Ligand-bound gold clusters, compositions and methods for treating liver cirrhosis

Abstract

The present invention relates to the technical field of liver cirrhosis treatment, in particular to ligand-bound gold clusters for treating liver cirrhosis, a composition comprising the ligand-bound gold clusters, and a method for treating liver cirrhosis using the ligand-bound gold clusters.

Description

[0001] This application is a divisional application of CN113398279A (application date October 28, 2020, application number 2020111723118, invention name Ligand-bound gold clusters, compositions and methods for treating cirrhosis). Technical Field

[0002] The present invention relates to the technical field of liver cirrhosis treatment, in particular to ligand-bound gold clusters for treating liver cirrhosis, a composition comprising the ligand-bound gold clusters, and a method for treating liver cirrhosis using the ligand-bound gold clusters. Background Art

[0003] The liver is the largest solid organ in the human body and has many important functions, including: making blood proteins that help with blood clotting, transport oxygen, and aid the immune system; storing excess nutrients and returning some of them to the blood; making bile to aid in the digestion of food; helping the body store sugar (glucose) in the form of glycogen; removing harmful substances from the body, including drugs and alcohol; and breaking down saturated fats and producing cholesterol.

[0004] Cirrhosis is a slowly progressive disease that develops over many years due to long-term, ongoing liver damage. As cirrhosis progresses, healthy liver tissue is gradually destroyed and replaced by scar tissue. This scar tissue blocks blood flow through the liver and slows the liver's ability to process nutrients, hormones, medications, and natural toxins. It also reduces the liver's production of proteins and other substances. Cirrhosis may eventually lead to liver failure and / or liver cancer, which may require a liver transplant.

[0005] In the early stages of cirrhosis, the liver's compensatory function is strong and there are no obvious symptoms. Late-stage symptoms include liver damage, portal hypertension, upper gastrointestinal bleeding, hepatic encephalopathy, secondary infection, hypersplenism, ascites, cancer, and other complications. The liver gradually deforms and hardens, progressing to cirrhosis. Histopathologically, cirrhosis manifests as extensive hepatocellular necrosis, nodular regeneration of residual hepatocytes, connective tissue proliferation, and the formation of fibrous septa, leading to the destruction of the hepatic lobule architecture and the formation of pseudolobules.

[0006] Cirrhosis has different causes. Some people with cirrhosis have liver damage from multiple causes. Common causes of cirrhosis include chronic alcohol abuse, chronic hepatitis B and C infection, fatty liver disease, toxic metals, genetic disorders, nutritional imbalances, industrial toxins, medications, circulatory and metabolic disorders, cholestasis, schistosomiasis, and many others.

[0007] Cirrhosis can be diagnosed using a number of tests / techniques. For example, blood tests may indicate cirrhosis if levels of liver enzymes, including alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP), and bilirubin are elevated, while levels of blood proteins are low.

[0008] Currently, there is no specific cure for cirrhosis, although treatment can slow its progression by eliminating the cause. Summary of the Invention

[0009] The present invention provides use of ligand-bound gold clusters in treating liver cirrhosis in patients, a method for treating liver cirrhosis in patients using the ligand-bound gold clusters, and use of the ligand-bound gold clusters in preparing a medicament for treating liver cirrhosis in patients.

[0010] Some embodiments of the present invention utilize ligand-bound gold clusters to treat liver cirrhosis in a patient; wherein the ligand-bound gold clusters comprise a gold core and a ligand bound to the gold core.

[0011] In some embodiments of this therapeutic use, the diameter of the gold core is 0.5-3 nm. In some embodiments, the diameter of the gold core is 0.5-2.6 nm.

[0012] In some embodiments of the therapeutic use, the ligand is one selected from L-cysteine and its derivatives, D-cysteine and its derivatives, cysteine-containing oligopeptides and their derivatives, and other thiol-containing compounds.

[0013] 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).

[0014] In some embodiments of the therapeutic use, the cysteine-containing oligopeptide and its derivatives are cysteine-containing dipeptides, cysteine-containing tripeptides, or cysteine-containing tetrapeptides.

[0015] In some embodiments of this therapeutic use, 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).

[0016] In some embodiments of this therapeutic use, 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) and L(D)-glutathione (GSH).

[0017] In some embodiments of this therapeutic use, 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).

[0018] In some embodiments of this therapeutic use, the other thiol-containing compound is selected from 1-[(2S)-2-methyl-3-thiol-1-oxopropyl]-L(D)-proline, thioglycolic acid, mercaptoethanol, thiophenol, D-3-mercaptovaline, N-(2-mercaptopropionyl)-glycine and dodecyl mercaptan.

[0019] Some embodiments of the present invention use ligand-bound gold clusters to prepare a medicament for treating liver cirrhosis in a subject, wherein the ligand-bound gold clusters comprise a gold core and a ligand bound to the gold core.

[0020] In some embodiments of the preparation use, the diameter of the gold core is 0.5-3 nm. In some embodiments, the diameter of the gold core is 0.5-2.6 nm.

[0021] In some embodiments of the preparation and use, the ligand is one selected from L-cysteine and its derivatives, D-cysteine and its derivatives, cysteine-containing oligopeptides and their derivatives, and other thiol-containing compounds.

[0022] In some embodiments of the preparation 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).

[0023] In some embodiments of the preparation use, the cysteine-containing oligopeptide and its derivatives are cysteine-containing dipeptides, cysteine-containing tripeptides or cysteine-containing tetrapeptides.

[0024] In some embodiments of the preparation use, 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).

[0025] In some embodiments of the preparation use, 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) and L(D)-glutathione (GSH).

[0026] In some embodiments of the preparation use, 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).

