Intestinal macrophage-targeting prodrug nanoassembly for treating inflammatory bowel disease

Abstract

Disclosed is an intestinal macrophage-targeting prodrug nanoassembly for treating inflammatory bowel disease. The nanoassembly comprises a complex in which an inflammation therapeutic agent having an amine group is bonded to one end of a peptide represented by SEQ ID NO: 1, and the nanoassembly is formed by self-assembly of the complex via intermolecular interactions. The nanoassembly targets intestinal macrophages, is accumulated in inflammatory colon tissue, and regulates immune response to eventually promote differentiation into anti-inflammatory M2 macrophages, thereby providing excellent therapeutic efficacy against IBD.

Description

Intestinal macrophage-targeting prodrug nanoassemblies for the treatment of inflammatory bowel disease

[0001] [Cross-reference to related applications]

[0002] This application claims priority to Republic of Korea Patent Application No. 10-2023-0185539, filed December 19, 2023, the entire contents of which are incorporated herein by reference.

[0003] [Description of Nationally Supported Research and Development]

[0004] This study was conducted at the Korea Institute of Science and Technology (KIST) under the management of the Korea Institute of Science and Technology (KIST) under the Ministry of Science and ICT. The research project name is KIST Research Operation Expense Support (Main Project Expense), and the research project name is Customized Diagnosis and Treatment, Regeneration, Rehabilitation, and New Drug Development (Project Identification Number: 1711196534).

[0005] The present disclosure discloses intestinal macrophage-targeting prodrug nanoassemblies for the treatment of inflammatory bowel disease.

[0006] Inflammatory bowel disease (IBD) is characterized by chronic, recurrent inflammation of the gastrointestinal tract, and is represented by Crohn's disease and ulcerative colitis. IBD is known to be complexly influenced by genetic, environmental, intestinal microbiota, and immune factors. Recently, it has also been recognized that it is caused by an imbalance in the gut microbiota and the resulting interaction with the immune system. The goal of treating IBD is to reduce inflammation in the intestinal mucosa and improve the patient's quality of life.

[0007] Mesalamine, also known as 5-aminosalicylic acid (5-ASA), is a leading drug for treating inflammatory bowel diseases, including ulcerative colitis. It is desirable for drugs for IBD to release their active ingredients within the affected intestinal tract. However, the small molecule structure of 5-ASA exhibits inadequate biodistribution due to rapid in vivo clearance and poor tissue delivery within the colon. 5-ASA is significantly absorbed in the upper gastrointestinal tract, reducing the amount of active compound reaching the target colonic tissue after oral administration, thereby reducing therapeutic efficacy and potentially increasing the risk of systemic exposure-related complications. Therefore, the development of a drug delivery system for 5-ASA remains a challenge for the effective treatment of IBD patients.

[0008] In one aspect, the present disclosure aims to provide intestinal macrophage-targeting nanoassemblies for treating inflammatory bowel diseases.

[0009] In another aspect, the present disclosure aims to provide a pharmaceutical composition for treating inflammatory bowel disease comprising the nanoassembly.

[0010] In one aspect, the present disclosure provides a nanoassembly comprising a complex in which an anti-inflammatory agent having an amine group at one end of a peptide represented by SEQ ID NO: 1 is bound, wherein the nanoassembly is formed by self-assembly of the complex through intermolecular interactions.

[0011] In an exemplary embodiment, the anti-inflammatory agent having the amine group may be 5-aminosalicylic acid.

[0012] In an exemplary embodiment, the complex may self-assemble through intermolecular interactions under aqueous conditions to form a nanoassembly exhibiting a (+) charge on the surface.

[0013] In an exemplary embodiment, the nanoassembly may be a prodrug for treating inflammatory bowel disease.

[0014] In an exemplary embodiment, the prodrug for treating inflammatory bowel disease may be administered orally.

[0015] In an exemplary embodiment, the prodrug for treating inflammatory bowel disease may be administered orally and accumulate in inflamed colonic tissue and promote M2 polarization of intestinal macrophages.

[0016] In an exemplary embodiment, the prodrug for treating inflammatory bowel disease may be administered orally at a dose of 10 to 500 mg / kg / day based on the weight of the anti-inflammatory agent having an amine group.

[0017] In an exemplary embodiment, the nanoassembly may have an average diameter of 100 to 200 nm.

[0018] In an exemplary embodiment, the nanoassembly may have an average molecular weight of 700 to 800 Da.

[0019] In an exemplary embodiment, the nanoassembly may comprise an anti-inflammatory agent having amine groups in an amount of at least 20 wt% based on the total weight of the nanoassembly.

[0020] In an exemplary embodiment, the nanoassembly may inhibit the production of one or more cytokines selected from the group consisting of TNF-α, IFN-γ, IL-1β, and IL-6, and increase the production of cytokines of IL-10.

[0021] In an exemplary embodiment, the nanoassembly may inhibit damage to tight junction-associated proteins, including at least one of ZO-1 (zonula occludens-1) and occludin-1.

[0022] In another aspect, the present disclosure provides a pharmaceutical composition for treating inflammatory bowel disease comprising the nanoassembly.

[0023] In one aspect, the technology disclosed in the present disclosure is effective in providing intestinal macrophage-targeting nanoassemblies for the treatment of inflammatory bowel disease. Many anti-inflammatory drugs, including 5-ASA, used to treat patients with intestinal diseases according to international guidelines are significantly absorbed in the upper gastrointestinal tract, which reduces therapeutic efficacy. Furthermore, administering large doses of the drug to address this issue can lead to systemic exposure and toxicity. The present disclosure provides a method for effectively delivering anti-inflammatory drugs to diseased sites in vivo by combining a peptide having a sequence number 1 with an amine-containing anti-inflammatory agent to induce nanostructure formation, thereby increasing adhesion to the intestinal mucus layer, thereby further enhancing the anti-inflammatory efficacy of the amine-containing anti-inflammatory agent.

[0024] In another aspect, the technology disclosed in the present disclosure is effective in providing a pharmaceutical composition for treating inflammatory bowel disease comprising the nanoassembly.

[0025] Figure 1 illustrates a schematic diagram of the fabrication of mesalamine prodrug nanoassemblies. (A) Chemical structure of FRRG-ASA and schematic of the mesalamine prodrug nanoassembly. (B) Schematic diagram of the mode of action of FRRG-ASA for the treatment of inflammatory bowel disease (IBD).

[0026] Figure 2 shows the results of physicochemical characterization of mesalamine prodrug nanoassemblies. (A) Molecular dynamics (MD) simulation of two FRRG-ASA molecules. (B) Molecular dynamics (MD) simulation of four FRRG-ASA molecules. (C) Hydrodynamic size and morphology of FRRG-ASA in aqueous conditions. (D) Particle size and polydispersity index (PDI) of FRRG-ASA in saline or SIF for 7 days.

