CN115400225A

CN115400225A - Ligand-bound gold clusters, compositions and methods for treating liver cirrhosis

Info

Publication number: CN115400225A
Application number: CN202211047265.8A
Authority: CN
Inventors: 孙涛垒
Original assignee: Wuhan Guanghang Scientific Research Co ltd
Current assignee: Wuhan Guanghang Scientific Research Co ltd
Priority date: 2020-03-16
Filing date: 2020-10-28
Publication date: 2022-11-29
Also published as: CN113398279B; CN113398279A

Abstract

The present invention relates to the technical field of liver cirrhosis treatment, and more particularly, 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

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

This application is a divisional application of CN113398279A (application date: 2020, 10/28, application No. 2020111723118, entitled ligand-bound gold clusters, compositions and methods for treating liver cirrhosis).

Technical Field

Background

The liver is the largest solid organ of the human body and has many important functions, including: manufacturing blood proteins that aid in clotting, oxygen delivery, and the immune system; and storing excess nutrients and returning a portion of the nutrients to the blood; making bile to aid in digesting food; help the body store sugar (glucose) in the form of glycogen; eliminating harmful substances in vivo, including drugs and alcohol; breakdown saturated fats and produce cholesterol.

Cirrhosis is a slowly progressive disease that develops over many years due to long-term, persistent liver damage. As cirrhosis progresses, healthy liver tissue is gradually destroyed and replaced by scar tissue. These scar tissues can prevent blood flow through the liver and slow the liver's ability to process nutrients, hormones, drugs and natural toxins. It also reduces the production of proteins and other substances produced by the liver. Cirrhosis may ultimately lead to liver failure and/or liver cancer that may require liver transplantation.

In the early stage of cirrhosis, liver compensation function is strong, and no obvious symptoms exist. The late stage symptoms comprise complications such as liver function damage, portal hypertension, upper gastrointestinal hemorrhage, hepatic encephalopathy, secondary infection, spleen hyperfunction, ascites, canceration, etc. The liver gradually becomes deformed and hardened, and cirrhosis progresses. Histopathologically, cirrhosis manifests itself as extensive hepatocyte necrosis, nodular regeneration of residual hepatocytes, connective tissue proliferation and fibroseptal formation, leading to destruction of hepatic lobular structures and formation of false lobules.

Cirrhosis has different causes. Some patients with cirrhosis have liver damage due to a variety of causes. Common causes of cirrhosis include chronic alcohol abuse, chronic hepatitis b and c infection, fatty liver disease, toxic metals, genetic disorders, nutritional disorders, industrial poisons, pharmaceuticals, blood circulation disorders, metabolic disorders, cholestasis, schistosomiasis, and the like.

Cirrhosis can be diagnosed by a number of tests/techniques. For example, if liver enzymes including alanine Aminotransferase (ALT), aspartate Aminotransferase (AST) and alkaline phosphatase (ALP) and bilirubin levels are elevated and blood protein levels are reduced, a blood test may indicate cirrhosis.

Currently, although treatment can delay the progression of cirrhosis by eliminating the cause of cirrhosis, there is no specific treatment for cirrhosis.

Disclosure of Invention

The present invention provides the use of ligand-bound gold clusters for the treatment of liver cirrhosis in a patient, a method of treating liver cirrhosis in a patient using ligand-bound gold clusters, and the use of ligand-bound gold clusters for the manufacture of a medicament for the treatment of liver cirrhosis in a patient.

Some embodiments of the invention utilize ligand-bound gold clusters to treat liver cirrhosis in a patient; wherein the ligand-bound gold cluster comprises a gold core, and a ligand bound to the gold core.

In some embodiments for this therapeutic use, the gold core has a diameter of 0.5-3nm. In some embodiments, the gold core has a diameter of 0.5 to 2.6nm.

In some embodiments of the therapeutic use, the ligand is one selected from the group consisting of L-cysteine and derivatives thereof, D-cysteine and derivatives thereof, cysteine-containing oligopeptides and derivatives thereof, and other thiol-containing compounds.

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).

In some embodiments of the therapeutic use, the cysteine-containing oligopeptide and derivative thereof is a cysteine-containing dipeptide, a cysteine-containing tripeptide, or a cysteine-containing tetrapeptide.

In some embodiments of this therapeutic use, the cysteine-containing dipeptide is selected from the group consisting of an L (D) -cysteine-L (D) -arginine dipeptide (CR), an L (D) -arginine-L (D) -cysteine dipeptide (RC), an L (D) -histidine-L (D) -cysteine dipeptide (HC), and an L (D) -cysteine-L (D) -histidine dipeptide (CH).

In some embodiments of this therapeutic use, the cysteine-containing tripeptide is selected from the group consisting of the glycine-L (D) -cysteine-L (D) -arginine tripeptide (GCR), the L (D) -proline-L (D) -cysteine-L (D) -arginine tripeptide (PCR), the L (D) -lysine-L (D) -cysteine-L (D) -proline tripeptide (KCP), and the L (D) -Glutathione (GSH).

In some embodiments of this therapeutic use, the cysteine-containing tetrapeptide is selected from the group consisting of 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).

In some embodiments of this therapeutic use, the other thiol-containing compound is selected from the group consisting of 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.

Some embodiments of the invention use ligand-bound gold clusters for the preparation of 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.

In some embodiments of this preparative use, the gold core has a diameter of 0.5 to 3nm. In some embodiments, the gold core has a diameter of 0.5 to 2.6nm.

