Gold clusters, compositions and methods for treating stroke

Abstract

Use of ligand-bound gold clusters and compositions containing ligand-bound gold clusters in the treatment of stroke and in the preparation of drugs for the treatment of stroke. Methods for treating ischemic stroke.

Description

Technical Field

[0001] This invention relates to the field of brain disease treatment, and more particularly to ligand-bound gold clusters (AuCs), compositions comprising ligand-bound gold clusters, and methods for preparing medicaments for treating stroke using the ligand-bound gold clusters and compositions. Background Technology

[0002] A stroke occurs when a blood vessel is blocked by a blood clot or ruptures. There are three types of stroke: hemorrhagic stroke, ischemic stroke, and transient ischemic attack (TIA).

[0003] A hemorrhagic stroke is caused by a ruptured blood vessel that stops blood flow to the brain. Common symptoms include sudden weakness, paralysis in any part of the body, inability to speak, vomiting, difficulty walking, coma, loss of consciousness, stiff neck, and dizziness. There is currently no effective treatment.

[0004] Ischemic stroke is one of the most common diseases in humans and a leading cause of death and disability. It accounts for approximately 87% of all strokes. An ischemic stroke is caused by a blockage, such as a blood clot or plaque, in the arteries supplying blood to the brain. This blockage can occur in the neck or skull, reducing blood flow and oxygen to the brain, leading to brain cell damage or death. If blood circulation is not restored quickly, the brain damage may be permanent.

[0005] The specific symptoms of an ischemic stroke depend on which area of ​​the brain is affected. Common symptoms of most ischemic strokes include vision problems, weakness or paralysis in the limbs, dizziness and vertigo, confusion, loss of coordination, and drooping of one side of the face. Once symptoms appear, it is crucial to seek treatment as soon as possible to minimize the likelihood of permanent damage.

[0006] Treatment options for ischemic stroke are limited. Currently used main medications, such as tissue plasminogen activator (tPA), work by breaking down blood clots; however, tPA must be administered intravenously within 4.5 hours of stroke onset to be effective. Furthermore, tPA can cause bleeding, therefore it should not be used in patients with a history of hemorrhagic stroke, cerebral hemorrhage, recent major surgery, or head injury. Long-term treatment includes aspirin or anticoagulants to prevent further thrombosis.

[0007] Amani et al. disclosed that OX26@GNPs, formed by combining OX26-PEG with the surface of 25nm colloidal gold nanoparticles, can significantly increase infarcted brain tissue, while exposed GNPs and PEGylated GNPs have no effect on infarct volume; the results indicate that OX26@GNPs are not suitable for the treatment of ischemic stroke.

[0008] Zheng et al. disclosed that in their OGD / R injury rat model, 20nm Au-NPs increased cell viability, reduced neuronal apoptosis and oxidative stress, and improved mitochondrial respiration. However, Zheng et al. also demonstrated that 5nm gold nanoparticles had the opposite effect and could not be used for the treatment of ischemic stroke.

[0009] Transient intraepithelial neoplasia (TIA) is caused by transient blood clots. Common symptoms include weakness, numbness or paralysis on one side of the body, slurred or confused speech, blindness, and dizziness. There is currently no specific medication for it.

[0010] Stroke treatment must be timely to avoid or alleviate neurological dysfunction or death caused by damage or death of brain nerve cells due to cerebral ischemia or hemorrhage. There are no specific drugs for hemorrhagic stroke and TIA; the main drugs for treating ischemic stroke, such as tPA, can cause bleeding, so they can only be used after a stroke patient has been diagnosed with ischemic stroke rather than hemorrhagic stroke; this diagnostic process consumes valuable time for timely treatment of ischemic stroke.

[0011] Better strategies and drugs are still needed to treat stroke, especially those effective against both hemorrhagic and ischemic strokes. However, because the causes of hemorrhagic and ischemic strokes are fundamentally different, all drug development in the literature targets either hemorrhagic or ischemic strokes. Therefore, developing a drug that can treat both hemorrhagic and ischemic strokes is an unimaginable challenge, or even a forbidden zone, in the field. Summary of the Invention

[0012] This invention provides the use of ligand-bound gold clusters in the treatment of stroke patients, a method for treating stroke patients using ligand-bound gold clusters, and the use of ligand-bound gold clusters in the preparation of medicaments for treating stroke patients.

