent (100 μL) was added to each sample or standard, followed by incubation at 60°C for 90 minutes. The hydroxyproline content was measured at an optical density of 450 nm using a Flash Multimode Reader (Varioskan TM , Thermo Scientific, USA).

[0275]

[0276] Example 10-14. Biomechanical Test

[0277] To confirm the presence of AT, biomechanical examinations were performed using the calcaneus-Achilles tendon selected from mice. Each sample was loaded onto an Instron Mechanical Tester (AG-10KNX, Shimadzu, Japan) using a specially designed device and then pulled in the sagittal direction at a crosshead speed of 5 mm / min with a preload of 1 N. Tensile strength and Achilles tendon stiffness were measured.

[0278]

[0279] Example 10-15. Analysis of Statistical Data

[0280] All quantitative results are presented as mean values and standard deviations. Graphs were drawn using Origin software. Statistical analysis was performed using Student’s t-test and expressed as *P<0.05, **P<0.01.

[0281]

[0282] Example 11. Synthesis and Characterization of PCNP Hydrogel

[0283] Temperature-sensitive PPZ nanoparticles (PNP) hydrogel was designed for in-situ gelation and hydrophobic drug encapsulation. PNP was synthesized by substituting hydrophobic IleOEt and hydrophilic AMPEG onto the PPZ backbone. The synthesized PNP structure was 1 confirmed by 1H NMR spectroscopy and shown in Figure 23. Carboxylic acid groups such as chondroitin sulfate and hyaluronic acid are replaced in the polymer backbone with negatively charged functional groups, which mimic the natural ECM and are known to induce interaction with cytokines during tissue regeneration. The carboxylic acid groups of PNP were 1Quantification was performed using ¹H NMR. The peaks at 2.0 and 2.5 indicated that the acidic groups were successfully bound to the PNP hydrogel backbone. The molecular weight of 62,920 Da for PNP was obtained using GPC with polystyrene as the standard substance (Fig. 23(c)). The ratio of the substituted side chains was calculated using the ¹H NMR spectrum, and the results showed the presence of side chains with IleOEt, AMPEG, EtOH, and carboxylic acid groups at ratios of 71%, 17%, 1%, and 11%, respectively. The high hydrophobic IleOEt content induced all hydrophobic gelation and tight hydrophobic interactions with CXB. The FTIR spectra of PCNP, PNP, and CXB were analyzed to confirm the CXB loading of PCNP, as shown in Fig. 24. The aromatic C═C peaks at 1530–1630 cm⁻¹ observed in the PCNP data indicated CXB loading. 1 The ¹H NMR spectrum was used for calculation, and the results showed the presence of side chains with IleOEt, AMPEG, EtOH, and carboxylic acid groups at ratios of 71%, 17%, 1%, and 11%, respectively. The high hydrophobic IleOEt content induced all hydrophobic gelation and tight hydrophobic interactions with CXB. The FTIR spectra of PCNP, PNP, and CXB were analyzed to confirm the CXB loading of PCNP, as shown in Fig. 24. The aromatic C═C peaks at 1530–1630 cm⁻¹ observed in the PCNP data indicated CXB loading.

[0284] CXB is generally used medicinally by dissolving it in an organic solvent or through chemical modification. However, CXB was successfully dissolved in PNP simply by mixing it in a solution state without a solvent or chemical modification. The gel state was maintained by the temperature-sensitive gelation properties of PNP, as shown in Fig. 25. PCNP was manufactured by encapsulating CXB in PNP by stirring CXB in a 10 wt% PNP solution at 4 °C. The temperature-dependent gelation of PCNPs was confirmed by injecting the polymer solution into water at 4 °C and 37 °C. Different from the polymer solution at 4 °C, when a group at 37 °C was injected into the aqueous solution, a gel-like 3D structure was observed, as shown in Fig. 26. This means that after in vivo injection, a stable 3D structure can be formed without dispersion because the sol–gel transition is likely to gel within seconds. The storage and loss elastic moduli of the PNP and PCNP hydrogels were monitored at precisely controlled temperatures in the range of 5–60 °C to confirm the temperature-dependent quantitative rheology of the gels. The sol–gel transition of the PNP and PCNP hydrogels occurred within a specific temperature range (20–25 °C) and maintained the gel state at body temperature (37 °C). The storage elastic modulus began to increase with temperature, and the highest value was observed at approximately 35–38 °C. This is shown in Fig. 27.