[0027] In some embodiments of the preparation use, the other thiol-containing compound is selected from 1-[(2S)-2-methyl-3-thiol-1-oxopropyl]-L(D)-proline, thioglycolic acid, mercaptoethanol, thiophenol, D-3-mercaptovaline, N-(2-mercaptopropionyl)-glycine and dodecyl mercaptan. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Preferred embodiments according to the present invention will now be described with reference to the accompanying drawings, wherein like reference numerals refer to like elements throughout.

[0029] Figure 1 Shown are ultraviolet-visible (UV) spectra, transmission electron microscopy (TEM) images, and particle size distribution of ligand L-NIBC-modified gold nanoparticles (L-NIBC-AuNPs) with different particle sizes.

[0030] Figure 2 Shown are ultraviolet-visible (UV) spectra, TEM images, and particle size distribution diagrams of ligand L-NIBC-bound gold clusters (L-NIBC-AuCs) with different particle sizes.

[0031] Figure 3 Shown are the infrared spectra of L-NIBC-AuCs with different particle sizes.

[0032] Figure 4 UV, IR, TEM, and particle size distribution images of ligand CR-conjugated gold clusters (CR-AuCs) are shown.

[0033] Figure 5 UV, IR, TEM, and size distribution images of ligand RC-conjugated gold clusters (RC-AuCs) are shown.

[0034] Figure 6 UV, infrared, TEM, and particle size distribution images of gold clusters (Cap-AuCs) bound to the ligand 1-[(2S)-2-methyl-3-thiol-1-oxopropyl]-L-proline (i.e., Cap).

[0035] Figure 7 UV, IR, TEM, and size distribution images of ligand GSH-bound gold clusters (GSH-AuCs) are shown.

[0036] Figure 8 UV, IR, TEM, and particle size distribution images of ligand D-NIBC-bound gold clusters (D-NIBC-AuCs) are shown.

[0037] Figure 9 UV, IR, TEM, and particle size distribution images of ligand L-cysteine-conjugated gold clusters (L-Cys-AuCs) are shown.

[0038] Figure 10 Shown are the effects of different doses of A-01 and A-02 on serum (A) ALT, (B) AST, (C) TBIL, (D) MAO, and (E) ALB levels in mice with cirrhosis model. The positive control group was sorafenib.

[0039] Figure 11 Shown are the effects of different doses of B-01 and B-02 on serum (A) ALT, (B) AST, (C) TBIL, (D) MAO, and (E) ALB levels in mice with cirrhosis model, where the positive control group was sorafenib.

[0040] Figure 12 Shown are the effects of high-dose drug C on serum (A) ALT, (B) AST, (C) TBIL, (D) MAO, and (E) ALB levels in mice with cirrhosis model, where the positive control group was sorafenib.

[0041] Figure 13 The results of HE staining pathological examination are shown: (A) blank control group; (B) model control group; (C) positive control group; (D) A-01 gold cluster low-dose administration group; (E) A-01 gold cluster high-dose administration group.

[0042] Figure 14Showing the effects of drug D, E, and F administration on serum (A) ALT, (B) AST, (C) TBIL, (D) MAO, and (E) ALB levels in mice with cirrhosis model. DETAILED DESCRIPTION

[0043] The present invention may be understood more readily by reference to the following detailed description of certain embodiments of the invention.

[0044] Where publications are cited, the disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains.

[0045] Ligand-bound gold clusters (AuCs) are a specialized form of gold that exists between gold atoms and gold nanoparticles. The gold cores of ligand-bound gold clusters are less than 3 nm in size and consist of only a few to a few hundred gold atoms, leading to 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 varying HOMO-LUMO gaps. This results in the disappearance of the surface plasmon resonance effect common to conventional gold nanoparticles and the corresponding plasmon resonance absorption band (520 ± 20 nm) in the UV-visible spectrum.

[0046] The present invention provides ligand-bound gold clusters.

[0047] In some embodiments, the ligand-bound gold cluster comprises a ligand and a gold core, wherein the ligand is bound to the gold core. Binding of the ligand to the gold core means that the ligand forms a stable complex with the gold core in solution 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.

[0048] In some embodiments, the ligand of the ligand-bound gold cluster is a thiol-containing compound or an oligopeptide. In some embodiments, the ligand is bonded to the gold core through an Au-S bond to form a ligand-bound gold cluster.

[0049] 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).

[0050] 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) 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) 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).

[0051] 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, thiophenol, D-3-mercaptovaline, or dodecyl mercaptan.

[0052] The present invention provides pharmaceutical compositions for treating cirrhosis of the liver in a subject. In some embodiments, the subject is a human. In some embodiments, the subject is a pet animal, such as a dog.

[0053] In some embodiments, the pharmaceutical composition comprises the ligand-bound gold clusters disclosed above and a pharmaceutically acceptable excipient. In some embodiments, the excipient is phosphate buffered saline or physiological saline.

[0054] The present invention provides use of the ligand-bound gold cluster disclosed above for the manufacture of a medicament for treating liver cirrhosis in a subject.

[0055] The present invention provides uses of the ligand-bound gold clusters disclosed above for treating liver cirrhosis in a subject, or methods of treating liver cirrhosis in a subject using the ligand-bound gold clusters disclosed above. In some embodiments, the treatment method comprises administering a pharmaceutically effective amount of the ligand-bound gold clusters to the subject. The pharmaceutically effective amount can be determined by conventional in vivo studies.

[0056] The following examples are provided merely to illustrate the principles of the present invention; they are in no way intended to limit the scope of the invention.