[0013] Some embodiments of the present invention utilize ligand-bound gold clusters to treat patients with stroke; wherein the ligand-bound gold clusters comprise a gold nucleus and a ligand bound to the gold nucleus; wherein stroke includes hemorrhagic stroke, ischemic stroke, and transient ischemic attack (TIA).

[0014] In some embodiments for this therapeutic use, the diameter of the gold nucleus is 0.5-3 nm. In some embodiments, the diameter of the gold nucleus is 0.5-2.6 nm.

[0015] In some embodiments of this therapeutic use, the ligand is selected from L-cysteine ​​and its derivatives, D-cysteine ​​and its derivatives, cysteine-containing oligopeptides and their derivatives, and other thiol-containing compounds.

[0016] 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).

[0017] In some embodiments of this therapeutic use, the cysteine-containing oligopeptide and its derivatives are cysteine-containing dipeptides, wherein the cysteine-containing dipeptide is selected from L(D)-cysteine-L(D)-arginine dipeptide (CR), L(D)-arginine-L(D)-cysteine ​​dipeptide (RC), L(D)-histidine-L(D)-cysteine ​​dipeptide (HC) and L(D)-cysteine-L(D)-histidine dipeptide (CH).

[0018] In some embodiments of this therapeutic use, the cysteine-containing oligopeptide and its derivatives are cysteine-containing tripeptides, wherein the cysteine-containing tripeptide is selected from glycine-L(D)-cysteine-L(D)-arginine tripeptide (GCR), L(D)-proline-L(D)-cysteine-L(D)-arginine tripeptide (PCR), L(D)-lysine-L(D)-cysteine-L(D)-proline tripeptide (KCP) and L(D)-glutathione (GSH).

[0019] In some embodiments of this therapeutic use, the cysteine-containing oligopeptide and its derivatives are cysteine-containing tetrapeptides, wherein the cysteine-containing tetrapeptide is selected from glycine-L(D)-serine-L(D)-cysteine-L(D)-arginine tetrapeptide (GSCR) and glycine-L(D)-cysteine-L(D)-serine-L(D)-arginine tetrapeptide (GCSR).

[0020] In some embodiments of this therapeutic use, the cysteine-containing oligopeptide and its derivatives are cysteine-containing pentapeptides, wherein the cysteine-containing pentapeptide is selected from cysteine-aspartic acid-glutamic acid-valine-aspartic acid (CDEVD) and aspartic acid-glutamic acid-valine-aspartic acid-cysteine ​​(DEVDC).

[0021] In some embodiments of this therapeutic use, other thiol-containing compounds are selected from 1-[(2S)-2-methyl-3-thiol-1-oxopropyl]-L(D)-proline, thioglycolic acid, mercaptoethanol, thiophene, D-3-mercaptovaline, N-(2-mercaptopropionyl)-glycine, dodecyl mercaptan, 2-aminoethanethiol, 3-mercaptopropionic acid, and 4-mercaptobenzoic acid.

[0022] Some embodiments of the present invention use ligand-bound gold clusters to prepare medicaments for treating stroke in subjects, wherein the ligand-bound gold clusters comprise a gold nucleus and a ligand bound to the gold nucleus; wherein stroke includes hemorrhagic stroke, ischemic stroke, and transient ischemic attack (TIA).

[0023] In some embodiments of this preparation, the diameter of the gold nucleus is 0.5-3 nm. In some embodiments, the diameter of the gold nucleus is 0.5-2.6 nm.

[0024] In some embodiments of this preparation, the ligand is selected from L-cysteine ​​and its derivatives, D-cysteine ​​and its derivatives, cysteine-containing oligopeptides and their derivatives, and other thiol-containing compounds.

[0025] In some embodiments of this preparation, L-cysteine ​​and its derivatives are selected from L-cysteine, N-isobutyryl-L-cysteine ​​(L-NIBC), and N-acetyl-L-cysteine ​​(L-NAC), and D-cysteine ​​and its derivatives are selected from D-cysteine, N-isobutyryl-D-cysteine ​​(D-NIBC), and N-acetyl-D-cysteine ​​(D-NAC).

[0026] In some embodiments of the preparation, the cysteine-containing oligopeptide and its derivatives are cysteine-containing dipeptides, wherein the cysteine-containing dipeptide is selected from L(D)-cysteine-L(D)-arginine dipeptide (CR), L(D)-arginine-L(D)-cysteine ​​dipeptide (RC), L(D)-histidine-L(D)-cysteine ​​dipeptide (HC) and L(D)-cysteine-L(D)-histidine dipeptide (CH).

