Gold clusters, compositions, and methods of treating inflammatory bowel disease

Abstract

Use of ligand-bound gold clusters (AuCs) and compositions containing ligand-bound gold clusters in the treatment of inflammatory bowel disease (IBD) and in the manufacture of a medicament for the treatment of inflammatory bowel disease (IBD). Methods of treating inflammatory bowel disease (IBD).

Description

Technical Field

[0001] This invention relates to ligand-bound gold clusters (AuCs), compositions comprising ligand-bound gold clusters, and methods for preparing medicaments for treating inflammatory bowel disease (IBD) using said ligand-bound gold clusters and compositions. Background Technology

[0002] Inflammatory bowel disease (IBD) is a chronic, nonspecific inflammatory bowel disease of unknown etiology. Its incidence has been increasing in recent years, with over 10 million people worldwide affected. IBD mainly includes two types: ulcerative colitis (UC) and Crohn's disease (CD). It is characterized by diarrhea, abdominal pain, bleeding, and systemic symptoms such as fever, anemia, and malnutrition. While significant progress has been made in IBD drug development over the past two decades, most patients still cannot achieve sustained disease remission with existing medications, making drug development a challenging endeavor.

[0003] Although the development of drugs for treating autoimmune diseases largely focuses on anti-inflammatory approaches, the pathogenesis and pathological manifestations of different autoimmune diseases vary greatly. Therefore, drug development strategies and treatments are difficult to expand from one indication to another, and treating one indication may even induce or worsen symptoms of another. For example, multiple sclerosis (MS) is an autoimmune disease affecting the central nervous system, characterized by damage to the central nervous system caused by immune cells attacking the myelin sheath of nerve cells. Drug development for MS primarily focuses on inhibiting T-cell or B-cell activity. Teriflunomide, for instance, targets dihydroorotate dehydrogenase (DHODH), inhibiting the proliferation of activated T lymphocytes and B lymphocytes, and has shown some effectiveness in inhibiting the progression of relapsing-remitting MS (RRMS). Ocrelizulimab, targeting the CD20 target on the surface of specific B cells, has achieved good results in inhibiting the progression of RRMS and primary progressive MS (PPMS), becoming the world's best-selling MS treatment. However, teriflunomide and ozretinoin have not yet demonstrated definitive therapeutic effects in the treatment of inflammatory bowel disease (IBD). There are even reports that teriflunomide may induce or exacerbate IBD-related symptoms or bowel disturbances in patients with multiple sclerosis (NZ Esfahani, G. von Geldern, MCRomba, DA Parikh, A. Wundes, Inflammatorycolitis associated with Teriflunomide. Multiple Sclerosis and Related Disorders 2020, 46, 102480; M. Ghiasian, A. R. Haghi, S. Borzouei, R. Bawand, Simultaneous onset of Crohn's disease and Psoriasis in a Multiple Sclerosis patient treated with Teriflunomide: A novel case report highlighting potential autoimmune interactions. Heliyon 2024, 10, e26195.).In addition, with the increasing time since ocrelizumab was launched and the increasing number of users, there have been more and more reports of ocrelizumab users experiencing inflammatory bowel disease-type skin disease side effects (S. Pfeuffer, Psorasisform dermatitis following anti-CD20therapies: Immunologic lessons and management dilemmas, Multiple Sclerosis Journal 2024, 30, 897-898; S. El Sankari, C. Van Essche, V. van Pesch, Cutaneous diseases related to a hyperactive T-cell response in ocrelizumab-treated multiple sclerosis patients, Journal of Neurology, 2021, 268, 4376-4378). Large-scale studies suggest that the use of ozoleizumab may increase the risk of inflammatory bowel disease in patients with multiple sclerosis (MHPorwal, D. Patel, M. Maynard, AZObeidat, Disproportional increase in psoriasis reports in association with B cell depleting therapies in patients with multiple sclerosis, Multiple Sclerosis and Related Disorders 2022, 63, 103832).

[0004] For example, etanercept is a biologic agent used to treat rheumatoid arthritis (RA). It reduces RA-related inflammation and joint damage by inhibiting TNF-α, but it cannot be used to treat IBD (including Crohn's disease and ulcerative colitis), and may even increase the risk of IBD (H. Mitoma, T. Horiuchi, H. Tsukamoto, N. Ueda, Molecular mechanisms of action of anti-TNF-α agents–Comparison among therapeutic TNF-α antaonists. Cytokine 2018, 101, 56-63; W. J. Sandborn, S. B. Hanauer, S. Katz, M. Safdi, D. G. Wolf, R. R. Baerg, W. J. Tremaine, T. Johnson, N. N. Diehl, R. R. Z. Insmeister, Etanercept for active Crohn's disease: A randomized, double-blind, placebo-controlled trial. Gastroenterology 2001, 121, 1088-1094).

[0005] For example, interferon-β1 (INF-β1) therapy is a common choice for treating multiple sclerosis, but there are reports that it may induce systemic lupus erythematosus (SLE) symptoms when used to treat multiple sclerosis (DMBahri, H. Khiari, A. Essouri, L. Laadhar, I. Zaraa, A. Mrabet, N. Meddeb, S. Sellami, Systemic lupus erythematosus induced by interferon-β1 therapy in a patient with multiple sclerosis. Fundamental & Clinical Pharmacology 2012, 26, 210-211; JCCrispín, E. Díaz-Jouanen, Systemic lupus erythematosus induced by therapy with interferon-β in a patient with multiple sclerosis. Lupus 2005, 14, 495-496).

[0006] Better strategies and medications are still needed to treat inflammatory bowel disease (IBD). Summary of the Invention

[0007] This invention provides the use of ligand-bound gold clusters in treating patients with inflammatory bowel disease (IBD), a method for treating patients with inflammatory bowel disease (IBD) using ligand-bound gold clusters, and the use of ligand-bound gold clusters in the preparation of medicaments for treating patients with inflammatory bowel disease (IBD).

[0008] Some embodiments of the present invention utilize ligand-bound gold clusters to treat patients with inflammatory bowel disease (IBD); wherein the ligand-bound gold clusters comprise a gold core and a ligand bound to the gold core.

[0009] In some embodiments for this therapeutic use, the diameter of the gold nucleus is 0.5-3 nm. In some embodiments, the diameter of the gold nucleus is 0.5-2.6 nm.

[0010] In some embodiments of this therapeutic use, the ligand is selected from L-cysteine ​​and its derivatives, D-cysteine ​​and its derivatives, cysteine-containing oligopeptides and their derivatives, and other thiol-containing compounds.

[0011] In some embodiments of this therapeutic use, L-cysteine ​​and its derivatives are selected from L-cysteine, N-isobutyryl-L-cysteine ​​(L-NIBC), and N-acetyl-L-cysteine ​​(L-NAC), and D-cysteine ​​and its derivatives are selected from D-cysteine, N-isobutyryl-D-cysteine ​​(D-NIBC), and N-acetyl-D-cysteine ​​(D-NAC).

[0012] In some embodiments of this therapeutic use, the cysteine-containing oligopeptide and its derivatives are cysteine-containing dipeptides, wherein the cysteine-containing dipeptide is selected from L / D-cysteine-L / D-arginine dipeptide (CR), L / D-arginine-L / D-cysteine ​​dipeptide (RC), L / D-histidine-L / D-cysteine ​​dipeptide (HC) and L / D-cysteine-L / D-histidine dipeptide (CH).

[0013] In some embodiments of this therapeutic use, the cysteine-containing oligopeptide and its derivatives are cysteine-containing tripeptides, wherein the cysteine-containing tripeptide is selected from glycine-L / D-cysteine-L / D-arginine tripeptide (GCR), L / D-proline-L / D-cysteine-L / D-arginine tripeptide (PCR), L / D-lysine-L / D-cysteine-L / D-proline tripeptide (KCP), L / D-lysine-L / D-cysteine-L / D-arginine (KCR), and L / D-glutathione (GSH).