In some embodiments of the preparative use, the ligand is one selected from the group consisting of L-cysteine and derivatives thereof, D-cysteine and derivatives thereof, cysteine-containing oligopeptides and derivatives thereof, and other thiol-containing compounds.

In some embodiments of this preparative 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).

In some embodiments of this preparative use, the cysteine-containing oligopeptide and derivative thereof is a cysteine-containing dipeptide, a cysteine-containing tripeptide, or a cysteine-containing tetrapeptide.

In some embodiments of this preparative use, the cysteine-containing dipeptide is selected from the group consisting of 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).

In some embodiments of this preparative use, the cysteine-containing tripeptide is selected from the group consisting of 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).

In some embodiments of this preparative use, the cysteine-containing tetrapeptide is selected from the group consisting of 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).

In some embodiments of this preparative use, the other thiol-containing compound is selected from the group consisting of 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.

Drawings

Preferred embodiments according to the present invention will now be described with reference to the accompanying drawings, in which like reference numerals refer to like elements.

Fig. 1 shows ultraviolet-visible (UV) spectra, transmission Electron Microscope (TEM) images, and particle size distribution profiles of ligand L-NIBC-modified gold nanoparticles (L-NIBC-AuNPs) having different particle sizes.

FIG. 2 shows ultraviolet-visible (UV) spectra, TEM images and particle size distribution plots of ligand L-NIBC-bound gold clusters (L-NIBC-AuCs) having different particle sizes.

FIG. 3 shows infrared spectra of L-NIBC-AuCs with different particle sizes.

FIG. 4 shows UV, infrared, TEM and particle size distribution plots of ligand CR-bound gold clusters (CR-AuCs).

Fig. 5 shows UV, infrared, TEM and particle size distribution plots of ligand RC-bound gold clusters (RC-AuCs).

FIG. 6 shows UV, infrared, TEM and particle size distribution plots of ligand 1- [ (2S) -2-methyl-3-thiol-1-oxopropyl ] -L-proline (i.e., cap) bound gold clusters (Cap-AuCs).

Figure 7 shows UV, infrared, TEM and particle size distribution profiles of ligand GSH-bound gold clusters (GSH-AuCs).

FIG. 8 shows UV, infrared, TEM and particle size distribution plots of ligand D-NIBC-bound gold clusters (D-NIBC-AuCs).

FIG. 9 shows UV, infrared, TEM and particle size distribution plots of ligand L-cysteine-bound gold clusters (L-Cys-AuCs).

FIG. 10 shows the effect of different doses of A-01 and A-02 on serum (A) ALT, (B) AST, (C) TBIL, (D) MAO and (E) ALB levels in cirrhosis model mice, with the positive control group being Sorafenib.

FIG. 11 shows the effect of different doses of B-01 and B-02 on serum (A) ALT, (B) AST, (C) TBIL, (D) MAO and (E) ALB levels in cirrhosis model mice, where the positive control group was sorafenib.

FIG. 12 shows the effect of high dose drug C administration on serum (A) ALT, (B) AST, (C) TBIL, (D) MAO and (E) ALB levels in cirrhosis model mice, where the positive control group was sorafenib.

Fig. 13 shows HE staining pathology detection results: (A) a blank control group; (B) a model control group; (C) a positive control group; (D) A-01 gold cluster low dose administration group; (E) A-01 gold cluster high dose administration group.

FIG. 14 shows the effect of drug administration of D, E, and F on serum (A) ALT, (B) AST, (C) TBIL, (D) MAO, and (E) ALB levels in cirrhosis model mice.

Detailed Description

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

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.

Ligand-bound gold clusters (AuCs) are a special form of gold that exists between gold atoms and gold nanoparticles. The gold core size of the ligand-bound gold clusters is less than 3nm, consisting of only a few to a few hundred gold atoms, leading to a collapse of the face-centered cubic stacked structure of gold nanoparticles. Thus, unlike the continuous or quasi-continuous energy levels of gold nanoparticles, the gold clusters exhibit molecular discrete electronic structures with different HOMO-LUMO gaps. This resulted in the disappearance of the surface plasmon resonance effect possessed by conventional gold nanoparticles and the corresponding plasmon resonance absorption band (520 ± 20 nm) in the ultraviolet-visible spectrum.

The present invention provides ligand-bound gold clusters.

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 means that the ligand forms a complex stable in solution with the gold core through covalent bonds, hydrogen bonds, electrostatic forces, hydrophobic forces, van der waals forces, and the like. In some embodiments, the gold core has a diameter of 0.5 to 3nm. In some embodiments, the gold core has a diameter in the range of 0.5 to 2.6nm.

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

In some embodiments, the ligand is, but is not limited to, L-cysteine, D-cysteine or cysteine derivatives. 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).

In some embodiments, the ligand is, but is not limited to, cysteine-containing oligopeptides and derivatives thereof. In some embodiments, the cysteine-containing oligopeptide is a cysteine-containing dipeptide. In some embodiments, the cysteine-containing dipeptide is an L (D) -cysteine-L (D) -arginine dipeptide (CR), an L (D) -arginine-L (D) -cysteine dipeptide (RC), or an 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 a 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).

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.

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, e.g., a dog.

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 buffered solution or physiological saline.