[0057] Example

[0058] Example 1: Preparation of ligand-bound gold clusters

[0059] 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;

[0060] 1.2 The ligand is dissolved in a solvent to obtain solution B, wherein the concentration of the ligand is 0.01 to 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), containing cysteine Oligopeptides of amino acids and their derivatives, including, but not limited to, dipeptides, tripeptides, tetrapeptides and other peptides containing cysteine, such as L(D)-cysteine-L(D)-arginine dipeptide (CR), L(D)-arginine-L(D)-cysteine dipeptide (RC), 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)-glutathione (GSH), 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), and other sulfhydryl-containing compounds such as 1-[(2S)-2-methyl-3-thiol-1-oxopropyl]-L(D)-proline, sulfhydryl one or more of acetic acid, mercaptoethanol, thiophenol, D-3-mercaptovaline and dodecyl mercaptan; the solvent is one or more of methanol, ethyl acetate, water, ethanol, n-propanol, pentane, formic acid, acetic acid, ethyl ether, acetone, anisole, 1-propanol, 2-propanol, 1-butanol, 2-butanol, amyl alcohol, butyl acetate, tributyl methyl ether, isopropyl acetate, dimethyl sulfoxide, ethyl formate, isobutyl acetate, methyl acetate, 2-methyl-1-propanol and propyl acetate;

[0061] 1.3 Mix solution A and solution B to a molar ratio of HAuCl4 to ligand of 1:(0.01-100), stir in an ice bath for 0.1-48 hours, add 0.025-0.8M NaBH4 in water, ethanol, or methanol, and continue stirring in an ice bath for 0.1-12 hours. The molar ratio of NaBH4 to ligand is 1:(0.01-100);

[0062] 1.4 After the reaction is complete, centrifuge the reaction solution at 8000-175 rpm for 10-100 minutes using a 3K-30K MWCO ultrafiltration tube to precipitate ligand-bound gold clusters of varying average particle sizes. The pore size of the filter membrane in the ultrafiltration tubes with varying MWCO directly determines the size of the ligand-bound gold clusters that can pass through the membrane. This step can be optionally omitted.

[0063] 1.5 Dissolve the ligand-bound gold cluster precipitates of different average particle sizes 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;

[0064] 1.6 After dialysis, freeze-dry the ligand-bound gold clusters for 12 to 24 hours to obtain a powder or flocculant substance, i.e., the ligand-bound gold clusters.

[0065] As detected, the particle size of the powder or flocculant obtained by the above method is less than 3 nm (usually distributed in the range of 0.5-2.6 nm). There is no obvious absorption peak at 520 nm. It is determined that the obtained powder or flocculant is a ligand-bound gold cluster.

[0066] Example 2: Preparation and identification of gold clusters bound by different ligands

[0067] 2.1 Preparation of L-NIBC-bound gold clusters, ie, L-NIBC-AuCs Taking the ligand L-NIBC as an example, the preparation and identification of the ligand L-NIBC-bound gold clusters are described in detail.

[0068] 2.1.1 Weigh 1.00 g of HAuCl4 and dissolve it in 100 mL of methanol to obtain 0.03 M solution A;

[0069] 2.1.2 Weigh 0.57 g of L-NIBC and dissolve it in 100 mL of glacial acetic acid (acetic acid) to obtain 0.03 M solution B.

[0070] 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 of HAuCl4 to L-NIBC is 1:0.5, 1:1, 1:2, 1:3, 1:4, or 1:5, respectively). Stir the mixture in an ice bath for 2 h. When the solution turns from bright yellow to colorless, quickly add 1 mL of freshly prepared 0.03 M NaBH4 ethanol solution (11.3 mg NaBH4 weighed and dissolved in 10 mL ethanol). After the solution turns dark brown, continue the reaction for 30 min. Terminate the reaction by adding 10 mL of acetone.

[0071] 2.1.4 After the reaction, the reaction solution was subjected to gradient centrifugation to obtain L-NIBC-AuCs powders of varying particle sizes. Specific method: After the reaction was complete, the reaction solution was transferred to a 50 mL ultrafiltration tube with a MWCO of 30K and centrifuged at 10,000 rpm for 20 min. The retentate in the inner tube was dissolved in ultrapure water. A powder with a particle size of approximately 2.6 nm was obtained. The mixed solution in the outer tube was then transferred to a 50 mL ultrafiltration tube with a MWCO of 10K and centrifuged at 13,000 rpm for 30 minutes. The retentate in the inner tube was dissolved in ultrapure water to obtain a powder with a particle size of approximately 1.8 nm. The mixed solution in the outer tube was then transferred to a 50 mL ultrafiltration tube with a MWCO of 3K and centrifuged at 17,500 rpm for 40 minutes. The retentate in the inner tube was dissolved in ultrapure water to obtain a powder with a particle size of approximately 1.1 nm.

[0072] 2.1.5 Precipitate the three powders of different particle sizes obtained by gradient centrifugation, remove the solvent separately, blow dry the crude product with N2, dissolve it in 5 mL of ultrapure water, place it in a dialysis bag (MWCO is 3 kDa), place the dialysis bag in 2 L of ultrapure water, change the water every other day, dialyze for 7 days, and freeze-dry it for use.

[0073] 2.2 Identification of L-NIBC-AuCs

[0074] The powder obtained above (L-NIBC-AuCs) was subjected to characterization experiments. Gold nanoparticles modified with the ligand L-NIBC (L-NIBC-AuNPs) were also used as a control. The preparation of gold nanoparticles with the ligand L-NIBC was described in the literature (W. Yan, L. Xu, C. Xu, W. Ma, H. Kuang, L. Wang and N. A Kotov, Journal of the American Chemical Society 2012, 134, 15114; X. Yuan, B. Zhang, Z. Luo, Q. Yao, DT Leong, N. Yan and J. Xie, Angewandte Chemie International Edition 2014, 53, 4623).