[0014] In some embodiments of this therapeutic use, the cysteine-containing oligopeptide and its derivatives are cysteine-containing tetrapeptides, wherein the cysteine-containing tetrapeptide is selected from glycine-L / D-serine-L / D-cysteine-L / D-arginine tetrapeptide (GSCR) and glycine-L / D-cysteine-L / D-serine-L / D-arginine tetrapeptide (GCSR).

[0015] In some embodiments of this therapeutic use, the cysteine-containing oligopeptide and its derivatives are cysteine-containing pentapeptides, wherein the cysteine-containing pentapeptide is selected from cysteine-aspartic acid-glutamic acid-valine-aspartic acid (CDEVD) and aspartic acid-glutamic acid-valine-aspartic acid-cysteine ​​(DEVDC).

[0016] In some embodiments of this therapeutic use, other thiol-containing compounds are selected from 1-[(2S)-2-methyl-3-thiol-1-oxopropyl]-L / D-proline, thioglycolic acid, mercaptoethanol, thiophene, D-3-mercaptovaline, N-(2-mercaptopropionyl)-glycine, dodecyl mercaptan, 2-aminoethanethiol (CSH), 3-mercaptopropionic acid (MPA), and 4-mercaptobenzoic acid (p-MBA).

[0017] Some embodiments of the present invention use ligand-bound gold clusters to prepare medicaments for treating inflammatory bowel disease (IBD) in subjects, wherein the ligand-bound gold clusters comprise a gold core and a ligand bound to the gold core.

[0018] In some embodiments for this preparation, the diameter of the gold nucleus is 0.5-3 nm. In some embodiments, the diameter of the gold nucleus is 0.5-2.6 nm.

[0019] In some embodiments of this preparation, the ligand is selected from L-cysteine ​​and its derivatives, D-cysteine ​​and its derivatives, cysteine-containing oligopeptides and their derivatives, and other thiol-containing compounds.

[0020] In some embodiments of this preparation, L-cysteine ​​and its derivatives are selected from L-cysteine, N-isobutyryl-L-cysteine ​​(L-NIBC), and N-acetyl-L-cysteine ​​(L-NAC), and D-cysteine ​​and its derivatives are selected from D-cysteine, N-isobutyryl-D-cysteine ​​(D-NIBC), and N-acetyl-D-cysteine ​​(D-NAC).

[0021] In some embodiments of the preparation, the cysteine-containing oligopeptide and its derivatives are cysteine-containing dipeptides, wherein the cysteine-containing dipeptide is selected from L / D-cysteine-L / D-arginine dipeptide (CR), L / D-arginine-L / D-cysteine ​​dipeptide (RC), L / D-histidine-L / D-cysteine ​​dipeptide (HC) and L / D-cysteine-L / D-histidine dipeptide (CH).

[0022] In some embodiments of this preparation, the cysteine-containing oligopeptide and its derivatives are cysteine-containing tripeptides, wherein the cysteine-containing tripeptide is selected from glycine-L / D-cysteine-L / D-arginine tripeptide (GCR), L / D-proline-L / D-cysteine-L / D-arginine tripeptide (PCR), L / D-lysine-L / D-cysteine-L / D-proline tripeptide (KCP), L / D-lysine-L / D-cysteine-L / D-arginine (KCR), and L / D-glutathione (GSH).

[0023] In some embodiments of the preparation, the cysteine-containing oligopeptide and its derivatives are cysteine-containing tetrapeptides, wherein the cysteine-containing tetrapeptide is selected from glycine-L / D-serine-L / D-cysteine-L / D-arginine tetrapeptide (GSCR) and glycine-L / D-cysteine-L / D-serine-L / D-arginine tetrapeptide (GCSR).

[0024] In some embodiments of the preparation, the cysteine-containing oligopeptide and its derivatives are cysteine-containing pentapeptides, wherein the cysteine-containing pentapeptide is selected from cysteine-aspartic acid-glutamic acid-valine-aspartic acid (CDEVD) and aspartic acid-glutamic acid-valine-aspartic acid-cysteine ​​(DEVDC).

[0025] In some embodiments of this preparation, other thiol-containing compounds are selected from 1-[(2S)-2-methyl-3-thiol-1-oxopropyl]-L / D-proline, thioglycolic acid, mercaptoethanol, thiophene, D-3-mercaptovaline, N-(2-mercaptopropionyl)-glycine, dodecyl thiol, 2-aminoethanethiol (CSH), 3-mercaptopropionic acid (MPA), and 4-mercaptobenzoic acid (p-MBA).

[0026] The objects and advantages of the present invention will become apparent from the following detailed description of preferred embodiments taken in conjunction with the accompanying drawings. Attached Figure Description

[0027] Preferred embodiments of the invention will now be described with reference to the accompanying drawings, wherein the same reference numerals denote the same elements.

[0028] Figure 1 The UV-Vis (UV) spectra, transmission electron microscopy (TEM) images, and particle size distribution maps of L-NIBC-modified gold nanoparticles (L-NIBC-AuNPs) with different particle sizes are shown. Specifically, the UV (A), TEM (B), and particle size distribution (C) maps of L-NIBC-AuNPs with an average diameter of 3.6 nm; the UV (D), TEM (E), and particle size distribution (F) maps of L-NIBC-AuNPs with an average diameter of 6.0 nm; the UV (G), TEM (H), and particle size distribution (I) maps of L-NIBC-AuNPs with an average diameter of 10.1 nm; and the UV (J), TEM (K), and particle size distribution (L) maps of L-NIBC-AuNPs with an average diameter of 18.2 nm.

[0029] Figure 2 The UV-Vis (UV) spectra, TEM images, and particle size distributions of L-NIBC-bound gold clusters (L-NIBC-AuCs) with different particle sizes are shown. Specifically: UV (A), TEM (B), and particle size distribution (C) images of L-NIBC-AuCs with an average diameter of 1.1 nm; UV (D), TEM (E), and particle size distribution (F) images of L-NIBC-AuCs with an average diameter of 1.8 nm; and UV (G), TEM (H), and particle size distribution (I) images of L-NIBC-AuCs with an average diameter of 2.6 nm.

[0030] Figure 3 Infrared spectra of L-NIBC-AuCs with different particle sizes are shown.

[0031] Figure 4 UV (A), IR (B), TEM (C), and particle size distribution (D) images of CR-bound gold clusters (CR-AuCs).

[0032] Figure 5 UV (A), IR (B), TEM (C), and particle size distribution (D) images of ligand RC-bound gold clusters (RC-AuCs).

[0033] Figure 6 UV (A), IR (B), TEM (C), and particle size distribution (D) plots of gold clusters (Cap-AuCs) bound to the ligand 1-[(2S)-2-methyl-3-thiol-1-oxopropyl]-L-proline (i.e., Cap).

[0034] Figure 7 UV (A), IR (B), TEM (C), and particle size distribution (D) images of gold clusters (GSH-AuCs) bound to ligand GSH.

[0035] Figure 8 UV (A), IR (B), TEM (C), and particle size distribution (D) plots of gold clusters bound to ligand D-NIBC- (D-NIBC-AuCs).

[0036] Figure 9 UV (A), IR (B), TEM (C), and particle size distribution (D) plots of gold clusters (L-Cys-AuCs) bound to L-cysteine ​​ligand.

[0037] Figure 10 The UV (A), IR (B), TEM (C), and particle size distribution (D) plots of the gold clusters (CSH-AuCs) bound to the ligand 2-aminoethanethiol are shown.

[0038] Figure 11 UV (A), IR (B), TEM (C), and particle size distribution (D) plots of gold clusters (MPA-AuCs) bound to ligand 3-mercaptopropionic acid.

[0039] Figure 12 UV (A), IR (B), TEM (C), and particle size distribution (D) plots of gold clusters (p-MBA-AuCs) bound to ligand 4-mercaptobenzoic acid.

[0040] Figure 13UV (A), TEM (B), and particle size distribution (C) plots of gold clusters (CDEVD-AuCs) bound to ligand 4-cysteine-aspartic acid-glutamic acid-valine-aspartic acid (CDEVD).