[0285] The storage and loss coefficients of PNP were affected by the CXB concentration ((b) to (d) in Fig. 27). The maximum storage modulus of the PNP hydrogel was 454.3 Pa at 35.1 °C. However, the maximum storage moduli of the PNP (CXB 1 mg) and PNP (CXB 4 mg) hydrogels were 655.0 Pa at 38.1 °C and 994.3 Pa at 37.6 °C, respectively. No significant differences were observed in the temperature and maximum storage elastic modulus between the PNP and PNP hydrogel groups. However, the maximum storage elastic modulus values increased after integrating CXB. This suggests that the hydrophobic interaction between the PNP hydrogel and CXB molecules increases the modulus because CXB acts as a hydrophobic crosslinking agent. Such data indicate a successful interaction between the PNP and CXB molecules.

[0286]

[0287] Example 12. Complexation of CXB and PNP

[0288] The hydrophobic CXB was clearly homogeneously dissolved and well distributed in the PNP solution. PCNP was formed after simple stirring at low temperature, which corresponds to an obvious advantage of this system. Toxic solvents or surfactants are generally hydrophobic and are required to improve the solubility of CXB in clinical trials. However, the fact that CXB can be easily integrated into PNP at low temperature without toxic solvents or surfactants is a considerable advantage.

[0289] The amphiphilic PNP hydrogel network was observed to be formed at a specific concentration and body temperature by hydrophobic interactions between polymer chains. However, the amphiphilic nature of PNP means that the polymer structure self-organizes to form polymer micelle particles at low concentration (0.5 wt%) via the folding of isolated chains. The morphologies of PNP and PCNP were characterized in both the gel state and the diluted state (Figure 28(a)), and the results indicate that the PNP and PCNP hydrogels have a porous structure with pores of sizes 6.2 and 4.9 μm, respectively. PCNP showed a rougher surface structure than PNP. However, no CXB aggregation was observed, indicating that the hydrophobic CXB was well integrated into the PCNP hydrogel matrix. After 20-fold dilution, micelle formation by self-organization occurred in both PNP and PCNP, and the particle structure could be observed. A wide range of particle sizes was measured. However, the particles in the PCNP group were smaller than those in the PNP group. Therefore, the hydrogel and particle morphologies of PNP and PCNP hydrogels suggest that CXB was successfully encapsulated in PNP and that the PCNP morphology was affected by the encapsulated CXB.

[0290] To further confirm the hydrophobic interaction between PNP and CXB, different concentrations of CXB were added to examine the size change of polymer nanoparticles before and after mixing ((b) in Figure 28). The experiment was conducted under precise temperature conditions so that the difference in particle size due to the hydrophobic interaction between PNP and CXB molecules could be observed. It was observed that the particle size of PNP gradually decreased from 240.0 nm to 126.2 nm as the temperature increased from 4 °C to 45 °C. This phenomenon was the result of the correlation between temperature and hydrophobicity. The hydrophobic moieties interacted tightly at high temperatures to form polymer nanoparticles in a contracted form. The sizes of PNP and PCNP particles were similar regardless of temperature. This indicates that the hydrogel network has stable temperature sensitivity and reversible physical properties due to non-covalent interactions, providing various advantages including simple drug loading and injectability. After CXB was integrated into PNP, the particle size decreased as the concentration of CXB increased at all temperatures. This means that the CXB molecules inside PNP act as hydrophobic cross-linking agents, inducing PCNP to form a more contracted structure. Therefore, the interaction between CXB and the PNP hydrogel means that CXB can be encapsulated in PNP at various concentrations without a surfactant, positively improving the therapeutic effect and reducing the side effects associated with this strategy.

[0291] The zeta potential was examined together with the particle size and is shown in (c) of Figure 28. The zeta potential of PNP was measured to be -19.9 mV due to the carboxyl groups of the PNP backbone. However, the zeta potential decreased significantly to -13.1 and -9.5 mV for PCNP (CXB 1 mg) and PCNP (CXB 4 mg), respectively, after CXB was included in PNP. This is because the hydrophobic CXB with a smaller particle size than PNP is encapsulated in the core of the PCNP micelle structure ((b) in Figure 28). This means that the hydrophilic PEG on the surface of PCNP covers the negative charge. Therefore, the hydrophobic interaction successfully bound CXB to PNP, forming PCNP with small pores and particle size.