[0075] 2.2.1 Morphology observation by transmission electron microscopy (TEM)

[0076] The test powders (L-NIBC-AuCs and L-NIBC-AuNPs) were dissolved in ultrapure water to a concentration of 2 mg / L. The samples were then prepared using the hanging drop method. Specifically, 5 μL of the sample was dropped onto an ultrathin carbon film and allowed to evaporate naturally until the droplet disappeared. The sample morphology was then observed using a JEM-2100F STEM / EDS field emission high-resolution TEM.

[0077] Four TEM images of L-NIBC-AuNP are shown in Figure 1 In panels B, E, H, and K of FIG; three TEM images of L-NIBC-AuCs are shown in FIG. Figure 2 In panels B, E and H.

[0078] Figure 2 The images in Figure 3 show that each L-NIBC-AuCs sample has uniform particle size and good dispersion, and the average diameters of L-NIBC-AuCs (referring to the diameter of the gold core) are 1.1 nm, 1.8 nm, and 2.6 nm, respectively, which are consistent with the results of the previous study. Figure 2 The results in panels C, F, and I are completely consistent. In contrast, the L-NIBC-AuNPs sample has a larger particle size. Their average diameters (referring to the diameter of the gold core) are 3.6nm, 6.0nm, 10.1nm, and 18.2nm, respectively, which are consistent with the results in panels C, F, and I. Figure 1 The results in panels C, F, I, and L are in good agreement.

[0079] 2.2.2 Ultraviolet (UV)-visible (vis) absorption spectrum

[0080] The test powders (L-NIBC-AuCs and L-NIBC-AuNPs) were dissolved in ultrapure water to a concentration of 10 mg·L⁻¹, and UV-vis absorption spectra were measured at room temperature. The scanning range was 190–1100 nm. The sample cell was a standard quartz cuvette with a 1 cm optical pathlength, and the reference cell was filled with ultrapure water.

[0081] The UV-vis absorption spectra of four L-NIBC-AuNP samples with different sizes are shown in Figure 1 The statistical distribution of particle size is shown in Figures A, D, G and J. 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 panels of FIG. Figure 2 The statistical distribution of particle size is shown in Figures A, D, and G. Figure 2 In panels C, F and I.

[0082] Figure 1The results show that L-NIBC-AuNPs exhibit an absorption peak at approximately 520 nm due to the surface plasmon effect. The location of the absorption peak is related to particle size. When the particle size is 3.6 nm, the UV absorption peak occurs at 516 nm; when the particle size is 6.0 nm, the UV absorption peak occurs at 517 nm; when the particle size is 10.1 nm, the UV absorption peak occurs at 520 nm; and when the particle size is 18.2 nm, the absorption peak occurs at 523 nm. None of the four samples exhibit any absorption peak above 560 nm.

[0083] Figure 2 The UV absorption spectra of L-NIBC-bound gold cluster samples of three different particle sizes show that the surface plasmon effect absorption peak at 520 nm disappears, and two distinct absorption peaks appear above 560 nm. The positions of the 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, resulting in a discontinuity in the gold cluster's density of states, energy level splitting, the disappearance of the plasmon resonance effect, and the appearance of new absorption peaks in the long-wavelength direction. It can be concluded that the three powder samples obtained above are all ligand-bound gold clusters.

[0084] 2.2.3 Fourier transform infrared spectroscopy

[0085] The infrared spectra were measured on a Bruker VERTEX80V Fourier transform infrared spectrometer in solid powder high vacuum total reflection mode with a scanning range of 4000–400 cm -1 , scanned 64 times. Taking the L-NIBC-bound gold cluster sample as an example, the test samples were three types of L-NIBC-bound gold cluster dry powders with different particle sizes, and the control sample was pure L-NIBC powder. The results are shown in Figure 3 .

[0086] Figure 3 The infrared spectra of gold clusters bound to L-NIBC of varying particle sizes are shown. Compared to pure L-NIBC (bottom curve), the SH stretching vibration between 2500 and 2600 cm⁻¹ is completely absent in the gold clusters bound to L-NIBC of varying particle sizes, while other characteristic peaks of L-NIBC are still observed. This demonstrates that L-NIBC molecules are successfully bound to the gold cluster surface via gold-sulfur bonds. The figure also shows that the infrared spectra of ligand-bound gold clusters are independent of their size.

[0087] Gold clusters bound by other ligands were prepared using a similar method as described above, except that the solvent of solution B, the feed ratio between HAuCl4 and the ligand, the reaction time, and the amount of added NaBH4 were slightly adjusted. For example, when L-cysteine, D-cysteine, N-isobutyryl-L-cysteine (L-NIBC), or N-isobutyryl-D-cysteine (D-NIBC) was used as the ligand, acetic acid was selected as the solvent; when dipeptide CR, dipeptide RC, or 1-[(2S)-2-methyl-3-mercapto-1-oxopropyl]-L-proline was used as the ligand, water was selected as the solvent, and so on. The other steps were similar, so no further details are provided here.

[0088] The present invention prepares and obtains a series of ligand-bound gold clusters by the above method. The parameters of the ligands and preparation methods are shown in Table 1.

[0089] Table 1. Preparation parameters of gold clusters bound to different ligands of the present invention

[0090]

[0091]

[0092] The samples listed in Table 1 were confirmed by the aforementioned method. The characteristics of the gold clusters bound by six different ligands 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 4-Figure 9 Shown are UV spectrum (panel A), IR spectrum (panel B), TEM image (panel C) and particle size distribution (panel D).

[0093] The results show that the diameters of the gold clusters bound by different ligands obtained in Table 1 are all less than 3nm. The UV spectrum also shows the disappearance of the peak at 520±20nm, and the appearance of absorption peaks at other positions. The position of the absorption peak varies with the ligand, particle size and structure. In some cases, no special absorption peak appears. This is mainly due to the formation of a mixture of gold clusters of different sizes and structures or the position of the absorption peak of some special gold clusters being outside the range of conventional UV-visible absorption spectrum measurement. At the same time, the Fourier transform infrared spectrum also shows that the infrared absorption peak of the thiol of the ligand disappears ( Figure 4-8The other infrared characteristic peaks are retained, indicating that the ligand molecules have successfully combined with the gold atoms to form ligand-bound gold clusters, indicating that the present invention has successfully obtained the gold clusters bound with the ligands listed in Table 1.