[0041] Figure 14 UV (A), TEM (B), and particle size distribution (C) plots of gold clusters (DEVDC-AuCs) bound to ligand 4-aspartic acid-glutamic acid-valine-aspartic acid-cysteine ​​(DEVDC).

[0042] Figure 15 This shows the weight changes in mice with a DSS-induced ulcerative colitis mouse model treated with five representative gold clusters.

[0043] Figure 16 The image shows mice with a DSS-induced ulcerative colitis mouse model treated with five representative gold clusters, along with the mice's DAI score.

[0044] Figure 17 The image shows the colon ratio of mice in a DSS-induced ulcerative colitis mouse model treated with five representative gold clusters.

[0045] Figure 18 The image shows the weight changes in mice with a TNBS-induced colitis mouse model treated with five representative gold clusters.

[0046] Figure 19 The image shows mice with a TNBS-induced colitis mouse model treated with five representative gold clusters, along with the mice's DAI scores.

[0047] Figure 20 The image shows mice with a TNBS-induced colitis mouse model treated with five representative gold clusters, and the colon ratio of the mice. Detailed Implementation

[0048] The present invention can be more readily understood by referring to the following detailed description of some embodiments thereof.

[0049] Where publications are cited, the entire contents of those publications are incorporated herein by reference in order to provide a more complete description of the prior art relating to this invention.

[0050] As used herein, “administration” means oral (“po”), suppository, local contact, intravenous (“iv”), intraperitoneal (“ip”), intramuscular (“im”), intralesional, intrahippocampal, lateral ventricle, nasal, or subcutaneous (“sc”) administration, or the implantation of a sustained-release device such as a microosmotic pump or a perishable implant into the subject. Administration can be performed via any route, including parenteral and transmucosal (e.g., oral, nasal, vaginal, rectal, or dermal). Parenteral administration includes, for example, intravenous, intramuscular, arteriolar, intradermal, subcutaneous, intraperitoneal, ventricular, and intracranial administration. Other delivery methods include, but are not limited to, the use of liposomal formulations, intravenous infusion, transdermal patches, etc.

[0051] The term "systemic administration" refers to the administration of a compound or composition to a mammal to deliver the compound or composition to sites within the body, including the target site of action, via the circulatory system. Systemic administration includes, but is not limited to, oral, intranasal, rectal, and parenteral administration (i.e., via routes other than the digestive tract, such as intramuscular, intravenous, arterial, percutaneous, and subcutaneous administration), provided that, as used herein, systemic administration does not include direct administration to brain regions via routes other than the circulatory system, such as intrathecal injection and intracranial administration.

[0052] As used herein, the term “treatment” means, after administration of a drug or systemic drug, delaying the onset of or slowing / reversing the development of a disease or symptom to which the term applies, or alleviating / preventing a disease or symptom.

[0053] The terms “patient,” “subject,” or “individual” can be used interchangeably to refer to mammals, such as humans or non-human mammals, including primates (e.g., macaques, pintailed monkeys, gibbons), domesticated mammals (e.g., felines, canines), agricultural mammals (e.g., cattle, sheep, pigs, horses), and laboratory mammals or rodents (e.g., rats, mice, lagomorphs, hamsters, guinea pigs).

[0054] Ligand-bound gold clusters (AuCs) are a special form of gold existing between gold atoms and gold nanoparticles. The gold nuclei of ligand-bound gold clusters are less than 3 nm in size and consist of only a few to a few hundred gold atoms, causing the collapse of the face-centered cubic stacking structure of gold nanoparticles. Therefore, unlike the continuous or quasi-continuous energy levels of gold nanoparticles, gold clusters exhibit a molecular-like discrete electronic structure with different HOMO-LUMO gaps. This leads to the disappearance of the surface plasmon resonance effect and the corresponding plasmon resonance absorption band (520 ± 20 nm) in the UV-Vis spectrum, which are present in conventional gold nanoparticles.

[0055] This invention provides ligand-bound gold clusters.

[0056] In some embodiments, the ligand-bound gold cluster comprises a ligand and a gold core, wherein the ligand is bound to the gold core. The binding of the ligand to the gold core indicates that the ligand forms a stable complex in solution with the gold core through covalent bonds, hydrogen bonds, electrostatic forces, hydrophobic forces, van der Waals forces, etc. In some embodiments, the diameter of the gold core is 0.5-3 nm. In some embodiments, the diameter of the gold core is in the range of 0.5-2.6 nm.

[0057] In some embodiments, the ligand for the ligand-bound gold cluster is a thiol-containing compound or an oligopeptide. In some embodiments, the ligand is bonded to the gold nucleus via an Au-S bond to form a ligand-bound gold cluster.

[0058] In some embodiments, the ligand is, but is not limited to, L-cysteine, D-cysteine, or a cysteine ​​derivative. In some embodiments, the cysteine ​​derivative is N-isobutyryl-L-cysteine ​​(L-NIBC), N-isobutyryl-D-cysteine ​​(D-NIBC), N-acetyl-L-cysteine ​​(L-NAC), or N-acetyl-D-cysteine ​​(D-NAC).

[0059] In some embodiments, the ligand is, but is not limited to, a cysteine-containing oligopeptide and its derivatives. In some embodiments, the cysteine-containing oligopeptide is a cysteine-containing dipeptide. In some embodiments, the cysteine-containing dipeptide is L / D-cysteine-L / D-arginine dipeptide (CR), L / D-arginine-L / D-cysteine ​​dipeptide (RC), L / D-histidine-L / D-cysteine ​​dipeptide (HC), or L / D-cysteine-L / D-histidine dipeptide (CH). In some embodiments, the cysteine-containing oligopeptide is a cysteine-containing tripeptide. In some embodiments, the cysteine-containing tripeptide is glycine-L / D-cysteine-L / D-arginine tripeptide (GCR), L / D-proline-L / D-cysteine-L / D-arginine tripeptide (PCR), L / D-lysine-L / D-cysteine-L / D-proline tripeptide (KCP), L / D-lysine-L / D-cysteine-L / D-arginine (KCR), or L / D-glutathione (GSH). In some embodiments, the cysteine-containing oligopeptide is a cysteine-containing tetrapeptide. In some embodiments, the cysteine-containing tetrapeptide is glycine-L / D-serine-L / D-cysteine-L / D-arginine tetrapeptide (GSCR) or glycine-L / D-cysteine-L / D-serine-L / D-arginine tetrapeptide (GCSR). In some embodiments, the cysteine-containing oligopeptide is a cysteine-containing pentapeptide. In some implementations, the cysteine-containing pentapeptide is cysteine-aspartic acid-glutamic acid-valine-aspartic acid (CDEVD) or aspartic acid-glutamic acid-valine-aspartic acid-cysteine ​​(DEVDC). "L / D" indicates either L or D.

[0060] In some embodiments, the ligand is a thiol-containing compound. In some embodiments, the thiol-containing compound is 1-[(2S)-2-methyl-3-thiol-1-oxopropyl]-L / D-proline, thioglycolic acid, mercaptoethanol, thiophene, D-3-mercaptovaline, N-(2-mercaptopropionyl)-glycine, dodecyl mercaptan, 2-aminoethanethiol, 3-mercaptopropionic acid, or 4-mercaptobenzoic acid.

[0061] This invention provides a pharmaceutical composition for treating inflammatory bowel disease in a subject. In some embodiments, the subject is a human being. In some embodiments, the subject is a pet animal, such as a dog.

[0062] In some embodiments, the pharmaceutical composition comprises a ligand-bound gold cluster as disclosed above and a pharmaceutically acceptable excipient. In some embodiments, the excipient is a phosphate buffer solution or physiological saline.

[0063] The present invention provides the use of the ligand-bound gold clusters disclosed above for manufacturing for treating inflammatory bowel disease in subjects.