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

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

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

Examples

Example 1 preparation of ligand-bound gold clusters

1.1 adding HAuCl ₄ Dissolving in methanol, water, ethanol, n-propanol or ethyl acetate to obtain solution A, in which HAuCl is present ₄ The concentration of (A) is 0.01-0.03M;

1.2 dissolving a ligand in a solvent to obtain a solution B, wherein the concentration of the ligand is 0.01-0.18M; ligands include, but are 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 derivatives thereof, including, but not limited to, dipeptides, tripeptides, tetrapeptides 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) -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 thiol-containing compounds, such as one or more of 1- [ (2S) -2-methyl-3-thiol-1-oxopropyl ] -L (D) -proline, thioglycolic 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, diethyl ether, acetone, anisole, 1-propanol, 2-propanol, 1-butanol, 2-butanol, pentanol, butyl acetate, tributylmethyl ether, isopropyl acetate, dimethyl sulfoxide, ethyl formate, isobutyl acetate, methyl acetate, 2-methyl-1-propanol and propyl acetate;

1.3 mixing solution A with solution B to make HAuCl ₄ With ligandsThe molar ratio is 1: (0.01-100), stirring for 0.1-48 h in ice bath, adding 0.025-0.8M NaBH ₄ Water, ethanol or methanol solution, and continuously stirring and reacting for 0.1-12 h in ice-water bath. NaBH ₄ The molar ratio of the ligand to the ligand is 1: (0.01 to 100);

1.4 after the reaction is finished, centrifuging the reaction solution for 10-100 min by using an MWCO 3K-30K ultrafiltration tube at the speed of 8000-175 r/min to obtain the ligand-bound gold cluster precipitates with different average particle diameters. The pores of the filtration membranes of the ultrafiltration tubes of different MWCO directly determine the size of the gold clusters that can be bound by the ligands of the membrane. This step may optionally be omitted;

1.5 dissolving the ligand-bound gold cluster precipitates with different average particle sizes obtained in the step (1.4) in water, placing the gold cluster precipitates in a dialysis bag, and dialyzing the gold cluster precipitates in the water for 1 to 7 days at room temperature;

and 1.6 after dialysis, freeze-drying the ligand-bound gold cluster for 12-24 hours to obtain a powdery or flocculant substance, namely the ligand-bound gold cluster.

As detected, the particle size of the powdered or flocculant substance obtained by the aforementioned method is less than 3nm (typically distributed between 0.5 and 2.6 nm). There was no significant absorption peak at 520 nm. The powder or floc obtained was determined to be ligand-bound gold clusters.

Example 2 preparation and identification of gold clusters bound with different ligands

2.1 preparation of L-NIBC-bound gold clusters, L-NIBC-AuCs the preparation and characterization of ligand L-NIBC-bound gold clusters is described in detail, using ligand L-NIBC as an example.

2.1.1 weighing 1.00g of HAuCl ₄ Dissolving the mixture in 100mL of methanol to obtain 0.03M solution A;

2.1.2 weighing 0.57g L-NIBC, dissolving it in 100mL glacial acetic acid (acetic acid) to obtain 0.03M solution B;

2.1.3 weigh 1mL of solution A and mix with 0.5mL, 1mL, 2mL, 3mL, 4mL, or 5mL of solution B (i.e., HAuCl) ₄ The molar ratio of the L-NIBC to the L-NIBC is 1:0.5, 1: 1. 1: 2. 1: 3. 1: 4. 1: 5) The reaction was stirred in an ice bath for 2h and when the solution turned from bright yellow to colorless, 1mL of freshly prepared 0.03M was added quickly (11.3 mg of NaBH weighed) ₄ And dissolved in 10mL of ethanol) NaBH ₄ Ethanol solution, after the solution turned dark brown, the reaction was continued for 30 minutes and stopped by adding 10mL of acetone.

2.1.4 after the reaction, carrying out gradient centrifugation on the reaction solution to obtain L-NIBC-AuCs powder with different particle sizes. The specific method comprises the following steps: after the reaction was completed, the reaction solution was transferred to an ultrafiltration tube of 50mL volume having MWCO of 30K, centrifuged at 10000r/min for 20min, and the retentate in the inner tube was dissolved in ultrapure water. A powder with a particle size of about 2.6nm was obtained. Then, the mixed solution in the outer tube was transferred to an ultrafiltration tube having a volume of 50mL and a MWCO of 10K, and centrifuged at 13,000r/min for 30 minutes. The retentate in the inner tube was dissolved in ultrapure water to give a powder having a particle size of about 1.8 nm. The mixed solution in the outer tube was then transferred to an ultrafiltration tube with a volume of 50mL and MWCO of 3K, and centrifuged at 17,500r/min for 40 minutes. The retentate in the inner tube was dissolved in ultrapure water to give a powder having a particle size of about 1.1 nm.

2.1.5 precipitating three powders with different particle sizes obtained by gradient centrifugation, respectively removing the solvent, blowing the crude product with N2, dissolving in 5mL of ultrapure water, placing into a dialysis bag (MWCO is 3 KDa), placing the dialysis bag into 2L of ultrapure water, changing water every other day, dialyzing for 7 days, and freeze-drying for later use.

2.2 identification of L-NIBC-AuCs

The powder obtained above (L-NIBC-AuCs) was subjected to an identification test. Meanwhile, ligand L-NIBC modified gold nanoparticles (L-NIBC-AuNP) were used as a control. References to the preparation of gold nanoparticles with L-NIBC as ligand (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, D.T.Leong, N.Yan and J.Xie, angewandte Chemie International Edition 2014,53, 4623).

2.2.1 Observation of morphology by Transmission Electron Microscopy (TEM)

Test powders (L-NIBC-AuCs samples and L-NIBC-AuNPs samples) were dissolved in ultrapure water to 2mg/L as a sample, and then the test sample was prepared by the pendant-drop method. More specifically, 5. Mu.L of the sample was dropped on an ultra-thin carbon film, and naturally volatilized until the water drop disappeared, and then the morphology of the sample was observed by JEM-2100F STEM/EDS field emission high resolution TEM.