[0094] Example 3

[0095] 3.1 Materials and Animals

[0096] 3.1.1 Test samples

[0097] A-01: ligand L-NIBC-bound gold clusters (L-NIBC-AuCs), 0.9±0.2 nm.

[0098] A-02: ligand L-NIBC-bound gold clusters (L-NIBC-AuCs), 1.9±0.5 nm.

[0099] B-01: ligand L-Cys-bound gold clusters (L-Cys-AuCs), 1.0±0.2 nm.

[0100] B-02: ligand L-Cys-bound gold clusters (L-Cys-AuCs), 1.7±0.3 nm.

[0101] C: L-NIBC-modified gold nanoparticles (L-NIBC-AuNPs), 6.3±1.5 nm.

[0102] All test samples were prepared by referring to the aforementioned method with slight modifications; their quality was verified by the above method.

[0103] 3.1.2 Positive control samples

[0104] Sorafenib.

[0105] 3.1.3 Experimental animals and groups

[0106] A total of 120 SPF male C57BL / 6N mice (purchased from Beijing Huafukang Experimental Animal Technology Co., Ltd. (production license number: SCXK (Beijing) 2019-0008), 6-8 weeks old, weighing 16-20 g, were randomly divided into 12 groups (n=10) according to body weight: blank control group, model group, positive control group, A-01 low-dose group, A-01 high-dose group, A-02 low-dose group, A-02 high-dose group, B-01 low-dose group, B-01 high-dose group, B-02 low-dose group, B-02 high-dose group, and C high-dose group.

[0107] 3.2 Modeling Method

[0108] Except for the blank control group, mice in other groups were treated with carbon tetrachloride (CCl4) induction to establish a cirrhosis model. The modeling method is as follows: (1) Each mouse was intraperitoneally injected with 10% CCl4 (diluted with olive oil) at 7 μL / g body weight twice a week for a total of 8 weeks; the blank control group mice were intraperitoneally injected with an equal amount of olive oil solvent. (2) Starting from the 6th week, 2 mice were selected and sacrificed 48 hours after the last injection each week. The appearance of the mouse liver was observed. When the appearance was consistent with the characteristics of cirrhosis (8th week), the liver tissue was formalin fixed and HE staining and Masson staining were performed to evaluate the establishment of the cirrhosis model.

[0109] 3.3 Administration

[0110] After successful model establishment, mice in the positive control group were gavaged with 25 mg / kg sorafenib. The low- or high-dose groups A-01, A-02, B-01, and B-02 were intraperitoneally injected with the corresponding test article at a dose of 2.5 or 10 mg / kg, respectively. The high-dose group C was intraperitoneally injected with the test article at a dose of 40 mg / kg. Mice in the blank control and model groups were intraperitoneally injected with normal saline at a dose of 10 mL / kg. Dosing was performed once daily for 20 consecutive days.

[0111] 3.4 Biochemical testing

[0112] After administration, blood was collected from the mice's orbits and serum was collected. Serum levels were measured using a biochemical analyzer (Siemens) using a Zhongsheng Beikong kit and five indicators: albumin (ALB), total bilirubin (TBil), alanine aminotransferase (ALT), aspartate aminotransferase (AST), and monoamine oxidase (MAO). The assays were performed strictly according to the kit instructions.

[0113] Table 2 shows the product information of the kits used for biochemical detection

[0114]

[0115] 3.5 Pathological examination

[0116] 3.5.1 HE staining

[0117] After euthanasia, liver tissue samples were fixed in 4% paraformaldehyde for at least 48 hours. Following fixation, they were dehydrated using a gradient of alcohol and cleared using xylene and ethanol. The liver tissue was then waxed and embedded. After the embedded material was trimmed, glued, and reshaped, the liver tissue was sectioned using a paraffin microtome at a thickness of 4 μm. The main procedure for HE staining was as follows: sections were oven-baked at 65°C, treated with xylene, and then dehydrated using a gradient of ethanol. After staining with hematoxylin, blueing solution, and 0.5% eosin solution, the sections were then treated with a gradient of ethanol and xylene and mounted with neutral gum. Liver fibrosis was observed microscopically.

[0118] 3.5.2 Masson staining

[0119] Mouse liver tissue sections were baked, dewaxed, and dehydrated. After chromatinization, sections were stained with Regaud's hematoxylin. After washing, sections were stained with Masson's Ponceau-Fuchsin solution. Sections were rinsed with 2% glacial acetic acid and differentiated with 1% phosphomolybdic acid. Sections were stained directly with aniline blue or light green solution and briefly rinsed with 0.2% glacial acetic acid. Sections were cleared with 95% alcohol, anhydrous alcohol, and xylene, and then mounted with neutral gum. Liver tissue was observed under a microscope.

[0120] 3.6 Experimental Results

[0121] 3.6.1 Successful modeling

[0122] The livers of the model mice were divided into circular or oval masses of varying sizes by proliferating fibrous septa. Serum ALT, TBil, and AST levels were significantly elevated compared to the blank control group, while ALB levels were significantly decreased. MAO levels were not significantly different from the blank control group, but were also elevated. These results indicate that the model was successfully established.