[0027] Figure 3 shows the results of evaluating the intracellular uptake of nanoassemblies in macrophages. (A) Representative confocal microscopy images of native or LPS-treated RAW 264.7 cells incubated with Cy5.5-FRRG-ASA. (B) Quantitative analysis of the amount of FRRG-ASA in native or LPS-treated RAW 264.7 cells after various incubation times.

[0028] Figure 4 shows the results of evaluating the intracellular uptake of nanoassemblies in macrophages. (A) Time-dependent quantitative analysis of the amounts of FRRG-ASA and free 5-ASA in native or LPS-treated RAW 264.7 cells as determined using HPLC. (B) Time-dependent ratio of free 5-ASA to FRRG-ASA in native or LPS-treated RAW 264.7 cells after treatment with FRRG-ASA.

[0029] Figure 5 shows the anti-inflammatory effect of nanoassemblies in macrophages. (A) Expression levels of p54 JNK, p46 JNK, and p38 in LPS-treated RAW 264.7 cells and cells treated with free 5-ASA or FRRG-ASA for 24 hours. (B) Proinflammatory cytokine levels in LPS-treated RAW 264.7 cells and cells treated with free 5-ASA or FRRG-ASA for 24 hours. (C, D) Expression levels of M1 or M2 macrophage markers in LPS-treated RAW 264.7 cells and cells treated with free 5-ASA or FRRG-ASA for 24 hours.

[0030] Figure 6 illustrates the anti-inflammatory effect of nanoassemblies in macrophages. (A) Schematic diagram of the co-culture assay methodology. (B) Proinflammatory cytokine levels in NCM460 cells after co-culture with LPS-treated RAW 264.7 cells or cells treated with FRRG-ASA for 24 hours.

[0031] Figure 7 illustrates the mucoadhesive properties of nanoassemblies. (A) Optical images of colon specimens treated with free Cy5.5 or Cy5.5-FRRG-ASA and then washed five times with SIF. (B) Relative amounts of free Cy5.5 or Cy5.5-FRRG-ASA collected from the flow-through solution across the colon specimen during SIF washing. (C) Representative confocal microscopy images of colon specimens stained with MUC2 after treatment with Cy5.5-FRRG-ASA and then washed five times with SIF.

[0032] Figure 8 shows the accumulation of nanoassemblies in the inflamed colon by the enhanced permeability and retection (eEPR) effect. (A) Whole-body NIRF images of IBD mice after oral administration of free Cy5.5 or Cy5.5-FRRG-ASA. (B) Fluorescence signals of free Cy5.5 or Cy5.5-FRRG-ASA in the gastrointestinal tract of normal or IBD mice after oral administration.

[0033] Figure 9 shows the accumulation of nanoassemblies in the inflamed colon by the eEPR (Enhanced Permeability and Retection) effect. Ex vivo NIRF images of major organs of IBD mice 9 hours after oral administration of free Cy5.5 or Cy5.5-FRRG-ASA.

[0034] Figure 10 illustrates the accumulation of nanoassemblies in inflamed colons by the enhanced permeability and retection (eEPR) effect. (A) Representative confocal microscopy images of colon tissue stained with F4 / 80 9 hours after oral administration of Cy5.5-FRRG-ASA. (B) Fluorescence signal profiles of F4 / 80 and Cy5.5-FRRG-ASA from line scans along the white line in image A of Figure 10 .

[0035] Figure 11 shows the therapeutic efficacy of nanoassemblies in an IBD model. (A, B) Changes in (A) body weight and (B) disease activity index (DAI) in DSS-treated or healthy mice during oral administration of PBS, free 5-ASA, or FRRG-ASA. (C, D) Optical image (C) and length (D) of the colon on day 9. (E) Colon tissue stained with H&E.

[0036] Figure 12 shows the therapeutic efficacy of nanoassemblies in an IBD model. (A, B) Levels of (A) pro-inflammatory and (B) anti-inflammatory cytokines in colon tissue. (C) Representative confocal microscopy images of colon tissue stained with CD163.

[0037] Figure 13 illustrates the restoration of the intestinal barrier, preventing systemic inflammation, through treatment with nanoassemblies. (A) Representative confocal microscopy images of cross-sectioned colon tissue stained with ZO-1 and occludin-1 antibodies on day 9. (B) Quantitative analysis of the fluorescence signals of ZO-1 and occludin-1 in colon tissue. (C) Levels of proinflammatory cytokines in serum. (D) Optical image and weight of the spleen.

[0038] Figure 14 illustrates restoration of the intestinal barrier, preventing systemic inflammation, through treatment with nanoassemblies. (A) Levels of hematological parameters. (B) Major organ tissues stained with H&E. Black arrows indicate structural abnormalities.

[0039] Hereinafter, the present disclosure is described in detail.

[0040] The present disclosure provides an intestinal macrophage-targeting nanoassembly that effectively delivers an amine group-containing anti-inflammatory agent, specifically 5-ASA (5-aminosalicylic acid), into the colon, thereby exhibiting excellent therapeutic effects for inflammatory bowel disease. The nanoassembly addresses the problem of low therapeutic efficacy of conventional 5-ASA. Upon oral administration, the nanoassembly accumulates in large quantities within inflamed colon tissue and exhibits excellent targeting ability to intestinal macrophages, thereby efficiently entering intestinal macrophages and providing an excellent intestinal inflammatory response suppression effect.

[0041] In one aspect, the present disclosure provides a nanoassembly comprising a complex in which an anti-inflammatory agent having an amine group at one end of a peptide represented by SEQ ID NO: 1 is bound, wherein the nanoassembly is formed by self-assembly of the complex through intermolecular interactions.

[0042] [Sequence number 1]

[0043] Phe-Arg-Arg-Gly

[0044] In an exemplary embodiment, the anti-inflammatory agent having the amine group may be 5-aminosalicylic acid.

[0045] In an exemplary embodiment, the anti-inflammatory agent having the amine group may be linked to a glycine of a peptide represented by SEQ ID NO: 1.

[0046] In an exemplary embodiment, the nanoassembly may be a peptide represented by SEQ ID NO: 1 and an anti-inflammatory agent having an amine group conjugated thereto through an amide bond reaction.

[0047] In an exemplary embodiment, the nanoassembly may be a nanoself-assembly.

[0048] In an exemplary embodiment, the complex may have an amphiphilic structure.