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

The image in FIG. 2 shows that each sample of L-NIBC-AuCs has uniform particle size and good dispersibility, and the average diameters (referring to the diameter of the gold core) of the L-NIBC-AuCs are 1.1nm, 1.8nm and 2.6nm, respectively, which are completely consistent with the results in panels C, F and I of FIG. 2. In contrast, the L-NIBC-AuNPs sample had a larger particle size. Their average diameters (referring to the diameter of the gold core) were 3.6nm, 6.0nm, 10.1nm and 18.2nm, respectively, in good agreement with the results in panel C, panel F, panel I and panel L of FIG. 1.

2.2.2 Ultraviolet (UV) -visible (vis) absorption Spectrum

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

The UV-vis absorption spectra of four L-NIBC-AuNP samples with different sizes are shown in panel a, panel D, panel G and panel J of fig. 1, and the statistical distribution of particle sizes is shown in panel C, panel F, panel I and panel L of fig. 1; the UV-vis absorption spectra of three samples of L-NIBC-AuCs with different sizes are shown in the A, D and G panels of FIG. 2, and the statistical distribution of particle sizes is shown in the C, F and I panels of FIG. 2.

FIG. 1 shows that L-NIBC-AuNP has an absorption peak at about 520nm due to the surface plasmon effect. The position of the absorption peak is related to the particle size. When the particle size was 3.6nm, a UV absorption peak appeared at 516 nm; when the particle size was 6.0nm, the UV absorption peak appeared at 517 nm; the UV absorption peak appears at 520nm when the particle size is 10.1nm, and at 523nm when the particle size is 18.2 nm. None of the four samples had any absorption peak above 560 nm.

Fig. 2 shows that in the ultraviolet absorption spectrum of the L-NIBC-bonded gold cluster samples with three different particle sizes, the surface plasma effect absorption peak at 520nm disappears, two obvious absorption peaks appear above 560nm, and the positions of the absorption peaks are slightly different from the particle sizes of the gold cluster. This is because the gold cluster exhibits a molecular-like property due to collapse of the face-centered cubic structure, which results in discontinuity of state density of the gold cluster, energy level splitting, disappearance of the plasmon resonance effect, and appearance of a new absorption peak in the long-wavelength direction. It can be concluded that the three different particle size powder samples obtained above are ligand-bound gold clusters.

2.2.3 Fourier transform Infrared Spectroscopy

The infrared spectrum is measured on a VERTEX80V type Fourier transform infrared spectrometer manufactured by Bruker by adopting a solid powder high vacuum total reflection mode, and the scanning range is 4000-400cm ^-1 And scanned 64 times. Taking the sample of the L-NIBC-bonded gold cluster as an example, the test sample is dry powder of the L-NIBC-bonded gold cluster with three different particle sizes, and the control sample is pure L-NIBC powder. The results are shown in FIG. 3.

FIG. 3 is an infrared spectrum of L-NIBC bound gold clusters with different particle sizes. Compared with pure L-NIBC (bottom curve), S-H stretching vibration of the L-NIBC combined gold cluster with different particle sizes between 2500-2600cm < -1 > completely disappears, and other characteristic peaks of the L-NIBC are still observed. The successful binding of the L-NIBC molecule to the surface of the gold cluster through a gold-sulfur bond was demonstrated. The figure also shows that the infrared spectrum of the ligand-bound gold clusters is independent of their size.

Gold clusters bound by other ligands were prepared in a similar manner as described above, except for the solvent of solution B, HAuCl ₄ Feed ratio to ligand, reaction time and NaBH added ₄ The amount of (a) is slightly adjusted, for example: when L-cysteine, D-cysteine, N-isobutyryl-L-cysteine (L-NIBC) or N-isobutyryl-D-cysteine (D-NIBC) is used as a ligand, acetic acid is selected as the solvent; when dipeptide CR, dipeptide RC or 1- [ (2S) -2-methyl-3-mercapto-1-oxopropyl is used]-when L-proline is the ligand, water is chosen as the solvent, and so on; the other steps are similar and therefore no further details are provided here.

The invention prepares and obtains a series of ligand-bonded gold clusters by the method. The ligand and preparation parameters are shown in table 1.

TABLE 1 preparation parameters of different ligand-bound gold clusters of the invention

The samples listed in table 1 were confirmed by the method described previously. The characteristics of the six different ligand-bound gold clusters are shown in FIG. 4 (CR-AuCs), FIG. 5 (RC-AuCs), FIG. 6 (Cap-AuCs) (Cap represents 1- [ (2S) -2-methyl-3-mercapto-1-oxopropyl ] -L-proline), FIG. 7 (GSH-AuCs), FIG. 8 (D-NIBC-AuCs), FIG. 9 (L-Cys-AuCs). FIGS. 4-9 show UV spectra (A panels), IR spectra (B panels), TEM images (C panels) and particle size distributions (D panels).

The results show that the different ligand-bound gold clusters obtained in table 1 all have diameters of less than 3nm. The ultraviolet spectrum also shows disappearance of the peak at 520 ± 20nm and appearance of an absorption peak at other positions, the position of which varies depending on the ligand and the particle diameter and structure, and there are cases where no specific absorption peak appears, mainly because the position of the absorption peak is out of the range of the conventional ultraviolet-visible absorption spectrometry due to the mixture of a plurality of gold clusters different in size and structure or some specific gold clusters. Meanwhile, the fourier transform infrared spectroscopy also showed that the thiol infrared absorption peak of the ligand disappeared (between the dotted lines in panel B in fig. 4-8), while the other infrared characteristic peaks remained, indicating that the ligand molecule has successfully combined with the gold atom to form a ligand-bound gold cluster, indicating that the present invention successfully obtained a ligand-bound gold cluster listed in table 1.