[0027] In some embodiments of the preparation, the cysteine-containing oligopeptide and its derivatives are cysteine-containing tripeptides, wherein the cysteine-containing tripeptide is selected from glycine-L(D)-cysteine-L(D)-arginine tripeptide (GCR), L(D)-proline-L(D)-cysteine-L(D)-arginine tripeptide (PCR), L(D)-lysine-L(D)-cysteine-L(D)-proline tripeptide (KCP) and L(D)-glutathione (GSH).

[0028] In some embodiments of the preparation, the cysteine-containing oligopeptide and its derivatives are cysteine-containing tetrapeptides, wherein the cysteine-containing tetrapeptide is selected from glycine-L(D)-serine-L(D)-cysteine-L(D)-arginine tetrapeptide (GSCR) and glycine-L(D)-cysteine-L(D)-serine-L(D)-arginine tetrapeptide (GCSR).

[0029] In some embodiments of the preparation, the cysteine-containing oligopeptide and its derivatives are cysteine-containing pentapeptides, wherein the cysteine-containing pentapeptide is selected from cysteine-aspartic acid-glutamic acid-valine-aspartic acid (CDEVD) and aspartic acid-glutamic acid-valine-aspartic acid-cysteine ​​(DEVDC).

[0030] In some embodiments of this preparation, other thiol-containing compounds are selected from 1-[(2S)-2-methyl-3-thiol-1-oxopropyl]-L(D)-proline, thioglycolic acid, mercaptoethanol, thiophene, D-3-mercaptovaline, N-(2-mercaptopropionyl)-glycine, dodecyl mercaptan, 2-aminoethanethiol, 3-mercaptopropionic acid, and 4-mercaptobenzoic acid.

[0031] The objects and advantages of the present invention will become apparent from the following detailed description of preferred embodiments taken in conjunction with the accompanying drawings. Attached Figure Description

[0032] Preferred embodiments of the invention will now be described with reference to the accompanying drawings, wherein the same reference numerals denote the same elements.

[0033] Figure 1The following images show the UV-Vis (UV) spectra, transmission electron microscopy (TEM) images, and particle size distribution maps of L-NIBC-modified gold nanoparticles (L-NIBC-AuNPs) with different particle sizes: (A) UV spectrum of 3.6 nm L-NIBC-AuNPs; (B) TEM image of 3.6 nm L-NIBC-AuNPs; (C) Particle size distribution map of 3.6 nm L-NIBC-AuNPs; (D) UV spectrum of 6.0 nm L-NIBC-AuNPs; (E) TEM image of 6.0 nm L-NIBC-AuNPs; (F) (G) Particle size distribution of 6.0 nm L-NIBC-AuNPs; (H) UV spectrum of 10.1 nm L-NIBC-AuNPs; (I) Particle size distribution of 10.1 nm L-NIBC-AuNPs; (J) UV spectrum of 18.2 nm L-NIBC-AuNPs; (K) TEM image of 18.2 nm L-NIBC-AuNPs; (L) Particle size distribution of 18.2 nm L-NIBC-AuNPs.

[0034] Figure 2 The UV-Vis (UV) spectra, TEM images, and particle size distributions of L-NIBC-bound gold clusters (L-NIBC-AuCs) with different particle sizes are shown; (A) UV spectrum of 1.1 nm L-NIBC-AuCs; (B) TEM image of 1.1 nm L-NIBC-AuCs; (C) Particle size distribution of 1.1 nm L-NIBC-AuCs; (D) UV spectrum of 1.8 nm L-NIBC-AuCs; (E) TEM image of 1.8 nm L-NIBC-AuCs; (F) Particle size distribution of 1.8 nm L-NIBC-AuCs; (G) UV spectrum of 2.6 nm L-NIBC-AuCs; (H) TEM image of 2.6 nm L-NIBC-AuCs; (I) Particle size distribution of 2.6 nm L-NIBC-AuCs.

[0035] Figure 3 Infrared spectra of L-NIBC-AuCs at 1.1 nm, 1.8 nm, and 2.6 nm are displayed.

[0036] Figure 4The UV, IR, TEM, and particle size distribution maps of CR-bound gold clusters (CR-AuCs) are shown; (A) shows the UV spectrum of CR-AuCs; (B) shows the IR spectrum of CR-AuCs; (C) shows the TEM image of CR-AuCs; (D) shows the particle size distribution map of CR-AuCs.