[0064] This invention provides methods for treating inflammatory bowel disease (IBD) in subjects using the disclosed ligand-bound gold clusters. In some embodiments, the treatment method includes administering a pharmaceutically effective amount of the ligand-bound gold cluster to the subject. The pharmaceutically effective amount can be determined through routine in vivo studies. In some embodiments, the pharmaceutically effective amount of the ligand-bound gold cluster is 0.001 mg / kg / day, 0.005 mg / kg / day, 0.01 mg / kg / day, 0.05 mg / kg / day, 0.1 mg / kg / day, 0.5 mg / kg / day, 1 mg / kg / day, 2 mg / kg / day, 3 mg / kg / day, 4 mg / kg / day, 5 mg / kg / day, 6 mg / kg / day, 7 mg / kg / day, 8 mg / kg / day, 9 mg / kg / day, 10 mg / kg / day, or 15 mg / kg / day. / day, 20 mg / kg / day, 30 mg / kg / day, 40 mg / kg / day, 50 mg / kg / day, 60 mg / kg / day, 70 mg / kg / day, 80 mg / kg / day, 90 mg / kg / day, 100 mg / kg / day, 200 mg / kg / day, 300 mg / kg / day, 400 mg / kg / day, 500 mg / kg / day, 600 mg / kg / day, 700 mg / kg / day, 800 mg / kg / day, 900 mg / kg / day, or 1000 mg / kg / day.

[0065] The following examples are provided only to illustrate the principles of the invention; they are by no means intended to limit the scope of the invention.

[0066] Example

[0067] Example 1: Preparation of ligand-bound gold clusters

[0068] 1.1 Dissolve HAuCl4 in methanol, water, ethanol, n-propanol or ethyl acetate to obtain solution A, wherein the concentration of HAuCl4 is 0.01-0.03M;

[0069] 1.2 The ligand is dissolved in a solvent to obtain solution B, wherein the concentration of the ligand is 0.01–0.18 M; the ligand includes, but is not limited to, L-cysteine, D-cysteine ​​and other cysteine ​​derivatives, such as N-isobutyryl-L-cysteine ​​(L-NIBC), N-isobutyryl-D-cysteine ​​(D-NIBC), N-acetyl-L-cysteine ​​(L-NAC) and N-acetyl-D-cysteine ​​(D-NAC), cysteine-containing oligopeptides and their derivatives, including, but not limited to, dipeptides, tripeptides, tetrapeptides, pentapeptides and other cysteine-containing peptides, such as L / D- Cysteine-L / D-arginine dipeptide (CR), L / D-arginine-L / D-cysteine ​​dipeptide (RC), L / D-histidine-L / D-cysteine ​​dipeptide (HC), L / D-cysteine-L / D-histidine (CH), glycine-L / D-cysteine-L / D-arginine tripeptide (GCR), L / D-proline-L / D-cysteine-L / D-arginine tripeptide (PCR), L / D-lysine-L / D-cysteine-L / D-proline tripeptide (KCP), L / D-lysine-L / D-cysteine-L / D-arginine (KCR) L / D-glutathione (GSH), glycine-L / D-serine-L / D-cysteine-L / D-arginine tetrapeptide (GSCR), glycine-L / D-cysteine-L / D-serine-L / D-arginine tetrapeptide (GCSR), cysteine-aspartic-glutamic-valine-aspartic-pentapeptide (CDEVD) and aspartic-glutamic-valine-aspartic-cysteine ​​pentapeptide (DEVDC), and other thiol-containing compounds such as 1-[(2S)-2-methyl-3-thiol-1-oxopropyl]-L / D-proline, mercaptoacetic acid, mercaptoethanol, One or more of thiophene, D-3-mercaptovaline, N-(2-mercaptopropionyl)-glycine, dodecyl mercaptan, 2-aminoethanethiol (CSH), 3-mercaptopropionic acid (MPA), and 4-mercaptobenzoic acid (p-MBA); the solvent is one or more of methanol, ethyl acetate, water, ethanol, n-propanol, pentane, formic acid, acetic acid, diethyl ether, acetone, anisole, 1-propanol, 2-propanol, 1-butanol, 2-butanol, pentanol, butyl acetate, tributyl methyl ether, isopropyl acetate, dimethyl sulfoxide, ethyl formate, isobutyl acetate, methyl acetate, 2-methyl-1-propanol, and propyl acetate;

[0070] 1.3 Mix solutions A and B to make the molar ratio of HAuCl4 to ligand 1:(0.01–100), stir in an ice bath for 0.1–48 h, add 0.025–0.8 M NaBH4 solution in water, ethanol, or methanol, and continue stirring in an ice-water bath for 0.1–12 h. The molar ratio of NaBH4 to ligand is 1:(0.01–100).

[0071] 1.4 After the reaction is complete, the reaction solution is centrifuged for 10–100 min at 8000–175 r / min using an MWCO 3K–30K ultrafiltration tube to obtain ligand-bound gold clusters with different average particle sizes. The pore size of the ultrafiltration membrane of different MWCO tubes directly determines the size of the ligand-bound gold clusters that can pass through the membrane. This step can be optionally omitted.

[0072] 1.5 Dissolve the gold clusters with different average particle sizes bound to ligands obtained in step (1.4) in water, place them in a dialysis bag, and dialyze them in water at room temperature for 1 to 7 days;

[0073] 1.6 After dialysis, the ligand-bound gold clusters were freeze-dried for 12–24 hours to obtain powdered or flocculant substances, i.e., ligand-bound gold clusters.

[0074] As detected, the particle size of the powdered or flocculant material obtained by the aforementioned method is less than 3 nm (typically distributed in the range of 0.5–2.6 nm). No significant absorption peak is observed at 520 nm. The obtained powder or flocculant is confirmed to be ligand-bound gold clusters.

[0075] Example 2: Preparation and identification of gold clusters bound to different ligands

[0076] 2.1 Preparation of L-NIBC-bound gold clusters, namely L-NIBC-AuCs

[0077] Taking ligand L-NIBC as an example, the preparation and identification of gold clusters bound by ligand L-NIBC are described in detail.

[0078] 2.1.1 Weigh 1.00 g of HAuCl4 and dissolve it in 100 mL of methanol to obtain a 0.03 M solution A;

[0079] 2.1.2 Weigh 0.57 g L-NIBC and dissolve it in 100 mL glacial acetic acid to obtain 0.03 M solution B;

[0080] 2.1.3 Take 1 mL of solution A and mix it with 0.5 mL, 1 mL, 2 mL, 3 mL, 4 mL, or 5 mL of solution B (i.e., the molar ratio between HAuCl4 and L-NIBC is 1:0.5, 1:1, 1:2, 1:3, 1:4, and 1:5, respectively). Stir the mixture in an ice bath for 2 h. When the solution changes from bright yellow to colorless, quickly add 1 mL of freshly prepared 0.03 M NaBH4 ethanol solution (weigh 11.3 mg NaBH4 and dissolve it in 10 mL of ethanol). After the solution turns dark brown, continue the reaction for 30 minutes and add 10 mL of acetone to terminate the reaction.

[0081] 2.1.4 After the reaction, the reaction solution was subjected to gradient centrifugation to obtain L-NIBC-AuCs powders of different particle sizes. Specific method: After the reaction was complete, the reaction solution was transferred to a 50 mL ultrafiltration tube with a MWCO of 30 K and centrifuged at 10,000 rpm for 20 min. The residue in the inner tube was dissolved in ultrapure water, yielding powder with a particle size of approximately 2.6 nm. Then, the mixed solution in the outer tube was transferred to a 50 mL ultrafiltration tube with a MWCO of 10 K and centrifuged at 13,000 rpm for 30 min. The residue in the inner tube was dissolved in ultrapure water, yielding powder with a particle size of approximately 1.8 nm. Then, the mixed solution in the outer tube was transferred to a 50 mL ultrafiltration tube with a MWCO of 3 K and centrifuged at 17,500 rpm for 40 min. The residue in the inner tube was dissolved in ultrapure water, yielding powder with a particle size of approximately 1.1 nm.