Example 3

3.1 materials and animals

3.1.1 test specimens

A-01: ligand L-NIBC-bound gold clusters (L-NIBC-AuCs) of 0.9 +/-0.2 nm.

A-02: ligand L-NIBC bound gold clusters (L-NIBC-AuCs) 1.9 +/-0.5 nm.

B-01: ligand L-Cys combined gold cluster (L-Cys-AuCs) is 1.0 +/-0.2 nm.

B-02: ligand L-Cys combined gold cluster (L-Cys-AuCs) is 1.7 +/-0.3 nm.

C: gold nanoparticles modified by L-NIBC (L-NIBC-AuNPs) with the particle size of 6.3 +/-1.5 nm.

All test sample preparation methods were slightly modified with reference to the aforementioned method; their quality was identified by the method described above.

3.1.2 Positive control samples

Sorafenib (Sorafenib).

3.1.3 test animals and groups

120 SPF male C57BL/6N mice (purchased from Beijing Huafukang laboratory animal technology Co., ltd. (production license number: SCXK (Jing) 2019-0008), 6-8 weeks old and 16-20g in body weight are randomly divided into 12 groups (N = 10) according to body weight, namely a blank control group, a model group, a positive control group, an A-01 low dose group, an A-01 high dose group, an A-02 low dose group, an A-02 high dose group, a B-01 low dose group, a B-01 high dose group, a B-02 low dose group, a B-02 high dose group and a C high dose group.

3.2 modeling method

Except for the blank control group, carbon tetrachloride (CCl) was used ₄ ) And (3) treating other groups of mice by an induction method to prepare a liver cirrhosis model. The molding method comprises the following steps: (1) Each mouse was intraperitoneally injected with 10% CCl at 7. Mu.L/g body weight ₄ (olive oil dilution) 2 times per week for 8 weeks; mice in the blank control group were injected intraperitoneally with an equal amount of olive oil solvent. (2) From week 6, 2 mice were selected for sacrifice after the last 48h weekly injection, and the appearance of the liver of the mice was observed, and after the appearance conformed to the characteristics of cirrhosis (week 8), the liver tissue was fixed with formalin, and HE staining, massson staining, etc. were performed to evaluate the modeling conditions of the cirrhosis model.

3.3 administration of drugs

After the model building is successful, the positive control group mice are gavaged with sorafenib 25 mg/kg; the corresponding test sample is respectively injected into the abdominal cavity of the low or high dosage groups A-01, A-02, B-01 and B-02 according to the dosage of 2.5 or 10 mg/kg; the test article is given to the C high-dose group by intraperitoneal injection according to the dose of 40 mg/kg; the mice in the control group and the model group were administered with normal saline by intraperitoneal injection at a rate of 10 mL/kg. The administration is 1 time per day for 20 days.

3.4 Biochemical assays

After the administration, the mouse was subjected to orbital blood collection, serum was collected, and 5 indexes of albumin (albumin, ALB), total bilirubin (TBil), alanine Aminotransferase (ALT), aspartate Aminotransferase (AST) and monoamine oxidase (MAO) were detected using a chinese north control kit and a biochemical analyzer (siemens). The detection method is carried out strictly according to the kit instructions.

Table 2 shows product information of the kit for biochemical detection

3.5 pathological examination

3.5.1HE staining

After the mice are euthanized, a liver tissue sample of the mice is fixed for more than 48 hours by using 4 percent paraformaldehyde fixing solution, and after the fixation, the liver tissue sample is subjected to gradient dehydration by using alcohol and is subjected to transparentization treatment by using dimethylbenzene and ethanol. Then the liver tissue is waxed and embedded. And (3) after the embedded material is subjected to block trimming, block sticking and trimming, the liver tissue is sliced by a paraffin slicer, and the slice thickness is 4 mu m. HE staining main procedure was as follows: baking the slices in an oven at 65 ℃, and then carrying out xylene treatment and ethanol gradient dehydration on the slices. And (3) dyeing with hematoxylin dyeing liquid, promoting blue liquid to return to blue and dyeing with 0.5% eosin liquid in sequence, then treating the slices with gradient ethanol and xylene, and sealing the slices with neutral gum. Fibrotic changes in liver tissue were observed with a microscope.

3.5.2Masson staining

The liver tissue slices of the mice are baked and dewaxed and dehydrated, and are nuclear-stained by Regaud hematoxylin staining solution after chromating treatment. After washing with water, the sections were stained with Masson ponceau acid reddening solution, and the sections were washed with 2% glacial acetic acid aqueous solution and then differentiated with 1% phosphomolybdic acid aqueous solution. Directly dyeing with aniline blue or light green solution, washing with 0.2% glacial acetic acid water solution for a moment, transparentizing with 95% alcohol, anhydrous alcohol and xylene, and sealing with neutral gum. Liver tissue was observed using a microscope.

3.6 results of the experiment

3.6.1 successful mold making

The liver of the mouse in the model group is divided into round or oval lumps with different sizes by the hyperplastic fiber partition, ALT, TBil and AST indexes in serum are obviously increased relative to a blank control group, ALB is obviously reduced relative to the blank control group, and the MAO index has no obvious difference with the blank control group, but the value is also increased. All the results prompt that the mold making of the experiment is successful.