[0292]

[0293] Example 13. Mass loss of PCNP hydrogels and sustained release of CXB in vitro and in vivo

[0294] To predict the maintenance requirements of PCNP hydrogels after in vivo injection, the mass loss of the hydrogels was measured and the release of CXB from the hydrogels was analyzed to confirm whether the PCNP hydrogels could maintain and release CXB over a long period, which is shown in Fig. 29. The in vitro degradation data showed the swelling and degradation patterns in an aqueous environment. The PNP hydrogels swelled to 147% in 3 days and then slowly dissolved over 5 weeks (Fig. 30). As shown in (a) of Fig. 29, the CXB release rate of the PCNP hydrogels was maintained almost constant for 29 days even when high swelling occurred due to the stable interaction between the PNP hydrogels and CXB. The CXB release rate was higher in the group with 1 mg of CXB, which is presumably because the increased hydrophobic interaction between CXB and PNP with a higher CXB concentration induced a denser structure.

[0295] PCNP was biodegraded by hydrolysis and CXB was released over 28 days. The correlation observed between the degradation and release profiles indicates that CXB release is related to the degradation of the PCNP hydrogels. Furthermore, the PCNP swelling observed at the beginning of the experiment (~7 days) did not lead to an explosive release of CXB, which indicates that the release is related to the dissolution of the PCNP hydrogels rather than the swelling.

[0296] The CXB release data was analyzed according to various kinetic models including zero-order, first-order, Higuchi, and Ritger-Peppas models (Table 5 below). Comparison of the correlation coefficient (R2) indicates that the Ritger-Peppas model is most suitable for both PCNP (CXB 1 mg) and PCNP (CXB 4 mg). When using the Ritger-Peppas model, a mixed release mechanism including swelling, erosion, and polymer porosity may be involved. This kinetic model is suitable for use with PCNP because CXB is released by the swelling and degradation of PCNP. Furthermore, the diffusion mechanism can be described by the diffusion exponent (n). The diffusion exponents (n) of PCNP (CXB 1 mg) and PCNP (CXB 4 mg), calculated to be 0.62 and 0.57 respectively, both indicate non-Fickian diffusion.

[0297]

Table 5

[0298]

[0299] To measure the in vivo biodegradation rate, PNP hydrogel was subcutaneously injected into the back of mice. The temperature-sensitive gelation properties of the PNP hydrogel mean that gelation occurs rapidly at body temperature, unlike the dispersion of the polymer solution observed at 4°C (Figure 26). Also, the PNP hydrogel was shown to maintain a stable modulus at angular frequencies in the range of 1 - 100 rad / s at 37°C (Figure 31). This indicates that the PNP hydrogel is suitable for injection and maintains its gel structure in vivo. The mass of the injected PNP hydrogel increased to 111% in 2 days in the in vivo environment and then continuously decreased for more than 28 days ((b) in Figure 29). This indicates that the PNP hydrogel in vivo began to gradually degrade after the initial water absorption into the PNP hydrogel network. Figure 32 shows the gradual decrease in the size of the PNP hydrogel in vivo. Such results indicate that CXB is slowly released after injection of the PCNP hydrogel into the body and that the PCNP hydrogel remains at the injection site for about 1 month.

[0300]

[0301] Example 14. Systemic Inflammatory Cytokine Suppression and Anti-inflammatory Effect of PCNP Hydrogel on Defective Tendons

[0302] Before in vivo experiments, the in vitro cytotoxicity of the PNP hydrogel was examined to confirm side effects ((a) in Fig. 33). The cell viability was maintained at 70% or more even at a high concentration of PNP (10 mg mL-1). The in vivo experiment mass loss data suggested that 200 μg of the injected PNP hydrogel was degraded within 30 days, indicating that about 6.6 μg of PNP was released per day in the injected hydrogel structure (Fig. 29). Therefore, it was confirmed that the PNP hydrogel is a biocompatible drug carrier without side effects, and the PNP hydrogel is degraded into non-toxic substances such as phosphate, poly(ethylene glycol), and ammonia by hydrolysis.