[0049] In an exemplary embodiment, the complex may be a nanoassembly that self-assembles through intermolecular interactions in aqueous conditions to form a nanoassembly exhibiting a (+) charge on its surface. The complex can form a nanoassembly having a positively charged surface that exhibits excellent particle stability and mucoadhesive properties to the intestinal mucus layer through intermolecular interactions without the use of additional polymers, organic / inorganic materials, or carrier materials. The nanoassembly has increased adhesiveness to the intestinal mucus layer, thereby exhibiting excellent delivery efficiency to intestinal macrophages, and ultimately providing an effect of enhancing the anti-inflammatory response of an anti-inflammatory agent having an amine group, specifically, 5-ASA.

[0050] In an exemplary embodiment, the nanoassembly may be a prodrug for treating inflammatory bowel disease.

[0051] In an exemplary embodiment, the prodrug for treating inflammatory bowel disease may be administered orally. The orally administered prodrug has the effect of targeting intestinal macrophages, modulating their immune response, and ultimately promoting their differentiation into anti-inflammatory M2 macrophages, thereby providing superior therapeutic efficacy for inflammatory bowel disease.

[0052] In an exemplary embodiment, the prodrug for treating inflammatory bowel disease may be administered orally, accumulate in inflamed colonic tissue, and promote M2 polarization of intestinal macrophages. Unlike when an anti-inflammatory agent having an amine group not bound to the peptide of SEQ ID NO: 1 is administered orally, the prodrug for treating inflammatory bowel disease inhibits degradation and elimination within the abdominal cavity and enhances accumulation and retention in inflamed colonic tissue, thereby providing significant therapeutic efficacy.

[0053] In an exemplary embodiment, the prodrug for treating inflammatory bowel disease may provide a therapeutic effect for inflammatory bowel disease without drug side effects such as weight loss.

[0054] In an exemplary embodiment, the prodrug for treating inflammatory bowel disease may be administered orally at a dose of 10 to 500 mg / kg / day based on the weight of the anti-inflammatory agent having an amine group. In another exemplary embodiment, the prodrug for treating inflammatory bowel disease is administered to a subject in an amount of at least 10 mg / kg / day, at least 20 mg / kg / day, at least 30 mg / kg / day, at least 40 mg / kg / day, at least 50 mg / kg / day, at least 100 mg / kg / day, at least 150 mg / kg / day, at least 200 mg / kg / day, at least 250 mg / kg / day, at least 300 mg / kg / day, at least 350 mg / kg / day, at least 400 mg / kg / day, or at least 450 mg / kg / day, and at most 500 mg / kg / day, at most 450 mg / kg / day, at most 400 mg / kg / day, at most 350 mg / kg / day, at most 300 mg / kg / day, at most 250 mg / kg / day, at most 200 It may be administered orally at a dose of 100 mg / kg / day or less, 150 mg / kg / day or less, 100 mg / kg / day or less, 90 mg / kg / day or less, 80 mg / kg / day or less, 70 mg / kg / day or less, 60 mg / kg / day or less, or 50 mg / kg / day or less.

[0055] In an exemplary embodiment, the inflammatory bowel disease may be ulcerative colitis or Crohn's disease.

[0056] In an exemplary embodiment, the inflammatory bowel disease may be acute or chronic.

[0057] In an exemplary embodiment, the nanoassembly may be spherical.

[0058] In one exemplary embodiment, the nanoassembly may have an average diameter of 100 to 200 nm. In another exemplary embodiment, the nanoassembly may have an average diameter of 100 nm or more, 110 nm or more, 120 nm or more, 130 nm or more, 140 nm or more, or 150 nm or more, and 200 nm or less, 190 nm or less, 180 nm or less, 170 nm or less, 160 nm or less, or 150 nm or less. Here, the diameter may refer to the longest diameter.

[0059] In one exemplary embodiment, the nanoassembly may have an average molecular weight of 700 to 800 Da. In another exemplary embodiment, the nanoassembly may have an average molecular weight of at least 700 Da, at least 710 Da, at least 720 Da, at least 730 Da, at least 740 Da, at least 750 Da, at least 760 Da, at least 770 Da, at least 780 Da, or at least 790 Da, and at most 800 Da, at most 790 Da, at most 780 Da, at most 770 Da, at most 760 Da, at most 750 Da, at most 740 Da, at most 730 Da, at most 720 Da, or at most 710 Da.

[0060] In an exemplary embodiment, the nanoassembly may comprise an anti-inflammatory agent having an amine group in an amount of greater than or equal to 20 wt%, from 20 to 80 wt%, from 20 to 70 wt%, from 20 to 60 wt%, from 20 to 50 wt%, from 20 to 40 wt%, from 20 to 30 wt%, or from 20 to 25 wt%, based on the total weight of the nanoassembly.

[0061] In an exemplary embodiment, the nanoassembly may inhibit pro-inflammatory cytokine production and increase anti-inflammatory cytokine production.

[0062] In an exemplary embodiment, the nanoassembly may inhibit the production of one or more cytokines selected from the group consisting of TNF-α, IFN-γ, IL-1β, and IL-6.

[0063] In one exemplary embodiment, the nanoassembly may increase cytokine production of IL-10.

[0064] In an exemplary embodiment, the nanoassembly may inhibit damage to tight junction-associated proteins. The nanoassembly may repair damage to tight junction-associated proteins, thereby restoring a damaged intestinal barrier. By restoring the intestinal barrier, the nanoassembly may prevent the leakage of inflammatory cytokines from inflamed colonic tissue into the systemic system, thereby providing a systemic inflammation-reducing effect.

[0065] In an exemplary embodiment, the tight junction-associated protein may include at least one of ZO-1 (zonula occludens-1) and occludin-1.

[0066] In another aspect, the present disclosure provides a pharmaceutical composition for treating inflammatory bowel disease comprising the nanoassembly.

[0067] In another aspect, the present disclosure provides a method for treating inflammatory bowel disease, comprising administering to a subject in need thereof an amount of the nanoassembly effective for treating inflammatory bowel disease.

[0068] In another aspect, the present disclosure provides the nanoassembly for use in the treatment of inflammatory bowel disease in a subject.

[0069] In another aspect, the present disclosure provides the use of the nanoassembly for the treatment of inflammatory bowel disease in a subject.

[0070] In another aspect, the present disclosure provides use of the nanoassembly in preparing a composition for treating inflammatory bowel disease.

[0071] In an exemplary embodiment, the nanoassembly may be administered to a subject in the form of a pharmaceutical composition.

[0072] In an exemplary embodiment, the inflammatory bowel disease may be ulcerative colitis or Crohn's disease.

[0073] In an exemplary embodiment, the inflammatory bowel disease may be acute or chronic.