[0082] 2.1.5 The three different particle sizes of the precipitate obtained by gradient centrifugation were separated by removing the solvent, the crude product was dried by blowing with N2, dissolved in 5 mL of ultrapure water, placed in a dialysis bag (MWCO of 3 kDa), and placed in 2 L of ultrapure water. The water was changed every other day, and the dialysis was performed for 7 days. After freeze-drying, it was ready for use.

[0083] 2.2 Identification of L-NIBC-AuCs

[0084] Identification experiments were performed on the powder (L-NIBC-AuCs) obtained above. Meanwhile, gold nanoparticles modified with L-NIBC ligand (L-NIBC-AuNP) were used as a control. The preparation method of gold nanoparticles with L-NIBC ligand is described in the references (W. Yan, L. Xu, C. Xu, W. Ma, H. Kuang, L. Wang and NAKotov, Journal of the American Chemical Society 2012, 134, 15114; X. Yuan, B. Zhang, Z. Luo, Q. Yao, D. Leong, N. Yan and J. Xie, Angewandte Chemie International Edition 2014, 53, 4623).

[0085] 2.2.1 Morphological observation using transmission electron microscopy (TEM)

[0086] The test powders (L-NIBC-AuCs and L-NIBC-AuNPs samples) were dissolved in ultrapure water to a concentration of 2 mg / L to prepare the samples, which were then prepared using the hanging drop method. More specifically, 5 μL of the sample was dropped onto an ultrathin carbon film and allowed to evaporate naturally until the water droplet disappeared. The morphology of the sample was then observed using a JEM-2100F STEM / EDS field emission high-resolution TEM.

[0087] Four TEM images of L-NIBC-AuNP are shown Figure 1 In the B, E, H, and K images; three TEM images of L-NIBC-AuCs are shown Figure 2 Among the B, E and H images.

[0088] Figure 2 The images show that each L-NIBC-AuCs sample has a uniform particle size and good dispersibility, and the average diameters (referring to the diameter of the gold core) of L-NIBC-AuCs are 1.1 nm, 1.8 nm, and 2.6 nm, respectively. Figure 2 The results in the C, F, and I images are completely consistent. In contrast, the L-NIBC-AuNPs samples have larger particle sizes. Their average diameters (referring to the diameter of the gold core) are 3.6 nm, 6.0 nm, 10.1 nm, and 18.2 nm, respectively. Figure 1 The results for the C, F, I, and L amplitudes are in good agreement.

[0089] 2.2.2 Ultraviolet (UV)-Vis absorption spectrum

[0090] The test powders (L-NIBC-AuCs and L-NIBC-AuNPs samples) were dissolved in ultrapure water until the concentration reached 10 mg·L⁻¹. -1 The UV-vis absorption spectrum was measured at room temperature. The scanning range was 190-1100 nm, the sample cell was a standard quartz cuvette with a path length of 1 cm, and the reference cell was filled with ultrapure water.

[0091] The UV-vis absorption spectra of four L-NIBC-AuNP samples of different sizes are shown in the figure. Figure 1 In images A, D, G, and J, the statistical distribution of granularity is shown in... Figure 1 The UV-vis absorption spectra of three L-NIBC-AuCs samples with different sizes are shown in the C, F, I, and L amplitudes. Figure 2 In images A, D, and G, the statistical distribution of granularity is shown in... Figure 2 In the C, F and I frames.

[0092] Figure 1This indicates that L-NIBC-AuNP exhibits an absorption peak at approximately 520 nm due to surface plasmon resonance. The position of the absorption peak is related to particle size. When the particle size is 3.6 nm, the UV absorption peak appears at 516 nm; when the particle size is 6.0 nm, the UV absorption peak appears at 517 nm; when the particle size is 10.1 nm, the UV absorption peak appears at 520 nm; and when the particle size is 18.2 nm, the absorption peak appears at 523 nm. None of the four samples exhibited any absorption peak above 560 nm.

[0093] Figure 2 The results show that in the UV absorption spectra of L-NIBC-bound gold clusters with three different particle sizes, the surface plasmon resonance absorption peak at 520 nm disappears, while two distinct absorption peaks appear above 560 nm. The positions of these absorption peaks vary slightly with the particle size of the gold clusters. This is because the collapse of the face-centered cubic structure causes the gold clusters to exhibit molecular-like properties, leading to discontinuities in the density of states, energy level splitting, the disappearance of the plasmon resonance effect, and the appearance of new absorption peaks in the longer wavelength directions. It can be concluded that the three powder samples with different particle sizes obtained above are all ligand-bound gold clusters.

[0094] 2.2.3 Fourier Transform Infrared Spectroscopy

[0095] Infrared spectroscopy was performed on a Bruker VERTEX 80V Fourier transform infrared spectrometer using solid powder high-vacuum total reflectance mode, with a scanning range of 4000-400 cm⁻¹. -1 The scan was performed 64 times. Taking L-NIBC-bound gold clusters as an example, the test samples consisted of three types of L-NIBC-bound gold cluster dry powders with different particle sizes, while the control sample was pure L-NIBC powder. Results are shown below. Figure 3 .

[0096] Figure 3 Infrared spectra of L-NIBC-bound gold clusters with different particle sizes are shown. Compared to pure L-NIBC (bottom curve), the L-NIBC-bound gold clusters with different particle sizes exhibit different infrared spectra in the 2500-2600 cm⁻¹ range. -1 The SH stretching vibrations between the ligands completely disappeared, while other characteristic peaks of L-NIBC were still observed. This demonstrates that L-NIBC molecules successfully bind to the surface of gold clusters via gold-sulfur bonds. The figure also shows that the infrared spectrum of the ligand-bound gold clusters is independent of their size.

[0097] Gold clusters bound to other ligands can be prepared using a similar method as described above, with slight adjustments to the solvent of solution B, the feed ratio of HAuCl4 to the ligand, the reaction time, and the amount of NaBH4 added. For example, when using L-cysteine, D-cysteine, N-isobutyryl-L-cysteine ​​(L-NIBC), or N-isobutyryl-D-cysteine ​​(D-NIBC) as ligands, acetic acid is chosen as the solvent; when using dipeptide CR, dipeptide RC, or 1-[(2S)-2-methyl-3-mercapto-1-oxopropyl]-L-proline as ligands, water is chosen as the solvent, and so on. Other steps are similar, so further details are not provided here.

[0098] This invention prepared and obtained a series of ligand-bound gold clusters using the above method. The parameters of the ligands and preparation method are shown in Table 1.

[0099] Table 1. Preparation parameters of gold clusters with different ligand bindings according to the present invention

[0100] The samples listed in Table 1 were confirmed using the aforementioned methods. The characteristics of the 11 different ligand-bound gold clusters are shown in... Figure 4 (CR-AuCs), Figure 5 (RC-AuCs), Figure 6 (Cap-AuCs) (Cap represents 1-[(2S)-2-methyl-3-mercapto-1-oxopropyl]-L-proline), Figure 7 (GSH-AuCs), Figure 8 (D-NIBC-AuCs), Figure 9 (L-Cys-AuCs), Figure 10 (CSH-AuCs), Figure 11 (MPA-AuCs) Figure 12 (p-MBA-AuCs), Figure 13 (CDEVD-AuCs) Figure 14 (DEVDC-AuCs). Figures 4-12 Displays UV spectrum (A), infrared spectrum (B), TEM image (C), and particle size distribution (D). Figures 13-14 Displays UV spectrum (A), TEM image (B), and particle size distribution (C).

[0101] The results showed that the diameters of the gold clusters bound by the different ligands obtained in Table 1 were all less than 3 nm. The UV-Vis spectrum also showed the disappearance of the peak at 520 ± 20 nm, and the appearance of absorption peaks at other positions. The positions of these absorption peaks varied with the ligands, particle size, and structure. In some cases, no specific absorption peak appeared, mainly because a mixture of gold clusters of different sizes and structures was formed, or certain special gold clusters caused the absorption peaks to be located outside the range of conventional UV-Vis absorption spectroscopy. Simultaneously, the Fourier transform infrared spectroscopy also showed the disappearance of the thiol infrared absorption peak of the ligands. Figure 4-8 The peaks between the dashed lines in the middle B amplitude are visible, while other infrared characteristic peaks are retained, indicating that the ligand molecules have successfully bound to the gold atoms to form ligand-bound gold clusters, indicating that the present invention has successfully obtained gold clusters bound to the ligands listed in Table 1.