[0037] Figure 5 (A) UV, IR, TEM and particle size distribution of RC-bound gold clusters (RC-AuCs); (B) UV spectrum of RC-AuCs; (C) TEM image of RC-AuCs; (D) Particle size distribution of RC-AuCs.

[0038] Figure 6 The UV, IR, TEM, and particle size distribution of gold clusters (Cap-AuCs) bound to the ligand 1-[(2S)-2-methyl-3-thiol-1-oxopropyl]-L-proline (i.e., Cap) are shown; (A) shows the UV spectrum of Cap-AuCs; (B) shows the IR spectrum of Cap-AuCs; (C) shows the TEM image of Cap-AuCs; (D) shows the particle size distribution of Cap-AuCs.

[0039] Figure 7 The UV, IR, TEM, and particle size distribution maps of GSH-bound gold clusters (GSH-AuCs) are shown; (A) shows the UV spectrum of GSH-AuCs; (B) shows the IR spectrum of GSH-AuCs; (C) shows the TEM image of GSH-AuCs; (D) shows the particle size distribution map of GSH-AuCs.

[0040] Figure 8 The UV, IR, TEM, and particle size distribution maps of gold clusters bound to ligand D-NIBC- (D-NIBC-AuCs) are shown; (A) shows the UV spectrum of D-NIBC-AuCs; (B) shows the IR spectrum of D-NIBC-AuCs; (C) shows the TEM image of D-NIBC-AuCs; (D) shows the particle size distribution map of D-NIBC-AuCs.

[0041] Figure 9 The UV, IR, TEM, and particle size distribution maps of L-Cys-AuCs are shown; (A) shows the UV spectrum of L-Cys-AuCs; (B) shows the IR spectrum of L-Cys-AuCs; (C) shows the TEM image of L-Cys-AuCs; (D) shows the particle size distribution map of L-Cys-AuCs.

[0042] Figure 10 The UV, IR, TEM, and particle size distribution maps of ligand-2-aminoethanethiol-bound gold clusters (CSH-AuCs) are shown; (A) shows the UV spectrum of CSH-AuCs; (B) shows the IR spectrum of CSH-AuCs; (C) shows the TEM image of CSH-AuCs; (D) shows the particle size distribution map of CSH-AuCs.

[0043] Figure 11 The UV, IR, TEM, and particle size distribution maps of ligand-3-mercaptopropionic acid-bound gold clusters (MPA-AuCs) are shown; (A) shows the UV spectrum of MPA-AuCs; (B) shows the IR spectrum of MPA-AuCs; (C) shows the TEM image of MPA-AuCs; (D) shows the particle size distribution map of MPA-AuCs.

[0044] Figure 12 The UV, IR, TEM, and particle size distribution maps of gold clusters bound to ligand 4-mercaptobenzoic acid (p-MBA-AuCs) are shown; (A) UV spectrum of p-MBA-AuCs is shown; (B) IR spectrum of p-MBA-AuCs is shown; (C) TEM image of p-MBA-AuCs is shown; (D) Particle size distribution map of p-MBA-AuCs is shown.

[0045] Figure 13 The UV, TEM, and particle size distribution of gold clusters (CDEVD-AuCs) bound to the ligand 4-cysteine-aspartic acid-glutamic acid-valine-aspartic acid (CDEVD) are shown; (A) UV spectrum of CDEVD-AuCs is shown; (B) TEM image of CDEVD-AuCs is shown; (C) Particle size distribution of CDEVD-AuCs is shown.

[0046] Figure 14 The UV, TEM, and particle size distribution of gold clusters bound to ligand 4-aspartic acid-glutamic acid-valine-aspartic acid-cysteine ​​(DEVDC) are shown; (A) UV spectrum of DEVDC-AuCs is shown; (B) TEM image of DEVDC-AuCs is shown; (C) Particle size distribution of DEVDC-AuCs is shown.

[0047] Figure 15 The neurobehavioral scores of rats in each group are shown in the bar chart at each time point, from left to right: sham-operated group (blank), model control group, A1 low-dose group, A1 high-dose group, A2 low-dose group, A2 high-dose group, A3 low-dose group, A3 high-dose group, A4 low-dose group, A4 high-dose group, B1 low-dose group, B1 high-dose group, B2 low-dose group, and B2 high-dose group.