[0303] The cytotoxicity of CXB and PCNP was also confirmed and shown in (b) and (c) of Fig. 33. The CXB group showed high cytotoxicity at 625 μg / ml, while no significant cytotoxicity was observed in the PCNP group at 40,000 μg / ml CXB. This is because the low solubility of CXB causes aggregation, so that cells are exposed to the drug at a high cytotoxicity level at a high concentration. Since PNP can deliver drugs without aggregation or cytotoxicity, it is regarded as a suitable carrier for CXB.

[0304] Since PCNP can effectively encapsulate various CXB dosages, it helps to control appropriate therapeutic dosages for successfully treating various inflammatory diseases. In the present invention, the effects of AT treatment were evaluated using PCNP (CXB 1 mg) and PCNP (CXB 4 mg), which are dosages used as injections.

[0305] CXB increases the expression of anti-inflammatory cytokines such as IL-4 and IL-10, and reduces inflammation and pain. CXB treatment is also known to reduce the expression of systemic inflammatory cytokines (TNF-α, IL-1, IL-6, COX-2, MMP-3 and MMP-13) in cartilage and synovial membrane. Such anti-inflammatory and inflammatory cytokines are directly related to tendon healing. Systemic inflammatory cytokines affect ECM homeostasis and tendon remodeling, but may also promote apoptosis of tendon cells. IL-4 increases the proliferation of tenocytes related to tendon healing, while IL-10 affects cell proliferation and survival via the STAT3 signaling pathway.

[0306] To analyze the suppression of systemic inflammatory cytokines and anti-inflammatory effects of the developed delivery vehicle, the PCNP hydrogel was injected into the Achilles tendon model with defects, and the levels of anti-inflammatory and inflammatory cytokines were measured using RT-PCR (Figure 34). The levels of anti-inflammatory cytokines (IL-4 and IL-10) in the blood were measured 4 weeks after treatment (Figures 34(a) and (b)). Compared with the control group, the expression of IL-4 increased 1.2-fold in the group treated with CXB once and 1.3-fold in the group treated 3 times. Compared with the control group, in the PCNP (1 mg and 4 mg CXB) injection groups, the expression of IL-4 increased 1.5-fold and 1.7-fold respectively, and IL-10 showed a similar pattern of increase, 1.3-fold and 1.5-fold respectively. The PCNP (1 and 4 mg CXB) groups showed increases of 1.8-fold and 2.2-fold respectively. Compared with the control group, the expression of anti-inflammatory cytokines did not change much in the untreated group, suggesting that no anti-inflammatory effect occurs without treatment. The expression level of the anti-inflammatory cytokine gene was higher in the group treated with the CXB solution, and the expression of the anti-inflammatory cytokine was higher after the injection of the PCNP hydrogel. This indicates that CXB injection caused an anti-inflammatory effect, and PCNP injection further enhanced the anti-inflammatory effect by the local and sustained release of CXB in the Achilles tendon with defects.

[0307] The expression of systemic inflammatory cytokines (COX-2, IL-1, IL-6, MMP-3, MMP13, and TNF-α) was also examined, which is shown in FIGS. 34(c)-(h). Severe inflammation in the untreated group was increased 7.6-, 6.4-, 7.1-, 7.1, 8.1-, and 7.8-fold, respectively, compared to the control group in the expression of COX-2, IL-1, IL-6, MMP-3, MMP13, and TNF-α. The expression of COX-2, IL-1, IL-6, MMP-3, MMP-13, and TNF-α of systemic inflammatory cytokines increased 6.3-, 5.4-, 6.0-, 5.9-, 7.2-, and 6.7-fold, respectively, in the CXB solution compared to the control group, which was significantly lower than that observed in the untreated group. In the PCNP (CXB 4 mg) hydrogel injection group, the numerical values of systemic inflammatory cytokines increased 5.6-fold, 4.7-fold, 5.2-fold, 5.1-fold, 6.6-fold, and 5.8-fold compared to the control group. All PCNP injection groups had lower expression levels of most inflammation-inducing cytokines than the CXB solution injection group. The pro-inflammatory cytokine and anti-inflammatory cytokine expression data indicate that single local injection of PCNP hydrogel produces a higher anti-inflammatory effect than multiple injections of CXB solution, which was evaluated to be because CXB is slowly released into the defect area again.