[0074] In an exemplary embodiment, the pharmaceutical composition may further contain pharmaceutical adjuvants such as preservatives, stabilizers, wetting or emulsifying agents, salts for osmotic pressure control, and / or buffers, and / or other therapeutically useful substances, and may be formulated into various oral dosage forms according to conventional methods.

[0075] The oral dosage forms include, for example, tablets, pills, hard and soft capsules, solutions, suspensions, emulsifiers, syrups, powders, granules, granules, pellets, etc., and these dosage forms may contain surfactants, diluents (e.g., lactose, dextrose, sucrose, mannitol, sorbitol, cellulose, and glycine), lubricants (e.g., silica, talc, stearic acid and its magnesium or calcium salts, and polyethylene glycol). Tablets may also contain binders such as magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, and polyvinylpyrrolidine, and optionally, pharmaceutical additives such as disintegrants such as starch, agar, alginic acid or its sodium salt, absorbents, coloring agents, flavoring agents, and sweetening agents. The above tablets can be manufactured by conventional mixing, granulating or coating methods.

[0076] Hereinafter, the present disclosure will be described in more detail through examples. These examples are intended solely to illustrate the present disclosure, and it will be apparent to those skilled in the art that the scope of the present disclosure is not limited by these examples.

[0077] Experimental Example 1. Fabrication and Characterization of Nanoassemblies

[0078] To prepare mesalamine prodrug nanoassemblies, FRRG (N-terminally acylated Phe-Arg-Arg-Gly) peptide (Peptron Co., Daejeon, South Korea) was conjugated to 5-ASA (Tokyo Chemical Industry, TCI; Toshima, Tokyo, Japan) via a one-step amide coupling reaction. Specifically, FRRG peptide (200 mg, 0.35 mmol, 1 eq), 5-ASA (53.48 mg, 0.35 mmol, 1 eq), and carbonyldiimidazole (CDI; 67.5 mg, 0.42 mmol, 1.2 eq) were dissolved in 1 mL anhydrous dimethylformamide (DMF, Sigma Aldrich, St. Louis, MO, USA). After 30 minutes of reaction, the final product, FRRG-ASA, was purified and its molecular weight was confirmed using LC / MS (Agilent 1200 Series HPLC system), and then freeze-dried for 3 days to obtain a white powder.

[0079] The hydrodynamic size and zeta potential of FRRG-ASA in saline at a concentration of 1 mg / mL were analyzed using a Zetasizer Nano ZS (Malvern Instruments, Worcestershire, UK). The morphology of the nanoassemblies in distilled water (1 mg / mL) was observed by transmission electron microscopy (TEM, CM-200, Philips, USA). To evaluate the particle stability, the hydrodynamic size and polydispersity index (PDI) in saline at a concentration of 1 mg / mL or simulated intestinal fluid (SIF) were analyzed for 7 days. The SIF solution was prepared according to a published method. A buffer containing 105.9 mM NaCl, 8.7 mM NaOH, and 28.4 mM NaH2PO4 was mixed with 5 mM sodium taurocholate (86339, Sigma Aldrich) to mimic the bile salt conditions in the intestine. Afterwards, distilled water containing 10% mucin II (M3895, Sigma Aldrich) was added to the buffer, and the pH was adjusted to 6.8.

[0080] Experimental Example 2. Evaluation of cellular uptake of nanoassemblies in macrophages

[0081] For NIRF imaging, FRRG-ASA conjugated with the fluorescent dye Cy5.5 was prepared and co-assembled with unmodified FRRG-ASA at a ratio of 1:99 wt% in saline. Specifically, Cy5.5 NHS ester (Lumiprobe, Wan Chai, Hong Kong; 100 mg, 0.13 mmol), NH2-FRRG-COOH peptide (Peptron Co.; 138.84 mg, 0.26 mmol), and N,N diisopropylethylamine (DIPEA, Sigma Aldrich; 20.12 mg, 0.156 mmol) were dissolved in anhydrous DMF. After 3 h of reaction, Cy5.5-FRRG-COOH was purified using RP-HPLC, and conjugation with 5-ASA was performed according to the method of Experimental Example 1. Afterwards, the generated Cy5.5-conjugated FRRG-ASA and unmodified FRRG-ASA were dispersed in saline solution at a ratio of 1:99 wt% to generate a nanoassembly of Cy5.5-FRRG-ASA.

[0082] The intracellular entry of FRRG-ASA was assessed using the murine macrophage cell line RAW 264.7 (American Type Culture Collection, ATCC, Manassas, VA, USA). RAW 264.7 cells were seeded at a density of 1 × 10 in glass-bottom confocal dishes (SPL life science, Gyeonggi-do, Republic of Korea). 5After seeding at a density of 10 μg / mL, cells were treated with lipopolysaccharide (LPS, L2630, Sigma Aldrich; 1 μg / mL) for 5 h to promote M1 phenotype differentiation. Cells were then treated with Cy5.5-FRRG-ASA (3.25 μM based on 5-ASA content), fixed with 4% paraformaldehyde for 10 min, and stained with 4',6-diamidino-2-phenylindole (DAPI; Invitrogen, Carlsbad, CA, USA) for 5 min in the dark. NIRF imaging was performed using a confocal laser scanning microscope (Leica, Germany), and the fluorescence intensity of the images was quantified using ImagePro Plus software (Media Cybernetic, Rockville, MD, USA).

[0083] To quantitatively analyze the intracellular uptake of FRRG-ASA and free 5-ASA, each compound was treated at a concentration of 3.25 μM based on 5-ASA content for various incubation times in LPS-treated RAW 264.7 cells. The cells were then washed twice with DPBS and dispersed in RIPA buffer (Thermo Fisher Scientific Inc. Rockford, IL, USA). After centrifugation at 14,000 rpm for 20 min to remove cell debris, the amount of FRRG-ASA or free 5-ASA in each sample was analyzed using HPLC.

[0084] Experimental Example 3. In vitro anti-inflammatory efficacy evaluation of nanoassemblies

[0085] Anti-inflammatory efficacy was evaluated in LPS-treated RAW 264.7 cells treated with FRRG-ASA or free 5-ASA at a concentration of 3.25 μM based on 5-ASA content for 24 h. After treatment, cells were washed twice with DPBS, resuspended in RIPA buffer, and centrifuged at 14,000 rpm for 20 min to remove cell debris. Proteins in each sample were then quantified using BCA assay (23221, Thermo Fisher Scientific), mixed with sodium dodecyl sulfate (SDS; NaraBio, Gyeonggi-do, South Korea) loading buffer, boiled for 5 min, and separated on a 10% SDS polyacrylamide gel. After transfer to polyvinylidene fluoride (PVDF) membranes, the membranes were further incubated with Tris-basic trisodium sulfate-T (TBS-T) buffer containing 5% bovine serum albumin (BSA, NaraBio) for 1 h at room temperature to prevent nonspecific IgG binding. Finally, the membranes were incubated with rabbit anti-mouse antibodies against phosphorylated JNK (MAB1205, R&D systems), p38 (ab195049, Abcam, Cambridge, UK), iNOS (ab15323, Abcam), CD206 (ab64693, Abcam), Arginase-1 (93668S, Cell Signaling), and β-actin (ab8227, Abcam) for 24 h at 4 °C, followed by goat anti-rabbit immunoglobulin-HRP (horseradish peroxidase) antibody (#7074, Cell Signaling) for 2 h. The HRP signal intensity of the membrane was detected via enhanced chemiluminescence (ECL) and quantified using ImagePro Plus software.