[0123] 3.6.2 Effects of the trial drug on alanine aminotransferase (ALT), total bilirubin (TBil), aspartate aminotransferase (AST), monoamine oxidase (MAO), and albumin (ALB)

[0124] 3.6.2.1 Investigational Drugs A-01 and A-02

[0125] Depend on Figure 10As shown in Figure 5, ALT activity in the model group showed a highly significant increase compared to that in the blank control group (from an average of 43.5±8.1 U / L to 188.5±4.9 U / L, P<0.01), suggesting that liver function in cirrhosis mice was impaired. Following administration of high and low doses of A-01 and A-02, ALT activity in all treatment groups decreased significantly, returning to the level of the blank control group or even lower (the highest was 41.5±5.4 U / L in the low-dose A-02 group, the lowest was 30.0±5.9 U / L in the high-dose A-01 group, and the positive control group had a significantly different ALT activity from that in the model group (P<0.01).

[0126] Depend on Figure 10 As shown in Figure 2, AST activity in the model group was significantly elevated compared to that in the blank control group (from 141.8±13.5 U / L to 192.0±11.3 U / L, P<0.05). After administration of A-01 and A-02, AST activity decreased in all groups, with high-dose A-01 and A-02 administration significantly reducing AST activity (130.4±12.8 U / L in the A-01 high-dose group and 131.3±9.9 U / L in the A-02 high-dose group, both P<0.01), significantly superior to the positive control group (165.5±11.6 U / L).

[0127] Depend on Figure 10 C shows that the TBil concentration in the model group was significantly elevated compared to that in the blank control group (from 1.02±0.20μmol / L to 2.91±0.39μmol / L), with a significant difference from the blank control group (P<0.01). Both high- and low-dose administration of A-01 and A-02 significantly decreased TBil (maximum 0.91±0.13μmol / L, minimum 0.78±0.26μmol / L), reaching the same level as the blank control group, but with a highly significant difference from the model group (P<0.01).

[0128] Depend on Figure 10D shows that the MAO activity in the model group was higher than that in the blank control group (blank control group: 18.8±2.9U / L, model group: 21.5±0.7U / L), but there was no statistical difference, suggesting that the changes in MAO activity indicators in mice with carbon tetrachloride-induced cirrhosis were not significant. A-01 and A-02 administration did not significantly affect the MAO activity of all drug-treated groups, but the MAO activity of all drug-treated groups decreased (maximum 19.3±1.5U / L, minimum 18.5±1.9U / L), which was at the same level as the blank control group. In contrast, the positive control group did not reduce MAO activity (21.3±2.1U / L). This result suggests that A-01 and A-02 may adjust the MAO activity of all drug-treated groups to the level of the blank control group, playing a role in the recovery of liver function in cirrhotic mice.

[0129] Depend on Figure 10 E shows that ALB levels in the model group were significantly lower than those in the blank control group (from 24.2±0.6 g / L to 22.1±1.3 g / L), with significant differences from the blank control group (P<0.05), indicating that carbon tetrachloride treatment significantly reduces serum ALB levels. Administration of A-01 and A-02 at different doses, as well as the positive control group, did not significantly affect serum ALB levels.

[0130] The positive control drug, sorafenib, significantly reduced ALT, AST, and TBIL levels, but may have no alleviating effect on MAO in cirrhotic mice. This result suggests that A-01 and A-02 have a repairing effect on liver function in cirrhotic mice, and their efficacy is superior to that of the positive control drug.

[0131] 3.6.2.2 Experimental Drugs B-01 and B-02

[0132] Depend on Figure 11 A shows that both low and high doses of B-01 and B-02 can significantly reduce ALT activity (maximum 46.3±7.4U / L, minimum 33.0±7.1U / L), which is at the same level as the blank control group and significantly different from the model group (188.5±4.9U / L) (P<0.01).

[0133] Depend on Figure 11As shown in Figure 2, compared with the model group (192.0±11.3 U / L), low- and high-dose B-01 (132.3±10.0 U / L, P<0.01; 129.7±26.6 U / L, P<0.01) and low-dose B-02 (149.6±21.8 U / L, P<0.05) significantly reduced AST activity to normal levels (P<0.01). However, high-dose B-02 also reduced AST activity to a certain extent, but the difference was not significant (P>0.05). In contrast, the positive drug sorafenib also reduced AST levels to 165.5±11.6 U / L (P<0.05), but the effect was not as good as low- and high-dose B-01 or low-dose B-02.

[0134] Depend on Figure 11 C shows that both low- and high-dose administration of B-01 and B-02 significantly reduced TBil (maximum 1.28±0.12μmol / L, minimum 0.96±0.15μmol / L), which was at the same level as the blank control group (1.02±0.20μmol / L) and significantly different from the model group (2.91±0.39μmol / L) (P<0.01).

[0135] Depend on Figure 11 D showed that compared with the model group (21.5±0.7U / L), low-dose B-01 (17.3±1.3U / L, P<0.01) and high-dose B-02 (18.3±0.6U / L, P<0.05) significantly reduced serum MAO levels to the level of the blank control group (18.8±2.9U / L), but the positive control sorafenib (21.3±2.1U / L) had no effect on serum MAO levels.

[0136] Depend on Figure 11 E shows that each drug-treated group and the positive control group had no significant effect on ALB levels.

[0137] The above results show that B-01 and B-02 significantly reduce ALT, AST, TBIL and MAO levels in a dose-dependent manner, and play a role in the recovery of liver function in cirrhotic mice. Their effects are better than those of positive control drugs at least in some indicators.

[0138] 3.6.2.3 Investigational Drug C

[0139] Depend on Figure 12 It can be seen that compared with the model group, high-dose administration of drug C had no significant improvement effect on the levels of (A) ALT, (B) AST, (C) TBIL, (D) MAO, and (E) ALB relative to the model control group, and even showed a certain deterioration trend, suggesting that drug C is ineffective in improving liver function in cirrhotic mice and may be toxic.