[0102] Example 3: Test samples for animal research

[0103] The gold clusters used in animal studies include 15 different ligand-modified gold clusters, namely: L-cysteine-modified gold clusters (L-Cys-AuCs) L-NIBC modified gold clusters (L-NIBC-AuCs) L-cysteine-L-arginine modified gold clusters (CR-AuCs) L-cysteine-L-histidine dipeptide-modified gold clusters (CH-AuCs) L-histidine-L-cysteine ​​dipeptide-modified gold clusters (HC-AuCs) Glutathione-modified gold clusters (GSH-AuCs) Gold clusters modified with glycine-L-cysteine-L-arginine tripeptide (GCR-AuCs) L-proline-L-cysteine-L-arginine tripeptide-modified gold clusters (PCR-AuCs) L-lysine-L-cysteine-L-proline tripeptide-modified gold clusters (KCP-AuCs) L-lysine-L-cysteine-L-arginine tripeptide-modified gold clusters (KCR-AuCs) Gold clusters modified with glycine-L-serine-L-cysteine-L-arginine tetrapeptides (GSCR-AuCs) Gold clusters modified with glycine-L-cysteine-L-serine-L-arginine tetrapeptide (GCSR-AuCs) 4-Mercaptobenzoic acid-modified gold clusters (MBA-AuCs), Thioglycolic acid-modified gold clusters (MA-AuCs), 1-[(2S)-2-methyl-3-thiol-1-oxopropyl]-L-proline-modified gold clusters (CAP-AuCs).

[0104] The gold core sizes of these 15 gold clusters are all between 0.5 and 3.0 nm.

[0105] The preparation methods for all test samples were the same as those described above, with slight modifications; their quality was verified by the aforementioned methods.

[0106] Example 4: Pharmacological study of gold clusters in a mouse model of ulcerative colitis induced by dextran sulfate sodium (DSS).

[0107] The dextran sulfate sodium (DSS)-induced mouse ulcerative colitis model is the most widely used chemically induced IBD model in mice. By dissolving DSS in drinking water and providing it to mice, the DSS, which cannot be directly absorbed by colonic epithelial cells but can damage the tight junctions and basement membrane of epithelial cells, leads to disruption of the intestinal mucosal barrier and increased intestinal permeability. This promotes the infiltration of gut microbiota and antigens, activates the immune system, and induces an inflammatory response, thus triggering ulcerative colitis symptoms in mice. Animals exhibit significant weight loss, loose stools, and bloody stools, remarkably similar to those in human ulcerative colitis. This model is one of the commonly used animal models for IBD research.

[0108] 4.1 Experimental Methods

[0109] Male C57BL / 6 mice were randomly divided into three groups of 10 mice each: a blank control group, a model control group (modeled and injected with an equal volume of saline daily), and treatment groups with different gold clusters (modeled and injected with an equal volume of gold clusters daily). Mice were acclimatized to the experimental facility for seven days prior to the experiment. The blank control group mice were normally fed and injected with 100 μL of saline daily during the experiment. Mice in the model control group and the gold cluster treatment groups received drinking water with 3% DSS added for 8 consecutive days from Day 0 to Day 7. Mice in the model control group received 100 μL of saline daily, while mice in the gold cluster treatment groups received an intraperitoneal injection of 5 mg / kg of the corresponding gold cluster saline solution daily, with an injection volume of 100 μL. To verify the therapeutic effects of individual ligand molecules and gold nanoparticles, we added four treatment groups (all at a dose of 5 mg / kg): L-Cys, L-NIBC, and CR, as well as L-NIBC-modified gold nanoparticles (L-NIBC-AuNPs, with diameters between 3.5 and 10 nm). All mice were weighed daily, and the Disease Activity Index (DAI) score for each mouse was recorded. The DAI score consisted of three parts, each scored from 0 to 4 points, and the scoring criteria are shown in Table 2.

[0110] Table 2. DAI Scoring Criteria score Weight loss (%) Stool characteristics Occult blood or bloody stool 0 0 normal Occult blood negative 1 1～5 soft stool Weak positive for occult blood 2 6～10 loose stool Occult blood positive 3 11～20 diarrhea bloody stool 4 >20 watery stool Severe bloody stools

[0111] On Day 8, after weighing the mice and recording their DAI scores, the animals were euthanized. The colon (from the anus to the cecum) of each mouse was collected, the fat and connective tissue around the colon were removed, the length was measured, the contents were removed, and the colon was weighed.

[0112] 4.2 Experimental Results

[0113] Figure 15-17 Representative experimental results for five of the gold clusters are shown.

[0114] Figure 15 These are the mouse body weight data. As can be seen, DSS modeling significantly reduced the body weight of the model control mice over time, reaching 83.9% ± 1.1% on Day 8, a significant difference compared to the blank control group (blank control group: 99.6% ± 0.9%, p < 0.001 vs. model control group, ***). Treatment with L-NIBC-AuCs and L-Cys-AuCs significantly inhibited this weight reduction (L-NIBC-AuCs: 95.1 ± 1.2%, p < 0.001 vs. model control group, ***; L-Cys-AuCs: 91.9 ± 1.5%, p < 0.001 vs. model control group, ***). CR-AuCs and GSH-AuCs also had significant effects, reducing body weight by 89.7 ± 1.8% (p < 0.01 vs. model control group, **) and 87.1% ± 0.9% (p < 0.05, *), respectively, on Day 8. MA-AuCs were also elevated compared to the model control group, but the difference was not statistically significant (MA-AuCs: 85.8±1.3%, p>0.05). Table 3 lists the mouse body weight data on Day 8 for the remaining 10 gold cluster treatment groups. Figure 15 As shown in Table 3, the percentage of body weight of mice in the L-NIBC-AuCs and L-Cys-AuCs treatment groups reached over 90% on Day 8, while the percentage of body weight of the rest was below 90%.

[0115] Table 3. Percentage of body weight of mice in the other 10 gold cluster treatment groups relative to Day 0 at Day 8.

[0116] Figure 16The results are the clinical DAI score (a key indicator for evaluating efficacy). The results showed that DSS modeling significantly increased the DAI score in mice over time, reaching 9.2±0.4 on Day 8, while the blank control group had a score of 0.7±0.4, a significant difference (p<0.001, ***). Treatment with L-NIBC-AuCs and L-Cys-AuCs significantly reduced the DAI score (L-NIBC-AuCs: 3.1±0.3, p<0.001 vs. model control group, ***; L-Cys-AuCs: 3.6±0.5, p<0.001 vs. model control group, ***). CR-AuCs, MA-AuCs, and GSH-AuCs also showed good results (CR-AuCs: 5.4±0.5, p<0.001 vs. model control group, ***; MA-AuCs: 7.8±0.7, p<0.05 vs. model control group, *; GSH-AuCs: 7.0±0.7, p<0.01 vs. model control group). Table 4 shows the DAI clinical scores of mice in the other 10 gold cluster treatment groups on Day 8. These data show that the average DAI clinical scores of mice in the L-NIBC-AuCs and L-Cys-AuCs treatment groups were below 4, while the scores of the other treatment groups were above 5. However, treatment with L-cys, L-NIBC, or CR alone, as well as L-NIBC-AuNPs treatment, did not show any significant effect. On Day 8, the mean DAI scores of the corresponding treatment groups were all above 8.8, and there was no statistically significant difference compared with the model control group, indicating that ligand treatment alone and gold nanoparticle treatment were ineffective in the DSS-induced mouse colitis model.