[0086] Proinflammatory cytokines, such as TNF-α (43907, San Diego, CA, USA), IFN-γ (430807, BioLegend), IL-1β (MLB00C, R&D systems), and IL-6 (M6000B, R&D systems), were evaluated by ELISA analysis in LPS-treated RAW 264.7 cells after 24 h of treatment with FRRG-ASA or free 5-ASA (3.25 μM based on 5-ASA content). To evaluate the anti-inflammatory effect in epithelial cells by 5-ASA cleaved from FRRG-ASA in macrophages, human colonic epithelial cells NCM460 were cultured at a density of 1 × 10 5 NCM460 cells were seeded at a density of 10 μM and co-cultured with LPS-treated RAW 264.7 cells or cells treated with FRRG-ASA (3.25 μM based on 5-ASA content) for 24 h. Subsequently, TNF-α and IFN-γ levels in NCM460 cells were assessed by ELISA analysis.

[0087] Experimental Example 4. Flow-through Evaluation of the Mucoadhesive Properties of Nanoassemblies

[0088] All experiments using animals were conducted in compliance with the relevant laws and institutional guidelines of the Institutional Animal Care and Use Committee (IACUC, Approval No. 2023-013) at the Korea Institute of Science and Technology (KIST). Mice (NaraBio, Gyeonggi-do, Republic of Korea) were raised under pathogen-free conditions at KIST. The mucoadhesive properties of FRRG-ASA were confirmed by flow analysis according to a published method. Specifically, colon specimens collected from 8-week-old BALB / c mice were opened longitudinally to the right and attached to glass slides using super glue. Cy5.5-FRRG-ASA (50 mg / kg based on 5-ASA content) or free Cy5.5 were incubated dropwise on the colon tissues at the same fluorescence intensity in a humidified atmosphere at 37°C for 10 min. The slides were then placed at a 45° angle and washed five times with SIF at a flow rate of 500 μL / min to mimic in vivo intestinal conditions. The fluorescence intensity of the flow-through solution collected from each wash cycle was analyzed using a fluorescence spectrophotometer (F-7000, Hitachi, Japan). Colonic specimens were washed five times with SIF, sectioned at 10 μm thickness, and stained with a mouse anti-mouse Alexa Fluor 488-conjugated antibody to MUC2 (NB120-11197AF488, Novus Biologicals, Centennial, CO, USA) at 4°C for 12 h. Subsequently, NIRF imaging was performed using a confocal laser scanning microscope after DAPI staining.

[0089] Experimental Example 5. Biodistribution Evaluation of Nanoassemblies

[0090] Accumulation of FRRG-ASA in the inflamed colon was assessed in a dextran sulfate sodium salt (DSS)-induced colitis model prepared by feeding water containing 3% DSS (0216011080, MP Biomedicals, Road Seven Hills, Australia) to 8-week-old male BALB / c mice. NIRF imaging was performed using an IVIS system (Lumina Series III, PerkinElmer, Waltham, MA, USA) 5 days after DSS treatment, following oral administration of Cy5.5-FRRG-ASA (50 mg / kg based on 5-ASA content) or free Cy5.5 at equivalent fluorescence intensities. Fluorescence intensity in the gastrointestinal (GI) tract was quantified using LivingImage software (PerkinElmer, USA). Ex vivo NIRF imaging was performed using major organs (liver, lung, spleen, kidney, heart, stomach, small intestine, and colon) collected from mice 9 h after treatment. Subsequently, colon tissues were sectioned at 10 μm thickness and stained with a rat anti-mouse FITC-conjugated antibody against F4 / 80 (123108, BioLegend) at 4°C for 12 h. NIRF imaging was performed using a confocal laser scanning microscope after DAPI staining, and the Pearson correlation coefficients of F4 / 80 (macrophages) and FRRG-ASA in colon tissues were determined using Prism10 software (GraphPad, Boston, MA, USA).

[0091] Experimental Example 6. Evaluation of the therapeutic efficacy of nanoprecursors in an IBD model.

[0092] To evaluate the therapeutic efficacy of the IBD model, 8-week-old male BALB / c mice were divided into control and colitis groups. Mice in the colitis group were fed water containing 3% DSS for 9 days, followed by daily oral administration of PBS, free 5-ASA (50 mg / kg), or FRRG-ASA (50 mg / kg based on 5-ASA content). Control mice were fed regular water throughout the period. The progression of colitis was scored daily using the Disease Activity Index (DAI), which is based on diarrhea, bleeding (maximum score: 4), and body weight loss. On day 9, colon tissues were collected from the mice, and their lengths were measured using a Vernier caliper. The collected colon tissues were then stained with H&E or rabbit anti-mouse Alexa Fluor 647-conjugated antibody to CD163 (ab313666, Abcam). A portion of colon tissue was dispersed in RIPA buffer, centrifuged at 14,000 rpm for 20 minutes to remove debris, and then analyzed for inflammation-related cytokines such as TNF-α, IFN-γ, IL-1β, IL-6, and IL-10 (DY217B, R&D systems) using ELISA analysis.

[0093] Experimental Example 7. Evaluation of Systemic Inflammation

[0094] Intestinal barrier restoration and prevention of systemic inflammation by FRRG-ASA were evaluated in an IBD model. Mice were treated with the same protocol described above. Colonic tissues were collected from mice on day 9 and stained with a rabbit anti-mouse Alexa Fluor 647-conjugated antibody to ZO-1 (98225S, Cell Signaling, Massachusetts, USA) and a mouse anti-mouse Alexa Fluor 488-conjugated antibody to occludin-1 (TFS-331588, Invitrogen, Carlsbad, USA). NIRF imaging was performed using a confocal laser scanning microscope, and the fluorescence intensity of the images was quantified using ImagePro Plus software. Serum IFN-γ and TNF-α levels were measured using ELISA assays. Blood samples were collected by cardiac puncture on day 9 under deep anesthesia and centrifuged at 2,200 rpm for 20 minutes to obtain serum from mice. On day 9, the following hematological parameters in serum were also evaluated: aspartate aminotransferase (AST), alanine transaminase (ALT), blood urea nitrogen (BUN), creatinine, lactate dehydrogenase (LDH), and creatinine kinase (CK). On day 9, major organs (liver, lungs, spleen, kidney, and heart) were collected from the mice, stained with H&E, and observed under a microscope (BX 51, Olympus, USA).