[0308]

[0309] Example 15. Regeneration of a Defective Achilles Tendon in Vivo

[0310] To analyze the in vivo tendon regeneration effect by the local sustained release of CXB from the PCNP hydrogel, a defective Achilles tendon was generated using collagenase, and the same concentration groups were used to examine the regeneration of the defective Achilles tendon (Figure 35). Hematoxylin and eosin (H&E) and Masson’s trichrome (MT) staining data showed the disruption of ECM alignment and collagen degradation in the collagenase 50 group, indicating that the tendon was completely damaged by collagenase (Figure 35(b) and (h)). The damaged tendon was partially regenerated in the CXB solution injection group after 4 weeks (Figure 35(c) and (d)). MT staining data clearly showed that more collagen was regenerated in the group administered with CXB solution three times than in the group injected with CXB only once (Figure 35(i) and (j)). This indicates that the time the defective tendon is exposed to CXB has a positive effect on tendon regeneration. The results of H&E staining showed that the tendon in the PCNP hydrogel treatment group was almost completely recovered with tissue alignment (Figure 35(e) and (f)). The results of MT staining showed that the PCNP hydrogel group exhibited improved collagen regeneration compared to the CXB solution injection group. The CXB concentration in the PCNP hydrogel also affected tendon regeneration (Figure 35(e)–(l)). Such results suggest that continuous CXB exposure of the defective tendon is necessary to induce long-term regeneration of the defective tendon.

[0311] The in vivo regeneration and functional recovery of tendon constructs were examined using hydroxyproline analysis, mechanical stiffness, and strength tests (Figure 36). Collagen, one of the main components of tendon, provides rigid mechanical properties. Collagen is mainly composed of hydroxyproline. Therefore, the amount of this substance present in the tissue indicates the amount of collagen present. The hydroxyproline content of normal tendon tissue at 35.5 μg / mg was shown to significantly decrease to 11.3 μg / mg in the untreated group (Figure 36(a)). Increased hydroxyproline content was observed in the CXB solution (once and three times per week injection) groups at values of 15.3 and 17.2 μg / mg, respectively, and further increases were observed in the hydroxyproline content of the PCNP hydrogel injection group (CXB 1 mg and 4 mg), reaching values of 21.4 and 24.4 μg / mg, respectively. The injection frequency of the CXB solution and the CXB concentration of the PCNP hydrogel affected the hydroxyproline content proportionally. The PCNP hydrogel provided a longer CXB exposure time due to Achilles tendon defects than the CXB solution, and encapsulation of various concentrations of CXB could directly affect the therapeutic effect.

[0312] The stiffness and tensile strength of the treated tendon were measured, and mechanical function regeneration was evaluated by comparison with normal tendons ((b) and (c) of FIG. 36). In the untreated group, the stiffness of the normal tendon showed a significant decrease from 36.3 N / mm to 13.7 N / mm. The groups treated with CXB solution (injections once and three times per week) showed higher stiffness values (16.4 and 18.6 N / mm), but the stiffness increased to 24.5 and 26.4 N / mm in the PCNP hydrogel-treated groups (CXB 1 and 4 mg), similar to the values obtained for normal tendons. The CXB solution-treated groups had improved tendon regeneration ability, but such an effect was much more prominent in the PCNP hydrogel-treated groups. The tensile strength data showed a pattern similar to the stiffness results, with the tensile strength of the normal tendon of 33.7 MPa decreasing to 11.4 MPa in the untreated group. The tensile strength was observed to be 12.5 and 14.7 MPa, respectively, in the CXB solution-treated groups (injections once and three times per week), and the PCNP hydrogel-treated groups (CXB 1 and 4 mg) showed recoveries of approximately 66% and 71% of normal Achilles tendon tissue, respectively. This indicates that the PCNP hydrogel induced both the regeneration of the aligned tendon ECM and the regeneration of the mechanical function of the tendon.

[0313]

[0314] As described above, the embodiments have been described with reference to the limited drawings. However, those with ordinary knowledge in the technical field can apply various technical modifications and variations based on the above. For example, the described technology can be carried out in a procedure different from the described method, and / or the components of the described system, structure, device, circuit, etc. can be combined or assembled in a form different from the described method, and can be opposed or replaced by other components or equivalents, and appropriate results can still be achieved.