[0117] Table 4. DAI scores of mice in the other 10 gold cluster treatment groups on Day 8

[0118] DSS-induced colitis may cause colonic shortening and increased colonic weight in animals. The colon ratio, the ratio of colonic weight to colonic length, is an important indicator of successful colitis modeling and the effectiveness of drug treatment. Our results show ( Figure 17In the model control group, the colon length of mice was significantly shorter (4.9±0.3cm) compared to the blank control group (7.1±0.2cm) (p<0.001, ***), while the weight was also significantly increased (model control group: 188.9±6.9mg, blank control group: 158.1±6.0mg, p<0.001). Correspondingly, the colon ratio (a key indicator for evaluating efficacy) was significantly increased (model control group: 39.2±1.6mg / cm, blank control group: 22.5±0.7mg / cm, p<0.001, ***). The colon length of mice in the L-NIBC-AuCs and L-Cys-AuCs treatment groups was significantly increased compared to the model control group (L-NIBC-AuCs: 6.4±0.3cm, p<0.001, ***; L-Cys-AuCs: 6.1±0.3cm, p<0.001, ***). The colon ratio was significantly lower in all groups compared to the model control group (L-NIBC-AuCs: 27.1±1.8 mg / cm, p<0.001, ***; L-Cys-AuCs: 28.7±1.6 mg / cm, p<0.001, ***). For the CR-AuCs, MA-AuCs, and GSH-AuCs treatment groups, the colon ratio was also significantly lower than the model control group (CR-AuCs: 31.4±1.4 mg / cm, p<0.01, **; MA-AuCs: 35.9±1.2, p<0.05, *; GSH-AuCs: 33.5±1.7, p<0.05, *). Table 5 shows the colon ratio data of mice in the other 10 gold cluster treatment groups at Day 8. These data show that the average colon ratio of mice treated with L-NIBC-AuCs and L-Cys-AuCs was below 30 mg / cm, while the ratio of the remaining gold clusters treated with L-NIBC-AuCs was above 30 mg / cm.

[0119] Table 5. Colon ratio of mice in the other 10 gold cluster treatment groups at Day 8

[0120] 4.3 Conclusion

[0121] The results above indicate that all gold clusters showed significant therapeutic effects in the DSS-induced mouse colitis model, although L-NIBC-AuCs and L-Cys-AuCs showed particularly significant effects.

[0122] Example 5: Pharmacological study of gold clusters in a TNBS-induced mouse model of colitis

[0123] Trinitrobenzenesulfonic acid (TNBS) is a hapten. It first disrupts the intestinal mucosal barrier through the chemical action of ethanol, then enters the local intestinal tissue. After binding with tissue proteins, it becomes a complete antigen, triggering an immune response and leading to intestinal inflammation. The pathological changes are mainly manifested as: obvious fissure-like ulcers on the surface of the colonic mucosa, transmural inflammation throughout the entire intestinal wall, thickening and hardening of the intestinal wall at the lesion site, severe edema of the intestinal mucosa, superficial ulcers resembling aphthous ulcers and cobblestone-like changes in the folds, and severe adhesion between the intestinal wall and surrounding tissues. These changes share many similarities with the pathological characteristics of human Crohn's disease, and this model is another commonly used animal model for studying intestinal dysplasia (IBD).

[0124] 5.1 Experimental Methods

[0125] Female C57BL / 6 mice were randomly assigned to groups of 10 each, including a blank control group, a model control group, and treatment groups with different gold clusters. Mice were acclimatized to the experimental facility for seven days prior to the experiment. The blank control group received 100 μL of 50% ethanol via rectal instillation, while the other groups received 100 μL of 1.5% TNBS solution (diluted with 50% ethanol) via rectal instillation. The gold cluster treatment groups received intraperitoneal injections of 5 mg / kg of gold clusters once daily from Day 1 to Day 4, with an injection volume of 100 μL. The blank control and model control groups received 100 μL of physiological saline intraperitoneally daily. To verify the therapeutic effects of individual ligand molecules and gold nanoparticles, we added four additional treatment groups: L-Cys, L-NIBC, and CR, as well as L-NIBC-modified gold nanoparticles (L-NIBC-AuNPs, with diameters between 3.5 and 10 nm) (all at a dose of 5 mg / kg). Mouse weight and DAI scores were recorded daily. The composition and scoring criteria of the DAI score are shown in Table 2 of Example 4. At the end of Day 5, the mice were euthanized, and the colons (from the anus to the cecum) were collected. The fat and connective tissue around the colon were removed, the length was measured, the contents were removed, and the colons were weighed.

[0126] 5.2 Experimental Results

[0127] Figure 18-20 Representative experimental results for five gold clusters are shown.

[0128] Figure 18This reflects the change in mouse body weight. Similar to Example 4, TNBS treatment also caused a significant decrease in body weight in the model control group mice. Although the body weight recovered slightly at the end, it was still significantly lower than the starting body weight on Day 5 (model control group: 82.4±1.5% of starting body weight; blank control group: 103.1±1.2% of starting body weight, p<0.001, ***). L-NIBC-AuCs and L-Cys-AuCs treatment significantly inhibited the weight loss induced by TNBS treatment (L-NIBC-AuCs: 96.5±1.4%, p<0.001 vs. model control group, ***; L-Cys-AuCs: 93.8±2.4%, p<0.001 vs. model control group, ***). The mean percentage of body weight in mice treated with CR-AuCs, MA-AuCs, and GSH-AuCs was significantly higher than that in the model control group (CR-AuCs: 89.6±2.0%, p<0.01,**; MA-AuCs: 86.2±1.5%, p<0.05,*; GSH-AuCs: 87.2±2.1%, p<0.05,*). Table 6 shows the body weight data of mice in the other 10 gold cluster treatment groups at Day 5. These results show that the average body weight ratio of mice in the L-NIBC-AuCs and L-Cys-AuCs treatment groups at Day 5 relative to Day 0 was greater than 90%, while the ratios in the other gold cluster treatment groups were less than 90%.

[0129] Table 6. Percentage of body weight of mice in the other 10 gold cluster treatment groups relative to Day 0 at Day 5.

[0130] Figure 19These are the results of the DAI clinical score. The results showed that TNBS modeling significantly increased the DAI score in mice. On Day 5, the DAI score of the model control group reached 9.0±0.4, while that of the blank control group was 0.0±0.0, a significant difference (p<0.001, ***). The L-NIBC-AuCs and L-Cys-AuCs treatment groups significantly reduced the Day 5 DAI score compared to the model control group (L-NIBC-AuCs: 3.7±0.6, p<0.001, ***; L-Cys-AuCs: 4.3±0.8%, p<0.001, ***). CR-AuCs, MA-AuCs, and GSH-AuCs also showed significant improvement (CR-AuCs: 6.2±0.7, p<0.01, **; MA-AuCs: 7.9±0.7, p<0.05, *; GSH-AuCs: 7.4±0.5, p<0.05, *). Table 7 shows the DAI clinical scores of mice in the other 10 gold cluster treatment groups at Day 5. These data show that the DAI of mice in the L-NIBC-AuCs and L-Cys-AuCs treatment groups was less than 5 at Day 5, while the DAI of the other gold cluster treatment groups was greater than 6. However, treatment with L-Cys, L-NIBC, or CR alone, as well as L-NIBC-AuNPs treatment, did not show any significant effect. On Day 5, the mean DAI scores of the corresponding treatment groups were all above 8.5, and there was no statistically significant difference compared with the model control group, indicating that the ligands alone and gold nanoparticles were ineffective in the TNBS-induced mouse colitis model.

[0131] Table 7. DAI scores of mice in the other 10 gold cluster treatment groups on Day 5.