[0140] 3.6.3 Pathological examination

[0141] Liver cirrhosis is characterized by diffuse fibrosis and pseudolobule formation in liver tissue. HE staining results show that Figure 13 As shown in A, the normal liver tissue structure of mice in the blank control group is clear, the liver lobules are intact, the liver cell cords are neatly arranged and radially arranged with the central vein as the center, the liver cell nuclei are normal, and there is only a small amount of fibrous tissue in the portal area; Figure 13 As shown in B, in the liver of the model control group mice, the hepatocytes were arranged in disorder, ballooning occurred, the hepatic lobules almost disappeared, and the pseudolobules ( Figure 13 The right arrow in B) appears in large numbers, and collagen fibers proliferate and form round or oval fiber septa ( Figure 13 Leftward arrow in B). Compared with the model control group, Figure 13 As shown in C, in the positive control group, the degree of liver damage was significantly reduced, the cells were arranged in a regular pattern, and although collagen fibers were proliferating, they were significantly weakened, no fibrous septa were formed, and pseudolobules almost disappeared. However, compared with normal liver tissue, the gaps between liver cells in the positive control group were significantly enlarged ( Figure 13 (Downward arrow in C). Compared with the model control group, the hepatocytes in the groups treated with the four gold cluster drugs (A-01, A-02, B-01, and B-02) showed extremely significant recovery from liver injury, as evidenced by a significant reduction in fibrosis and pseudolobules in the liver, which was dose-dependent.

[0142] Figure 13 D and Figure 13 E shows the HE staining images of the liver tissue damage and repair effects of low-dose and high-dose administration represented by A-01 gold clusters. Figure 13 As shown in D, in the A-01 gold cluster low-dose administration group, the hepatocytes were arranged more neatly, the pseudolobules almost disappeared, and the collagen fiber proliferation was significantly weakened. However, the intercellular spaces between the liver cells were enlarged to a certain extent compared with normal liver tissue ( Figure 13 D). Figure 13 As shown in Figure E, the high-dose A-01 gold cluster group showed a more pronounced improvement than the low-dose group. Pseudolobules completely disappeared, collagen fiber proliferation was not observed, and the enlargement of the intercellular spaces between liver cells was almost invisible, showing no significant difference from normal liver cells. This demonstrates that the A-01 gold cluster drug demonstrated a superior effect on repairing liver tissue damage compared to the positive control drug.

[0143] The results of Masson staining were consistent with those of HE staining.

[0144] The other three gold cluster drugs also showed similar effects to the A-01 drug and will not be described in detail here.

[0145] In summary, the four gold cluster test drugs, A-01, A-2, B-01, and B-02, significantly reduced liver fibrosis and pseudolobules. Liver function test results also showed signs of liver recovery, with the most significant changes in alanine aminotransferase (ALT) and total bilirubin (TBil). Aspartate aminotransferase (AST) and monoamine oxidase (MAO) also showed significant recovery, while albumin (ALB) showed no significant changes. The four gold cluster test drugs significantly improved liver function and liver pathology in cirrhotic mice, with overall efficacy superior to that of the positive control, sorafenib, providing experimental evidence for further application. However, drug C had no significant therapeutic effect, indicating that it cannot be used for the treatment of cirrhosis.

[0146] Example 4

[0147] 4.1 Materials and Animals

[0148] 4.1.1 Test samples

[0149] D: Ligand L-NAC-bound gold clusters (L-NAC-AuCs), size 0.5-3 nm.

[0150] E: Ligand CR-bound gold clusters (CR-AuCs), size 0.5-3 nm.

[0151] F: Ligand RC-bound gold clusters (RC-AuCs), size 0.5-3 nm.

[0152] All test samples were prepared by referring to the aforementioned method with slight modifications; their quality was verified by the above method.

[0153] 4.1.2 Experimental animals and groups

[0154] Fifty SPF male C57BL / 6N mice (purchased from Beijing Huafukang Experimental Animal Technology Co., Ltd. (production license number: SCXK (Beijing) 2019-0008), 6-8 weeks old, weighing 16-20 g, were randomly divided into 5 groups (n=10) according to body weight: blank control group, model control group, D drug group, E drug group, and F drug group.

[0155] 4.2 Modeling Method

[0156] Except for the blank control group, mice in other groups were treated with carbon tetrachloride (CCl4) induction to establish a cirrhosis model. The modeling method is as follows: (1) Each mouse was intraperitoneally injected with 10% CCl4 (diluted with olive oil) at 7 μL / g body weight twice a week for a total of 8 weeks; the blank control group mice were intraperitoneally injected with an equal amount of olive oil solvent. (2) Starting from the 6th week, 2 mice were selected and sacrificed 48 hours after the last injection each week. The appearance of the mouse liver was observed. When the appearance was consistent with the characteristics of cirrhosis (8th week), the liver tissue was formalin fixed and HE staining and Masson staining were performed to evaluate the establishment of the cirrhosis model.

[0157] 4.3 Administration

[0158] After successful modeling, all three treatment groups received an intraperitoneal injection of the corresponding gold cluster drug at a dose of 40 mg / kg. The blank control and model control groups received an intraperitoneal injection of normal saline at a dose of 10 mL / kg. The drugs were administered once daily for 20 consecutive days.

[0159] 4.4 Biochemical testing

[0160] The reagents and methods are the same as those in Section 3.4.

[0161] 4.5 Experimental Results

[0162] 4.5.1 Successful modeling

[0163] The livers of the model mice were divided into circular or oval masses of varying sizes by proliferating fibrous septa. Serum ALT, TBil, and AST levels were significantly elevated compared to the blank control group, while ALB levels were significantly decreased. MAO levels were not significantly different from the blank control group, but were also elevated. These results indicate that the model was successfully established.