3.6.2 testing the Effect of drugs on alanine Aminotransferase (ALT), total bilirubin (TBil), aspartate Aminotransferase (AST), monoamine oxidase (MAO) and Albumin (ALB)

3.6.2.1 test drugs A-01 and A-02

As can be seen from FIG. 10A, the ALT activity in the model group showed a very significant increase (from a mean of 43.5. + -. 8.1U/L to 188.5. + -. 4.9U/L, P < 0.01) relative to the ALT activity in the blank control group, suggesting that liver function in the liver cirrhosis model mice was diseased. After the A-01 and the A-02 are administrated at high and low doses, ALT activity of all the administration groups is obviously reduced, and the ALT activity is recovered to the level of a blank control group and is even lower (the highest is 41.5 +/-5.4U/L of the low dose group of the A-02, the lowest is 30.0 +/-5.9U/L of the A-01 high dose group, and the lowest is 42.8 +/-5.4U/L of the positive control group), and the ALT activity is very different from that of a model group (P < 0.01).

As can be seen from FIG. 10B, the AST activity of the model group showed a significant increase (from 141.8. + -. 13.5U/L to 192.0. + -. 11.3U/L, P < 0.05) relative to that of the blank control group. After the A-01 and A-02 are administrated, the AST activity of all the administration groups is reduced, wherein the AST activity is obviously reduced by the administration of high doses A-01 and A-02 (130.4 +/-12.8U/L of the A-01 high dose group, 131.3 +/-9.9U/L of the A-02 high dose group and less than 0.01 of P), and the AST activity is obviously better than that of a positive control group (165.5 +/-11.6U/L).

As can be seen from FIG. 10C, the TBil concentration of the model group showed a significant increase (from 1.02. + -. 0.20. Mu. Mol/L to 2.91. + -. 0.39. Mu. Mol/L) relative to that of the blank control group, and was significantly different from that of the blank control group (P < 0.01). TBil was significantly reduced after both high and low doses of a-01 and a-02 (maximum 0.91 ± 0.13 μmol/L, minimum 0.78 ± 0.26 μmol/L), at the same level as the blank control group, but with a very significant difference from the model group (P < 0.01).

As shown in FIG. 10D, the MAO activity in the model group was increased compared to that in the blank control group (18.8. + -. 2.9U/L for the blank control group and 21.5. + -. 0.7U/L for the model group), but there was no statistical difference, indicating that the carbon tetrachloride-induced change in the MAO activity index was not significant in the liver cirrhosis mice. The MA0 activity of all the groups was not significantly affected by the A-01 and A-02 administration, but the MAO activity of all the groups was decreased (19.3 + -1.5U/L and 18.5 + -1.9U/L), and was at the same level as that of the blank control group, compared with the positive control group, which did not decrease the MAO activity (21.3 + -2.1U/L), suggesting that the A-01 and A-02 administration may adjust the MAO activity of all the groups to the level of the blank control group, and exert an effect on the recovery of liver function of the liver cirrhosis mice.

As can be seen in fig. 10E, the ALB level of the model group showed a significant decrease (from 24.2 ± 0.6g/L to 22.1 ± 1.3 g/L) relative to the ALB level of the blank group, which was significantly different from that of the blank group (P < 0.05), indicating that carbon tetrachloride treatment could significantly reduce serum ALB level. The ALB level in serum was not significantly affected by the different doses of A-01 and A-02, as well as by administration of the positive control group.

The positive medicament sorafenib obviously reduces the levels of ALT, AST and TBIL, but has no relieving effect on the cirrhosis mouse on MAO index. The results suggest that A-01 and A-02 have a repairing effect on liver function of the liver cirrhosis mice, and the effect is superior to that of the positive control medicament.

3.6.2.2 test drugs B-01 and B-02

As can be seen in FIG. 11A, both low and high doses of B-01 and B-02 significantly reduced ALT activity (46.3. + -. 7.4U/L maximum and 33.0. + -. 7.1U/L minimum), at the same level as the blank control group, with significant differences (P < 0.01) from the model group (188.5. + -. 4.9U/L).

As can be seen from fig. 11B, both low or high dose administration of B-01 (132.3 ± 10.0U/L, P <0.01, 129.7 ± 26.6U/L, P < 0.01) and low dose administration of B-02 (149.6 ± 21.8U/L, P < 0.05), respectively, significantly reduced AST activity to normal levels (P < 0.01) compared to the model group (192.0 ± 11.3U/L), but high dose administration of B-02 somewhat reduced AST activity but did not differ significantly (P > 0.05). Compared with the positive drug Sorafenib, the positive drug Sorafenib can also reduce the AST level to 165.5 +/-11.6U/L (P < 0.05), but the effect is not as good as that of B-01 when the high dose is low and B-02 when the low dose is administered.

As can be seen in FIG. 11C, both the low and high doses of B-01 and B-02 significantly reduced TBil (1.28. + -. 0.12. Mu. Mol/L maximum and 0.96. + -. 0.15. Mu. Mol/L minimum), at the same level as the blank control (1.02. + -. 0.20. Mu. Mol/L) and significantly different (P < 0.01) from the model (2.91. + -. 0.39. Mu. Mol/L) groups.

As can be seen in FIG. 11D, both the low dose of B-01 (17.3. + -. 1.3U/L, P < 0.01) and the high dose of B-02 (18.3. + -. 0.6U/L, P < 0.05) significantly reduced serum MAO levels to the blank control (18.8. + -. 2.9U/L) compared to the model (21.5. + -. 0.7U/L), but the positive control sorafenib (21.3. + -. 2.1U/L) had no effect on serum MAO levels.