[0048] Figure 16 The percentage of cerebral infarction area in each group of rats is shown in the bar chart (from left to right: sham-operated group (blank), model control group, A1 low-dose group, A1 high-dose group, A2 low-dose group, A2 high-dose group, A3 low-dose group, A3 high-dose group, A4 low-dose group, A4 high-dose group, B1 low-dose group, B1 high-dose group, B2 low-dose group, B2 high-dose group).

[0049] Figure 17 Representative images of TTC staining in brain tissue of MCAO rats after administration of gold cluster drugs and gold nanoparticles are presented, including (1) sham-operated group; (2) model control group; (3) low-dose A1 group; (4) high-dose A1 group; (5) low-dose B1 group; and (6) high-dose B1 group.

[0050] Figure 18 The results of mNSS neurological function scores at different time points after modeling are presented, including: normal control group (NC); sham operation group (SHAM); model control group (IVH); low-dose drug A group (AL); high-dose drug A group (AH); low-dose drug B group (BL); high-dose drug B group (BH); (A) mNSS neurological function score 12 hours after modeling; (B) mNSS neurological function score 1 day after modeling; (C) mNSS neurological function score 2 days after modeling; (D) mNSS neurological function score 4 days after modeling; (E) mNSS neurological function score 7 days after modeling; ***: P < 0.001 compared to the IVH group.

[0051] Figure 19 This study presents the Evans blue content in the supernatant of rat brain tissue homogenate in each group during the Evans blue staining assay for blood-brain barrier (BBB) ​​permeability detection. The groups included: normal control (NC); sham-operated group (SHAM); model control group (IVH); low-dose drug A group (AL); and high-dose drug A group (AH). *: P < 0.05 relative to the IVH group; **: P < 0.01 relative to the IVH group; ***: P < 0.001 relative to the IVH group.

[0052] Figure 20 The study presented the water content of rat brain tissue measured by the wet-dry weight method; the groups included: normal control group (NC); sham-operated group (SHAM); model control group (IVH); low-dose drug A group (AL); and high-dose drug A group (AH). *: P < 0.05 relative to IVH group rats; **: P < 0.01 relative to IVH group rats; ***: P < 0.001 relative to IVH group rats.

[0053] Figure 21 Representative H&E staining images of rat brain tissue from different groups are presented. (A) NC group; (B) SHAM group; (C) IVH group; (D) AL group; (E) AH group.

[0054] Figure 22 Representative images of iNOS immunofluorescence staining of rat brain tissue from different groups are presented. (A) NC group; (B) SHAM group; (C) IVH group; (D) AL group; (E) AH group.

[0055] Figure 23 The expression of MMP9 protein in rat brain tissue during Western blotting (WB) was presented in the following groups: normal control group (NC); sham-operated group (SHAM); model control group (IVH); low-dose drug A group (AL); and high-dose drug A group (AH). (A) MMP9 protein bands in 6 parallel samples; (B) relative protein expression of MMP9 (with GAPDH as reference). *: P < 0.05 compared to the IVH group rats.

[0056] Figure 24 The MDA levels in rat brain tissue were presented in each group, including the normal control group (NC), sham-operated group (SHAM), model control group (IVH), low-dose drug A group (AL), and high-dose drug A group (AH). ***: P < 0.001 relative to the IVH group; **: P < 0.01 relative to the IVH group.

[0057] Figure 25 The SOD levels in the brain tissue of rats in each group were presented, including the normal control group (NC), sham-operated group (SHAM), model control group (IVH), low-dose drug A group (AL), and high-dose drug A group (AH). ***: P < 0.001 compared to the IVH group.

[0058] Figure 26 Representative transmission electron micrographs of ultrathin sections of brain tissue from each group are presented: (A) NC group; (B) SHAM group; (C) IVH group; (D) AL group; (E) AH group. Detailed Implementation

[0059] The present invention can be more readily understood by referring to the following detailed description of some embodiments thereof.

[0060] Where publications are cited, the entire contents of those publications are incorporated herein by reference in order to provide a more complete description of the prior art relating to this invention.