[0164] 4.5.2 Effects of the trial drug on alanine aminotransferase (ALT), total bilirubin (TBil), aspartate aminotransferase (AST), monoamine oxidase (MAO), and albumin (ALB)

[0165] Depend on Figure 14 As shown in Figure A, the ALT activity in the model group was significantly elevated compared to that in the blank control group (P<0.01, **), indicating that liver function in cirrhosis model mice was impaired. After administration in Figures D, E, and F, ALT activity in all treatment groups decreased significantly, returning to the level of the blank control group and exhibiting a significantly different ALT activity from that in the model control group (P<0.01).

[0166] Depend on Figure 14As shown in B, the serum AST activity of the model control group was significantly higher than that of the blank control group (P<0.05, *). After administration in D, E, and F, the AST activity of all the groups was significantly decreased (P<0.05, *).

[0167] Depend on Figure 14 As shown in C, the TBil concentration in the model control group was significantly higher than that in the blank control group, and there was a significant difference between the two groups (P < 0.01, **). After administration in D, E, and F, TBil levels were significantly reduced to the level of the blank control group, but there was a significant difference between the two groups (P < 0.01, **).

[0168] Depend on Figure 14 As shown in D, MAO activity in the model control group increased compared to that in the blank control group, but the difference was not statistically significant (P>0.5), indicating that changes in MAO activity in mice with carbon tetrachloride-induced cirrhosis were not significant. Administration of D, E, and F did not significantly affect MAO activity in any of the groups, but MAO activity in all groups decreased to the level of the blank control group.

[0169] Depend on Figure 14 E shows that the ALB level in the model control group was lower than that in the blank control group, but the difference was not significant (P>0.05). However, administration of D, E, and F increased the ALB level in serum, but the difference was not significant (P>0.05).

[0170] In summary, the test drugs in clusters D, E, and F significantly improved liver function. Changes in alanine aminotransferase (ALT) and total bilirubin (TBil) were most pronounced. Aspartate aminotransferase (AST) and monoamine oxidase (MAO) also showed significant recovery, while albumin (ALB) also improved, albeit less significantly. This provides experimental evidence for further application.

[0171] Gold clusters of different sizes bound to L-cysteine, L-NIBC, L-NAC, CR, and RC, as well as gold clusters of different sizes bound to other ligands, also have the same effect, though their effects may vary. These will not be described in detail here.

[0172] Industrial Applicability

[0173] Ligand-bound gold clusters can be used to treat liver cirrhosis. They are suitable for industrial applications.

[0174] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. Use of ligand-bound gold clusters in the preparation of a medicament for treating patients with liver cirrhosis, characterized in that: The ligand-bound gold clusters comprise: Gold core; and A ligand bound to the gold core; the ligand is selected from thiol-containing compounds; The preparation method of the gold cluster comprises the following steps: 1) dissolving HAuCl4 in a solvent including methanol, water, ethanol, n-propanol or ethyl acetate, wherein the concentration of HAuCl4 is at least 0.01 to 0.03 M, to obtain solution A; 2) dissolving a ligand in a solvent to obtain a solution B, wherein the ligand is selected from a thiol-containing compound, wherein the thiol-containing compound is selected from 1-[(2S)-2-methyl-3-thiol-1-oxopropyl]-L(D)-proline, thioglycolic acid, mercaptoethanol, thiophenol, D-3-mercaptovaline, N-(2-mercaptopropionyl)-glycine, and dodecyl mercaptan; 3) mixing solution A and solution B so that the molar ratio of HAuCl4 to ligand is 1:(0.01-100), stirring for 0.1-48 hours, adding water, ethanol or methanol solution containing at least 0.025-0.8M NaBH4, and continuing to stir the reaction for 0.1-24 hours to obtain a reaction solution; 4) After the reaction is completed, the reaction solution is centrifuged and dialyzed or ultrafiltered to obtain the ligand-bound gold clusters.

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

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

4. The use according to claim 1, characterized in that The concentration of the ligand is 0.01-0.18M.

5. The use according to claim 1, characterized in that The molar ratio of NaBH4 to the ligand is 1:(0.01-100).

6. A pharmaceutical composition for treating liver cirrhosis, characterized in that: The pharmaceutical composition comprises ligand-bound gold clusters and a pharmaceutically acceptable excipient; The ligand-bound gold clusters comprise: Gold core; and A ligand bound to the gold core; the ligand is selected from thiol-containing compounds; The preparation method of the gold cluster comprises the following steps: 1) dissolving HAuCl4 in a solvent including methanol, water, ethanol, n-propanol or ethyl acetate, wherein the concentration of HAuCl4 is at least 0.01 to 0.03 M, to obtain solution A; 2) dissolving a ligand in a solvent to obtain a solution B, wherein the ligand is selected from a thiol-containing compound, wherein the thiol-containing compound is selected from 1-[(2S)-2-methyl-3-thiol-1-oxopropyl]-L(D)-proline, thioglycolic acid, mercaptoethanol, thiophenol, D-3-mercaptovaline, N-(2-mercaptopropionyl)-glycine, and dodecyl mercaptan; 3) mixing solution A and solution B so that the molar ratio of HAuCl4 to ligand is 1:(0.01-100), stirring for 0.1-48 hours, adding water, ethanol or methanol solution containing at least 0.025-0.8M NaBH4, and continuing to stir the reaction for 0.1-24 hours to obtain a reaction solution; 4) After the reaction is completed, the reaction solution is centrifuged and dialyzed or ultrafiltered to obtain the ligand-bound gold clusters.

7. The pharmaceutical composition according to claim 6, wherein The excipient is phosphate buffer solution or physiological saline.

8. Use of the pharmaceutical composition according to claim 6 in the preparation of a medicament for treating patients with liver cirrhosis.

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