[0095] Experimental Example 8. Statistical Analysis

[0096] Statistical significance between two groups was analyzed using the Student's t test. For groups with more than two, one-way analysis of variance (ANOVA) was used, and multiple comparisons were performed using the Tukey-Kramer post-hoc test. Survival results were plotted using Kaplan-Meier curves and analyzed using the log-rank method. All results were expressed as mean ± SD, and P values ​​of <0.05*, <0.01**, and <0.001*** were considered statistically significant.

[0097] Experimental Results 1. Analysis of Physicochemical Properties of Mesalamine Prodrug Nanoassemblies

[0098] Mesalamine prodrug nanoassemblies were prepared by conjugating the FRRG peptide with 5-ASA via a one-step amide coupling reaction. After the reaction, 99% of the FRRG-ASA was purified using high-performance liquid chromatography (HPLC). The successful synthesis was further confirmed by LC / MS analysis, and the exact molecular weight of FRRG-ASA was calculated to be 711.78 Da, with measured m / z [M] and m / z [M / 2] of 712.6 and 356.8, respectively. The FRRG-ASA conjugate spontaneously formed nanoassemblies in aqueous conditions due to intermolecular interactions, resulting in a high drug loading content of 100%, with the prodrug containing 21.6% 5-ASA.

[0099] Molecular dynamics (MD) simulations of two FRRG-ASA molecules showed that the 5-ASA moieties of each molecule consistently maintained a close distance over 20 ns (see Figure 2A). As a control, RRG-ASA, a conjugate of the RRG peptide and 5-ASA without the F sequence, did not form nanoassemblies. These results indicate that the main driving forces promoting the self-assembly of FRRG-ASA are hydrogen bonds and hydrophobic interactions between the aromatic rings within the 5-ASA molecules or phenylalanine, respectively. Furthermore, all-atom MD simulations of four FRRG-ASA molecules showed that the positively charged R peptide (blue) was predominantly exposed on the nanoassembly surface (see Figure 2B). As a result, FRRG-ASA was found to have an average size of 148.21 ± 6.53 nm with a spherical morphology in aqueous conditions, as confirmed by dynamic light scattering (DLS) and transmission electron microscopy (TEM), respectively (see Figure 2C). In addition, FRRG-ASA maintained its hydrodynamic size when cultured in saline and SIF, with no significant change over 7 days (see Figure 2D). The particle stability was confirmed to be high, as the PDI value was maintained below 0.2 in both buffers without significant size change over 7 days. The excellent particle stability of the nanoassemblies in both solutions provides the effect of enhancing accumulation in inflamed colonic tissues via the eEPR effect after oral administration. The zeta potential value of the nanoassemblies was determined to be 24.73 ± 2.27 mV, which is much higher than that of conventional nanoparticles that promote mucoadhesive properties through electrostatic interactions with the negatively charged mucus layer.

[0100] Experimental Results 2. Actual Entry and Anti-Inflammatory Effects of FRRG-ASA in Macrophages

[0101] Enhanced cellular uptake of FRRG-ASA occurred in activated M1 phenotype macrophages, which are abundant at sites of inflammation, because exogenous nanoparticles are recognized and actively phagocytosed by antigen-presenting cells. To assess the cellular uptake of FRRG-ASA, FRRG-ASA conjugated with 1 wt% Cy5.5 fluorescent dye was co-assembled with unmodified FRRG-ASA to create Cy5.5-FRRG-ASA. When LPS-treated RAW 264.7 cells were incubated with Cy5.5-FRRG-ASA, robust cellular uptake (red) was clearly observed (see Figure 3A). The amount of nanoassemblies within macrophages gradually increased with incubation time but remained almost the same after 6 or 24 h of treatment. Furthermore, LPS-treated RAW 264.7 cells more actively phagocytosed Cy5-5-FRRG-ASA compared to the intact cells after 24 h of culture, showing 6.77- to 7.04-fold higher cell entry, confirming enhanced cell entry of the nanoassemblies in M1 macrophages (see Figure 3B). Additionally, enhanced cell entry of 5-ASA was observed after formulating FRRG-ASA as nanoassemblies. Investigating the intrinsic cell entry of free 5-ASA using fluorescence imaging was difficult because its molecular weight was too low to introduce a fluorescent dye. Therefore, a comparison between FRRG-ASA and free 5-ASA was performed using HPLC analysis. FRRG-ASA (3.25 μM based on 5-ASA content) showed significantly greater cell entry in LPS-treated RAW 264.7 cells compared to an equivalent dose of free 5-ASA (see Figure 4A). The amount of free 5-ASA in LPS-treated RAW 264.7 cells was similar to that in the original cells after 24 h of culture, and there was a marked difference in the cellular uptake of FRRG-ASA, which was significantly increased in macrophages after LPS treatment.These results indicate that the recognition and phagocytosis of free 5-ASA by M1 macrophages was enhanced after formulation of nanoassemblies. Furthermore, quantitative analysis using HPLC confirmed that the ratio of free 5-ASA to FRRG-ASA gradually increased with incubation time (see Figure 4B). After 24 h of incubation, approximately 99% of FRRG-ASA was cleaved into free 5-ASA in LPS-treated RAW 264.7 cells, whereas free 5-ASA was reduced to half in intact cells.

[0102] The anti-inflammatory effects of FRRG-ASA were evaluated in M1 macrophages. Multiple mechanisms of action for the anti-inflammatory effects of 5-ASA were demonstrated, including a distinct mechanism within macrophages that suppresses mitogen-activated protein kinases (MAPKs)-mediated regulatory genes implicated in colitis, such as c-Jun N-terminal kinases (JNKs) and p38. RAW 264.7 cells significantly upregulated the levels of phosphorylated JNK and p38 after LPS treatment (Fig. 5A). Treatment with FRRG-ASA (3.25 μM based on 5-ASA content) resulted in a significant downregulation of inflammatory mediators in LPS-treated RAW 264.7 cells after 24 h of incubation, with p54 JNK, p46 JNK, and p38 levels being reduced by 63.2%, 60.5%, and 65.7%, respectively, compared to the free 5-ASA-treated group. Furthermore, a significant decrease in the levels of proinflammatory cytokines, such as IL-1β, IL-6, TNF-α, and IFN-γ, was additionally observed in LPS-treated RAW 264.7 cells 24 h after treatment with FRRG-ASA compared to free 5-ASA (see Figure 5B). Consequently, FRRG-ASA induced the differentiation of LPS-treated RAW 264.7 cells from the M1 phenotype to the M2 phenotype, and its effect in promoting macrophage differentiation was significantly higher than that of free 5-ASA. These changes were accompanied by an increase in anti-inflammatory markers, such as CD206 and arginase-1, and a decrease in the proinflammatory marker iNOS in LPS-treated RAW 264.7 cells (see Figures 5C and 5D).