As can be seen from fig. 11E, the ALB levels were not significantly affected by each administration group and the positive control group.

The results show that B-01 and B-02 obviously reduce the levels of ALT, AST, TBIL and MAO, have certain dose dependence, play a role in restoring the liver function of a cirrhosis mouse, and have the effect which is at least superior to that of a positive control medicament on partial indexes.

3.6.2.3 test drug C

As can be seen from fig. 12, compared to the model group, the high dose of the drug C did not significantly improve the levels of (a) ALT, (B) AST, (C) TBIL, (D) MAO, and (E) ALB, even with a certain tendency to deteriorate, compared to the model control group, suggesting that the drug C is not effective in improving liver functions of cirrhosis mice, and may be toxic.

3.6.3 pathological examination

Cirrhosis is a pathological feature of diffuse fibrosis of liver tissue and the formation of pseudolobules. The HE staining pathological examination results showed that, as shown in fig. 13A, the blank control group mice had clear normal liver tissue structure, intact liver lobules, well-arranged hepatocyte chords, radially arranged with central vein as the center, normal liver cell nuclei, and had only a small amount of fibrous tissue in the zone of confluence; as shown in fig. 13B, in the liver of the mouse of the model control group, the arrangement of hepatocytes was disturbed, ballooning occurred, hepatic lobules were nearly disappeared, pseudolobules (right arrow in fig. 13B) appeared in large numbers, collagen fibers were proliferated in large numbers and formed into circular or oval fibrous spaces (left arrow in fig. 13B). Compared with the model control group, as shown in fig. 13C, the positive control group had significantly reduced liver damage, significantly aligned cells, collagen fibers with hyperplasia but significantly reduced, no fibrous septa formed, and almost no false leaflets; however, the gap between liver cells of the positive control group was significantly increased compared to the normal liver tissue (downward arrow in fig. 13C). Compared with a model control group, the liver cells of the four gold cluster drug (A-01, A-02, B-01 and B-02) administration groups all show extremely remarkable liver injury recovery phenomena, show that the fibroplasia and false lobule are obviously reduced in the liver, and show certain dose dependence.

FIGS. 13D and 13E show graphs of HE staining of the effects of low and high dose administration, represented by A-01 gold clusters, on liver tissue damage repair, respectively. As shown in fig. 13D, in the a-01 gold cluster low dose group, the hepatocytes were aligned, the pseudolobules were almost disappeared, the collagen fibril proliferation was also significantly reduced, and the hepatic intercellular space was increased to some extent with respect to the normal hepatic tissue (indicated by the downward arrow in fig. 13D). As shown in fig. 13E, the a-01 gold cluster high dose group showed more significant improvement effect than the low dose group, the pseudolobules disappeared completely, no collagen fiber proliferation was observed, and the phenomenon of increased liver cell gap was hardly visible and was not significantly different from that of normal liver cells. From this, it can be seen that the a-01 gold cluster drug exhibited better effect of repairing liver tissue damage than the positive control drug.

Masson staining was identical to HE staining.

The other three gold cluster drugs also show similar effects to the A-01 drug, and are not described in detail herein.

In conclusion: four gold cluster test medicaments of A-01, A-2, B-01 and B-02 obviously reduce liver fibroplasia and false lobule of liver. Liver function index test results also show the phenomenon of liver function recovery, with alanine Aminotransferase (ALT) and total bilirubin (TBil) changes being the most significant. Aspartate Aminotransferase (AST) and monoamine oxidase (MAO) also recovered significantly, and the change in Albumin (ALB) was not significant. The four gold cluster test drugs can obviously improve the liver function and the pathological structure of the liver cirrhosis mouse in the liver cirrhosis mouse, and the overall effect is superior to that of the positive control sorafenib, thereby providing experimental basis for further application in the future. However, the C drug has no obvious therapeutic effect, and thus cannot be used for treating liver cirrhosis.

Example 4

4.1 materials and animals

4.1.1 test specimens

D: ligand L-NAC-bound gold clusters (L-NAC-AuCs) with a size of 0.5-3nm.

E: ligand CR-bound gold clusters (CR-AuCs) of size 0.5-3nm.

F: ligand RC-bonded gold clusters (RC-AuCs) with the size of 0.5-3nm.

All test sample preparation methods were as described above with minor modifications; their quality was determined by the above-mentioned method.

4.1.2 Experimental animals and groups

SPF-grade male C57BL/6N mice (purchased from Beijing Huafukang laboratory animal technology Co., ltd. (production license number: SCXK (Jing) 2019-0008)) are 6-8 weeks old, and the body weight is 16-20g, and are randomly divided into 5 groups (N = 10) according to the body weight, namely a blank control group, a model control group, a D administration group, an E administration group and an F administration group.

4.2 modeling method

Carbon tetrachloride (CCl) was used in addition to the blank control group ₄ ) Other groups of mice are treated by an induction method to prepare a cirrhosis model. The molding method comprises the following steps: (1) Each mouse was intraperitoneally injected with 10% CCl at 7. Mu.L/g body weight ₄ (olive oil dilution) 2 times a week for 8 weeks; mice of blank control group are injected with the same amount of olive oil in the abdominal cavityAnd (3) a solvent. (2) From week 6, 2 mice were selected for sacrifice after the last 48h weekly injection, and the appearance of the liver of the mice was observed, and after the appearance conformed to the characteristics of cirrhosis (week 8), the liver tissue was fixed with formalin, and HE staining, massson staining, etc. were performed to evaluate the modeling conditions of the cirrhosis model.