[0132] Figure 20This is a result of the colon ratio. Similar to the results of the DSS-induced ulcerative colitis experiment, TNBS treatment led to a significant increase in the colon ratio in mice (model control group: 54.8±2.7 mg / cm, blank control group: 20.9±1.3 cm / mg, p<0.001, ***). L-NIBC-AuCs and L-Cys-AuCs treatment significantly reduced the colon ratio compared to the model control group (L-NIBC-AuCs: 30.7±2.4 mg / cm, p<0.001, ***; L-Cys-AuCs: 34.8±2.2 mg / cm, p<0.001, ***). CR-AuCs and GSH-AuCs also showed good effects (CR-AuCs: 41.9±2.6 mg / cm, p<0.01, ***; GSH-AuCs: 46.2±2.1 mg / cm, p<0.05, *). The MA-AuCs treatment group also showed a decrease compared to the model control group, but the difference was not statistically significant (51.1±1.9, p>0.05). Table 8 shows the colon ratio data of mice in the other 10 gold cluster treatment groups on Day 5. These data show that the colon ratio of mice in the L-NIBC-AuCs and L-Cys-AuCs treatment groups was less than 35 mg / cm, while the other gold cluster treatment groups had a colon ratio greater than 40 mg / cm.

[0133] Table 8. Colon ratio of mice in the other 10 gold cluster treatment groups at Day 5

[0134] 5.3 Conclusion

[0135] The results above indicate that all gold clusters showed significant therapeutic effects in the TNBS-induced mouse colitis model, although L-NIBC-AuCs and L-Cys-AuCs were particularly effective.

[0136] The results of the two examples above demonstrate that all gold clusters exhibit significant therapeutic effects in both IBD animal models and can be used for the development of IBD-related therapeutic drugs.

[0137] Industrial applicability

[0138] Ligand-bound gold clusters can be used to treat inflammatory bowel disease. They are also suitable for industrial applications.

Claims

1. The use of ligand-bound gold clusters in the treatment of patients with inflammatory bowel disease (IBD), characterized in that, The ligand-bound gold clusters mentioned above include: Gold core; and Ligands that bind to the gold nucleus.

2. The therapeutic use according to claim 1, characterized in that, The diameter of the gold core is 0.5-3 nm.

3. The therapeutic use according to claim 1, characterized in that, The diameter of the gold core is 0.5-2.6 nm.

4. The therapeutic use according to claim 1, characterized in that, The ligand is selected from L-cysteine ​​and its derivatives, D-cysteine ​​and its derivatives, cysteine-containing oligopeptides and their derivatives, and other thiol-containing compounds.

5. The therapeutic use according to claim 4, characterized in that, The L-cysteine ​​and its derivatives are selected from L-cysteine, N-isobutyryl-L-cysteine ​​(L-NIBC) and N-acetyl-L-cysteine ​​(L-NAC), and the D-cysteine ​​and its derivatives are selected from D-cysteine, N-isobutyryl-D-cysteine ​​(D-NIBC) and N-acetyl-D-cysteine ​​(D-NAC).

6. The therapeutic use according to claim 4, characterized in that, The cysteine-containing oligopeptides and their derivatives are cysteine-containing dipeptides, and the cysteine-containing dipeptides are selected from L / D-cysteine-L / D-arginine dipeptide (CR), L / D-arginine-L / D-cysteine ​​dipeptide (RC), L / D-histidine-L / D-cysteine ​​dipeptide (HC) and L / D-cysteine-L / D-histidine dipeptide (CH).

7. The therapeutic use according to claim 4, characterized in that, The cysteine-containing oligopeptide and its derivatives are cysteine-containing tripeptides, which are selected from glycine-L / D-cysteine-L / D-arginine tripeptide (GCR), L / D-proline-L / D-cysteine-L / D-arginine tripeptide (PCR), L / D-lysine-L / D-cysteine-L / D-proline tripeptide (KCP), L / D-lysine-L / D-cysteine-L / D-arginine (KCR), and L / D-glutathione (GSH).

8. The therapeutic use according to claim 4, characterized in that, The cysteine-containing oligopeptide and its derivatives are cysteine-containing tetrapeptides, and the cysteine-containing tetrapeptides are selected from glycine-L / D-serine-L / D-cysteine-L / D-arginine tetrapeptide (GSCR) and glycine-L / D-cysteine-L / D-serine-L / D-arginine tetrapeptide (GCSR).

9. The therapeutic use according to claim 4, characterized in that, The cysteine-containing oligopeptides and their derivatives are cysteine-containing pentapeptides, and the cysteine-containing pentapeptides are selected from cysteine-aspartic acid-glutamic acid-valine-aspartic acid (CDEVD) and aspartic acid-glutamic acid-valine-aspartic acid-cysteine ​​(DEVDC).

10. The therapeutic use according to claim 4, characterized in that, The other thiol-containing compounds are selected from 1-[(2S)-2-methyl-3-thiol-1-oxopropyl]-L / D-proline, thioglycolic acid, mercaptoethanol, thiophene, D-3-mercaptovaline, N-(2-mercaptopropionyl)-glycine, dodecyl thiol, 2-aminoethanethiol, 3-mercaptopropionic acid, and 4-mercaptobenzoic acid.

11. The use of ligand-bound gold clusters in the preparation of medicaments for treating patients with inflammatory bowel disease (IBD), characterized in that, The ligand-bound gold clusters comprise: Gold core; and Ligands that bind to the gold nucleus.

12. The preparation and use according to claim 11, characterized in that, The diameter of the gold core is 0.5-3 nm.

13. The preparation and use according to claim 11, characterized in that, The diameter of the gold core is 0.5-2.6 nm.

14. The preparation and use according to claim 11, characterized in that, The ligand is selected from L-cysteine ​​and its derivatives, D-cysteine ​​and its derivatives, cysteine-containing oligopeptides and their derivatives, and other thiol-containing compounds.

15. The preparation and use according to claim 14, characterized in that, The L-cysteine ​​and its derivatives are selected from L-cysteine, N-isobutyryl-L-cysteine ​​(L-NIBC) and N-acetyl-L-cysteine ​​(L-NAC), and the D-cysteine ​​and its derivatives are selected from D-cysteine, N-isobutyryl-D-cysteine ​​(D-NIBC) and N-acetyl-D-cysteine ​​(D-NAC).

16. The preparation and use according to claim 14, characterized in that, The cysteine-containing oligopeptides and their derivatives are cysteine-containing dipeptides, and the cysteine-containing dipeptides are selected from L / D-cysteine-L / D-arginine dipeptide (CR), L / D-arginine-L / D-cysteine ​​dipeptide (RC), L / D-histidine-L / D-cysteine ​​dipeptide (HC) and L / D-cysteine-L / D-histidine dipeptide (CH).

17. The preparation and use according to claim 14, characterized in that, The cysteine-containing oligopeptide and its derivatives are cysteine-containing tripeptides, which are selected from glycine-L / D-cysteine-L / D-arginine tripeptide (GCR), L / D-proline-L / D-cysteine-L / D-arginine tripeptide (PCR), L / D-lysine-L / D-cysteine-L / D-proline tripeptide (KCP), L / D-lysine-L / D-cysteine-L / D-arginine (KCR), and L / D-glutathione (GSH).

18. The preparation and use according to claim 14, characterized in that, The cysteine-containing oligopeptide and its derivatives are cysteine-containing tetrapeptides, and the cysteine-containing tetrapeptides are selected from glycine-L / D-serine-L / D-cysteine-L / D-arginine tetrapeptide (GSCR) and glycine-L / D-cysteine-L / D-serine-L / D-arginine tetrapeptide (GCSR).

19. The preparation and use according to claim 14, characterized in that, The cysteine-containing oligopeptides and their derivatives are cysteine-containing pentapeptides, and the cysteine-containing pentapeptides are selected from cysteine-aspartic acid-glutamic acid-valine-aspartic acid (CDEVD) and aspartic acid-glutamic acid-valine-aspartic acid-cysteine ​​(DEVDC).

20. The preparation and use according to claim 14, characterized in that, The other thiol-containing compounds are selected from 1-[(2S)-2-methyl-3-thiol-1-oxopropyl]-L / D-proline, thioglycolic acid, mercaptoethanol, thiophene, D-3-mercaptovaline, N-(2-mercaptopropionyl)-glycine, dodecyl thiol, 2-aminoethanethiol, 3-mercaptopropionic acid, and 4-mercaptobenzoic acid.

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