[0103] We assessed whether free 5-ASA, cleaved from FRRG-ASA in macrophages, could also be delivered to adjacent epithelial cells to potentially restore the intestinal barrier in inflamed colonic tissue in vivo. LPS-treated RAW 264.7 cells were cultured with FRRG-ASA for 24 h, after which residual material was removed and co-cultured with human colonic epithelial NCM460 cells for 24 h using a transwell system (Fig. 6A). Despite not being directly exposed to FRRG-ASA, NCM460 cells showed significantly reduced TNF-α and IFN-γ levels after co-culture with FRRG-ASA-treated macrophages compared to cells treated with LPS alone (Fig. 6B). These results indicate that FRRG-ASA efficiently enters M1 macrophages and releases free 5-ASA, thereby promoting their differentiation into an anti-inflammatory M2 phenotype. Additionally, we found that these active compounds can also be delivered to nearby epithelial cells, potentially restoring the intestinal barrier destroyed as the disease progresses.

[0104] Experimental results 3. Accumulation of inflamed colon through mucoadhesive properties and eEPR effect

[0105] As mentioned above, after confirming the unique mechanism of FRRG-ASA exhibiting excellent anti-inflammatory effects in a culture system, we further evaluated its mucoadhesive properties and eEPR effect, which enhance the in vivo properties of 5-ASA. Free Cy5.5 was used as a model drug for 5-ASA, as its molecular weight is too low to directly introduce the fluorescent dye. In vitro flow assays demonstrated higher uptake and residence of Cy5-FRRG-ASA on the surface of mouse colonic specimens compared to free Cy5.5 (see Figure 7A). Moreover, after washing with SIF, the fluorescence intensity of the flow solution was significantly lower in the Cy5.5-FRRG-ASA-treated group than in the free Cy5.5 group, confirming the mucoadhesive properties of the nanoassemblies in colonic tissue (see Figure 7B). Histological analysis of colonic specimens after five washes with SIF revealed a stronger fluorescence signal of Cy5.5-FRRG-ASA (red) in the mucus layer (green) stained with mucin 2 (MUC2) compared to free Cy5.5 (see Figure 7C). This mucoadhesive property of FRRG-ASA appears to be due to the electrostatic interaction between the negatively charged mucus layer and the positively charged nanoassembly surface.

[0106] Near-infrared fluorescence (NIRF) images of BALB / c mice demonstrated sustained retention of Cy5.5-FRRG-ASA in the gastrointestinal tract compared to free Cy5.5 after oral administration, confirming its mucoadhesive properties in vivo. Detectable fluorescence signals of Cy5.5-FRRG-ASA in the gastrointestinal tract were clearly observed 9 hours after treatment, while most of the free Cy5.5 disappeared from the body due to rapid in vivo clearance. Furthermore, NIRF imaging was additionally performed in an IBD model prepared by feeding BALB / c mice with 3% DSS for 5 days. The amount of Cy5.5-FRRG-ASA accumulated in the gastrointestinal tract was significantly increased in IBD mice compared to the control mice after oral administration, whereas free Cy5.5 exhibited similar biodistribution in both models (see Figures 8A and 8B). These results demonstrate that the accumulation of nanoassemblies in inflamed colonic tissue results from their mucoadhesive properties and the eEPR effect (a phenomenon in which nanoparticles passively penetrate through the destroyed epithelial layer of target tissues due to disease progression).

[0107] Ex vivo fluorescence imaging of major organs in IBD mice further confirmed significant accumulation of Cy5.5-FRRG-ASA in the gastrointestinal tract 9 h after oral administration compared to free Cy5.5 (see Figure 9). Furthermore, substantial uptake of Cy5.5-FRRG-ASA into intestinal macrophages was demonstrated. Strong co-localization of the nanoassemblies (red) with the macrophage marker F4 / 80 (green) with a Pearson correlation coefficient of 0.9041 was clearly observed in the colonic tissue of IBD mice 9 h after treatment (see Figures 10A and 10B). These results demonstrate that FRRG-ASA efficiently accumulates within inflamed colonic tissue and is ultimately internalized by intestinal macrophages after oral administration in the IBD model due to its mucoadhesive properties and the eEPR effect.

[0108] Experimental Results 4. Therapeutic Efficacy of FRRG-ASA in IBD Model

[0109] The in vivo anti-inflammatory effects of FRRG-ASA were evaluated in a DSS-induced colitis model. For this investigation, mice were divided into four groups: (i) untreated healthy mice (control), (ii) DSS + PBS (PBS), (iii) DSS + free 5-ASA (5-ASA), and (iv) DSS + FRRG-ASA (FRRG-ASA). As an acute colitis model, BALB / c mice were administered 3% DSS in water for 9 days, and then treated with PBS, free 5-ASA, or FRRG-ASA by oral administration daily, starting on day 5 of DSS treatment. Oral administration of FRRG-ASA at a daily dose of 50 mg / kg 5-ASA protected animals from DSS-induced weight loss and increased disease activity index (DAI) compared to the same dose of free 5-ASA (see Figures 11A and 11B). The colon length (6.04 ± 0.36 cm) of the FRRG-ASA-treated group was similar to that of the control group (7.39 ± 0.69 cm), whereas free 5-ASA-treated group (4.78 ± 0.28 cm) failed to prevent the decrease in colon length in the acute colitis model (PBS-treated group; 3.87 ± 0.27 cm) on day 9 (see Figures 11C and 11D). Furthermore, histological analysis of H&E-stained colon tissues showed a significant reduction in colitis-related pathophysiological parameters, such as goblet cell loss, proliferation, ulceration, and inflammatory cell infiltration, in IBD mice after treatment with FRRG-ASA (see Figure 11E). In contrast, mice in the PBS- and 5-ASA-treated groups exhibited substantial structural abnormalities in the colon tissues due to severe inflammation. This remarkable therapeutic efficacy of FRRG-ASA is due to its enhanced accumulation and retention in inflamed colonic tissue due to its mucoadhesive properties and eEPR effect.