4.3 administration of drugs

After the molding is successful, the three administration groups inject corresponding gold cluster medicines into the abdominal cavity according to the dosage of 40 mg/kg; the mice in the blank control group and the model control group are given physiological saline by intraperitoneal injection at the rate of 10 mL/kg. The administration is 1 time per day for 20 days.

4.4 Biochemical assays

Reagents and methods are the same as in section 3.4.

4.5 results of the experiment

4.5.1 successful Molding

The liver of the mouse in the model group is divided into round or oval masses with different sizes by the hyperplastic septa, ALT, TBil and AST indexes in serum are obviously increased relative to a blank control group, ALB is obviously reduced relative to the blank control group, and MAO indexes have no obvious difference with the blank control group, but the numerical value is also increased. All the results prompt that the mold making of the experiment is successful.

4.5.2 Effect of test drugs on alanine Aminotransferase (ALT), total bilirubin (TBil), aspartate Aminotransferase (AST), monoamine oxidase (MAO) and Albumin (ALB)

As can be seen from fig. 14A, the ALT activity of the model group showed a very significant increase (P <0.01 ×) relative to the ALT activity of the blank control group, suggesting that liver function of the cirrhosis model mice was diseased. D. After E and F are administrated, ALT activity of all the administration groups is obviously reduced, the ALT activity is recovered to the level of a blank control group, and the ALT activity is very different from that of a model control group (P < 0.01).

As can be seen from fig. 14B, the serum AST activity of the model control group showed a significant increase (P < 0.05;) relative to the AST activity of the blank control group. D. After E and F, AST activity of all the administration groups is remarkably reduced (P is less than 0.05).

As can be seen from fig. 14C, the TBil concentration of the model control group showed a significant increase relative to the TBil concentration of the blank control group, with a significant difference from the blank control group (P < 0.01). D. After E and F administration, TBil decreased significantly to the level of the blank control group, but there was a significant difference from the model control group (P < 0.01.

As shown in fig. 14D, the MAO activity of the model control group was increased compared to the MAO activity of the blank control group, but there was no statistical difference (P > 0.5), which suggests that the change of MAO activity index of carbon tetrachloride-induced cirrhosis mouse was not significant. D. The E and F administration did not significantly affect the MA0 activity of each administration group, but the MAO activity of all administration groups decreased to the level of the blank control group.

As can be seen from fig. 14E, the ALB level of the model control group showed a decrease relative to the ALB level of the blank control group, but the difference was not significant (P > 0.05). However, D, E, F administration increased ALB levels in serum, but the difference was not significant (P > 0.05).

In conclusion, the tested drugs of the three gold clusters D, E and F significantly improve liver functions. Of these, alanine Aminotransferase (ALT) and total bilirubin (TBil) are the most common. Aspartate Aminotransferase (AST) and monoamine oxidase (MAO) were also significantly restored, and Albumin (ALB) was also improved, but not significantly. And experimental basis is provided for further application in the future.

The same effect was obtained with different sizes of L-cysteine-bound gold clusters, L-NIBC-bound gold clusters, L-NAC-bound gold clusters, CR-bound gold clusters and RC-bound gold clusters, and other ligand-bound gold clusters of different sizes, which were different from each other. They are not described in detail here.

Industrial applicability

The ligand-bound gold clusters can be used for treating liver cirrhosis. They are suitable for industrial use.

Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.

Claims

1. Use of ligand-bound gold clusters for the manufacture of a medicament for the treatment of a patient with liver cirrhosis, wherein the ligand-bound gold clusters comprise:

a gold core; and

a ligand that binds to the gold core; the ligand is selected from thiol-containing compounds.

2. Use according to claim 1, characterized in that the gold core has a diameter of 0.5-3nm.

3. Use according to claim 1, characterized in that the gold core has a diameter of 0.5-2.6nm.

4. Use according to claim 1, characterized in that 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.

5. Use according to claim 1, characterized in that the preparation method of the gold clusters comprises the following steps:

1) Adding HAuCl ₄ Dissolving in methanol, water, ethanol, n-propanol or ethyl acetate, and making into HAuCl ₄ The concentration of (A) is 0.01-0.03M to obtain a solution A;

2) Dissolving a ligand selected from thiol-containing compounds 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 dodecylmercaptan in a solvent to obtain a solution B;

3) Mixing solution A and solution B to obtain HAuCl ₄ The molar ratio of the ligand to the ligand is 1: (0.01-100), stirring for 0.1-48 h in ice bath, adding 0.025-0.8M NaBH ₄ Water, ethanol or methanol solution, and continuously stirring and reacting in ice water bath for 0.1-24 h to obtain reaction solution; (ii) a

4) And after the reaction is finished, centrifuging the reaction solution, and dialyzing to obtain the ligand-bound gold cluster.

6. Use according to claim 5, characterized in that the ligand concentration is between 0.01 and 0.18M.

7. Use according to claim 5, characterized in that the NaBH is ₄ The molar ratio of the ligand to the ligand is 1: (0.01-100).

8. A pharmaceutical composition for the treatment of liver cirrhosis, wherein the pharmaceutical composition comprises ligand-bound gold clusters and a pharmaceutically acceptable excipient;

the ligand-bound gold cluster comprises:

a gold core; and

a ligand bound to the gold core; the ligand is selected from thiol-containing compounds.

9. The pharmaceutical composition of claim 8, wherein the excipient is a phosphate buffered solution or physiological saline.

10. Use of a pharmaceutical composition according to claim 8 in the manufacture of a medicament for the treatment of a patient with cirrhosis of the liver.