[0315] Therefore, other embodiments, other examples, and those equivalent to the claims also belong to the claims described below.

[0316] This invention was completed with the support of the projects of "Development of Intelligent Solution Technology for Disaster Safety (Project Number 2E31600, Project Period: January 1, 2022 to December 31, 2022)", "Development of Local Drug Delivery Platform Technology for the Treatment of Generalized Musculoskeletal Diseases (Project Specific Number 1711056093, Project Period: April 1, 2017 to December 31, 2017)", and "Development of Hydrogel Engineering Technology for the Reconstruction of Neural Networks (Project Specific Number 1711134231, Project Period: July 1, 2021 to December 31, 2021)".

Claims

1. On the phosphorus atom of the polyphosphazene skeleton represented by the following chemical formula 1, a first moiety of an amino acid ester represented by the following chemical formula 2, a second moiety of polyethylene glycol represented by the following chemical formula 3, and a third moiety containing aminoethanol and an organic acid, are contained in a molar ratio of a:b:c, respectively, and a therapeutically effective amount of a drug for inflammation control and treatment A composition for preventing or treating an inflammatory disease, comprising 【Chemical 1】 【Chemical 2】 [Chemical Formula 3] (In the above chemical formula, R １ is alkyl of C １－６ , alkenyl of C １－６ , or aryl-C ６－１０ alkyl of １－６ and R ２ is hydrogen, methyl, isopropyl, 1-methylpropyl, 2-methylpropyl, thiomethyl, methylthioethyl, benzyl, hydroxybenzyl, or 2-indolylmethyl, R ３ is an alkyl of C １－６ and n is an integer from 3 to 100,000, p is an integer from 1 to 20, The molar ratio of a:b is from 5.5:1 to 7.5:1, a is 70 to 80 mol%.

2. The organic acid in the third moiety contains glutaric acid. The composition for preventing or treating an inflammatory disease according to claim 1.

3. Said R １ is methyl, ethyl, propyl, butyl, benzyl, or 2-propenyl, and the composition for preventing or treating an inflammatory disease according to claim 1.

4. Said R ３ is methyl, and the composition for preventing or treating an inflammatory disease according to claim 1.

5. The drug for treating the inflammatory disease is hydrophobic. The composition for preventing or treating an inflammatory disease according to claim 1.

6. The hydrophobic drug for treating the inflammatory disease is any one or more selected from the group consisting of triamcinolone acetonide, methylprednisolone, dexamethasone, celecoxib, ibuprofen, naproxen, indomethacin, ketoprofen, etodolac, meloxicam, rofecoxib, etoricoxib, valdecoxib, lumiracoxib, and diclofenac. The composition for preventing or treating an inflammatory disease according to claim 5.

7. The polymer is characterized in that it sustains the release of the drug for treating the inflammatory disease. The composition for preventing or treating an inflammatory disease according to claim 1.

8. The sustained release releases the drug at a drug concentration within the therapeutically effective amount range. The composition for preventing or treating an inflammatory disease according to claim 1.

9. The polyphosphazene-based polymer is dissolved in a solvent at a concentration of 1 to 50% by weight. The composition for preventing or treating an inflammatory disease according to claim 1.

10. The solvent is one or more selected from the group consisting of water, buffer solution, acidic solution, basic solution, salt solution, physiological saline, water for injection, cell culture medium, and glucose saline solution. The composition for preventing or treating an inflammatory disease according to claim 9.

11. The polymer exhibits sol-gel transition behavior in the range of 5 to 70 °C and forms a hydrogel at a predetermined temperature. The composition for preventing or treating an inflammatory disease according to claim 1.

12. The polymer according to claim 1, which gels when applied to a living body or in an in vitro environment, loses its temperature sensitivity at a predetermined temperature, and maintains a gel state regardless of temperature changes, for preventing or treating an inflammatory disease.

13. The composition for preventing or treating an inflammatory disease according to claim 1, wherein the inflammatory disease is any one selected from the group consisting of osteoarthritis, rheumatoid arthritis, rheumatoid arthritis, osteoporosis, and Achilles tendinitis.

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