[0110] The anti-inflammatory effects of FRRG-ASA were assessed at the molecular level by analyzing inflammation-related cytokines in the colon on day 9. Enzyme-linked immunosorbent assays (ELISAs) confirmed that FRRG-ASA treatment significantly downregulated the levels of proinflammatory cytokines, such as TNF-α, IFN-γ, IL-1β, and IL-6, compared to the other groups (see Figure 12A). In contrast, the anti-inflammatory cytokine IL-10 was significantly increased in the colon tissue of the FRRG-ASA-treated group (see Figure 12B). Furthermore, confocal microscopy images of the colon tissue of mice in the FRRG-ASA-treated group showed a significant upregulation of the M2 macrophage marker CD163 compared to the other groups (see Figure 12C). These results demonstrate that orally administered nanoassemblies induced superior therapeutic efficacy in an IBD model by targeting intestinal macrophages, modulating immune responses, and ultimately promoting their differentiation into anti-inflammatory M2 macrophages.

[0111] Experimental Results 5. Restoration of the Intestinal Barrier to Prevent Systemic Inflammation by FRRG-ASA

[0112] We investigated whether oral administration of FRRG-ASA suppresses the inflammatory response within epithelial cells and ultimately restores the disrupted intestinal barrier in inflamed colons. Oral administration of DSS has been reported to extensively disrupt tight junctions between epithelial cells in inflamed colonic tissue, leading to the leakage of proinflammatory cytokines through the disrupted intestinal barrier, resulting in systemic inflammation. Therefore, two major tight junction-related proteins in colonic tissue, zonula occludens-1 (ZO-1) and occludin-1, were evaluated in IBD mice on day 9 after treatment using the same protocol as in Experiment 4. Treatment with free 5-ASA failed to prevent DSS-induced tight junction disruption, and the expression of ZO-1 (red) and occludin-1 (green) in the colon was significantly reduced compared to healthy mice, similar to that in the PBS-treated group (Fig. 13A). FRRG-ASA treatment significantly restored the levels of tight junction-related proteins in inflamed colons. Indeed, there was no significant difference in the expression levels of ZO-1 and occludin-1 between the control and FRRG-ASA-treated groups (see Figure 13B). These results demonstrate that the nanoassemblies have a unique mode of action, allowing them to be internalized by intestinal macrophages, releasing active compounds across a wide range of inflammatory sites, and ultimately restoring the damaged intestinal barrier.

[0113] The therapeutic efficacy of FRRG-ASA in suppressing systemic inflammation in a DSS-induced colitis model was further evaluated. After 9 days of 3% DSS administration, significant increases in serum levels of TNF-α and IFN-γ were clearly observed in mice. FRRG-ASA treatment significantly reduced serum levels of proinflammatory cytokines, whereas expression levels remained similar in the PBS and 5-ASA-treated groups (see Figure 13C). Furthermore, mice in the FRRG-ASA-treated group exhibited similar spleen size and weight compared to healthy mice, whereas a marked decrease in systemic inflammation was observed in the other groups (see Figure 13D). The reduction in systemic inflammation in FRRG-ASA-treated mice may be attributed to its ability to restore the intestinal barrier in the IBD model, thereby preventing the leakage of inflammatory cytokines from inflamed colonic tissue into the systemic system.

[0114] The beneficial effects of FRRG-ASA were further confirmed by hematological and histological analyses. Several hematological parameters indicated severe hepatic, renal, and cardiac toxicity in the PBS and 5-ASA-treated groups, as evidenced by significant changes in the blood concentrations of aspartate aminotransferase (AST), alanine transaminase (ALT), blood urea nitrogen (BUN), creatinine, lactate dehydrogenase (LDH), and creatinine kinase (CK) compared to healthy mice (see Figure 14A). In contrast, these parameters in the FRRG-ASA-treated group were within the normal range and similar to the control group. Consistent with the above analysis results related to systemic inflammation, major organs stained with H&E showed significant structural abnormalities with damaged areas in the PBS and 5-ASA-treated groups, whereas such histological toxicity was not observed in the FRRG-ASA-treated group (see Figure 14B). Therefore, we found that the nanoassemblies effectively suppressed local and systemic inflammation associated with the disease, providing significant therapeutic efficacy in an IBD model.

[0115] While specific aspects of the present disclosure have been described in detail, it will be apparent to those skilled in the art that these specific descriptions are merely preferred embodiments and do not limit the scope of the present disclosure. Accordingly, the substantial scope of the present disclosure is defined by the appended claims and their equivalents.

[0116] Sequence number 1 (FRRG peptide): Phe-Arg-Arg-Gly

Claims

1. It is a nano assembly, The above nanoassembly comprises a complex in which an anti-inflammatory agent having an amine group at one end of a peptide represented by sequence number 1 is bound, The above nanoassembly is a nanoassembly formed by self-assembly of the above complex through intermolecular interactions.

2. In paragraph 1, The anti-inflammatory agent having the above amine group is 5-aminosalicylic acid, nanoassembly.

3. In paragraph 1, The above complex is a nanoassembly that self-assembles through intermolecular interactions under aqueous conditions to form a nanoassembly exhibiting a (+) charge on the surface.

4. In paragraph 1, The above nanoassembly is a nanoassembly that is a prodrug for treating inflammatory bowel disease.

5. In paragraph 4, A nanoassembly, wherein the prodrug for treating the above inflammatory bowel disease is administered orally.

6. In paragraph 5, A nanoassembly wherein the prodrug for treating the above inflammatory bowel disease is administered orally, accumulates in inflamed colon tissue, and promotes M2 polarization of intestinal macrophages.

7. In paragraph 5, A nanoassembly, wherein the prodrug for treating the above inflammatory bowel disease is orally administered at a dose of 10 to 500 mg / kg / day based on the weight of the anti-inflammatory agent having an amine group.

8. In paragraph 1, A nanoassembly having an average diameter of 100 to 200 nm.

9. In paragraph 1, A nanoassembly having an average molecular weight of 700 to 800 Da.

10. In paragraph 1, A nanoassembly comprising an anti-inflammatory agent having amine groups in an amount of at least 20 wt% based on the total weight of the nanoassembly.

11. In paragraph 1, The nanoassembly is a nanoassembly that inhibits the production of one or more cytokines selected from the group consisting of TNF-α, IFN-γ, IL-1β, and IL-6, and increases the production of cytokines of IL-10.

12. In paragraph 1, The above nanoassembly is a nanoassembly that inhibits damage to tight junction-related proteins including at least one of ZO-1 (zonula occludens-1) and occludin-1.

13. A pharmaceutical composition for treating inflammatory bowel disease, comprising a nanoassembly according to any one of claims 1 to 12.

Images