[0061] As used herein, “administration” means oral (“po”), suppository, local contact, intravenous (“iv”), intraperitoneal (“ip”), intramuscular (“im”), intralesional, intrahippocampal, lateral ventricle, nasal, or subcutaneous (“sc”) administration, or the implantation of a sustained-release device such as a microosmotic pump or a perishable implant into the subject. Administration can be performed via any route, including parenteral and transmucosal (e.g., oral, nasal, vaginal, rectal, or dermal). Parenteral administration includes, for example, intravenous, intramuscular, arteriolar, intradermal, subcutaneous, intraperitoneal, ventricular, and intracranial administration. Other delivery methods include, but are not limited to, the use of liposomal formulations, intravenous infusion, transdermal patches, etc.

[0062] The term "systemic administration" refers to the administration of a compound or composition to a mammal to deliver the compound or composition to sites within the body, including the target site of action, via the circulatory system. Systemic administration includes, but is not limited to, oral, intranasal, rectal, and parenteral administration (i.e., via routes other than the digestive tract, such as intramuscular, intravenous, arterial, percutaneous, and subcutaneous administration), provided that, as used herein, systemic administration does not include direct administration to brain regions via routes other than the circulatory system, such as intrathecal injection and intracranial administration.

[0063] As used in this article, the term “treatment” means, for the disease or symptom to which the term applies, delaying its onset or slowing / reversing its development, or alleviating / preventing the disease or symptom.

[0064] The terms “patient,” “subject,” or “individual” can be used interchangeably to refer to mammals, such as humans or non-human mammals, including primates (e.g., macaques, pintailed monkeys, gibbons), domesticated mammals (e.g., felines, canines), agricultural mammals (e.g., cattle, sheep, pigs, horses), and laboratory mammals or rodents (e.g., rats, mice, lagomorphs, hamsters, guinea pigs).

[0065] Ligand-bound gold clusters (AuCs) are a special form of gold existing between gold atoms and gold nanoparticles. The gold nuclei of ligand-bound gold clusters are less than 3 nm in size and consist of only a few to a few hundred gold atoms, causing the collapse of the face-centered cubic stacking structure of gold nanoparticles. Therefore, unlike the continuous or quasi-continuous energy levels of gold nanoparticles, gold clusters exhibit a molecular-like discrete electronic structure with different HOMO-LUMO gaps. This leads to the disappearance of the surface plasmon resonance effect and the corresponding plasmon resonance absorption band (520 ± 20 nm) in the UV-Vis spectrum, which are present in conventional gold nanoparticles.

[0066] This invention provides ligand-bound gold clusters.

[0067] In some embodiments, the ligand-bound gold cluster comprises a ligand and a gold core, wherein the ligand is bound to the gold core. The binding of the ligand to the gold core indicates that the ligand forms a stable complex in solution with the gold core through covalent bonds, hydrogen bonds, electrostatic forces, hydrophobic forces, van der Waals forces, etc. In some embodiments, the diameter of the gold core is 0.5-3 nm. In some embodiments, the diameter of the gold core is in the range of 0.5-2.6 nm.

[0068] In some embodiments, the ligand for the ligand-bound gold cluster is a thiol-containing compound or an oligopeptide. In some embodiments, the ligand is bonded to the gold nucleus via an Au-S bond to form a ligand-bound gold cluster.

[0069] 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).

[0070] In some implementations, the ligand is, but is not limited to, cysteine-containing oligopeptides and their derivatives.

[0071] 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).

[0072] 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). In some embodiments, the cysteine-containing oligopeptide is a cysteine-containing pentapeptide. In some implementations, the cysteine-containing pentapeptide is cysteine-aspartic acid-glutamic acid-valine-aspartic acid (CDEVD) or aspartic acid-glutamic acid-valine-aspartic acid-cysteine ​​(DEVDC).

[0073] In some embodiments, the ligand is a thiol-containing compound. In some embodiments, the thiol-containing compound is 1-[(2S)-2-methyl-3-thiol-1-oxopropyl]-L(D)-proline, thioglycolic acid, mercaptoethanol, thiophene, D-3-mercaptovaline, dodecyl mercaptoethanol, 2-aminoethanethiol, 3-mercaptopropionic acid, or 4-mercaptobenzoic acid.

[0074] This invention provides a pharmaceutical composition for treating a subject with ischemic stroke, wherein stroke includes hemorrhagic stroke, ischemic stroke, and transient ischemic attack (TIA). In some embodiments, the subject is a human. In some embodiments, the subject is a pet animal, such as a dog.

[0075] In some embodiments, the pharmaceutical composition comprises a ligand-bound gold cluster as disclosed above and a pharmaceutically acceptable excipient. In some embodiments, the excipient is a phosphate buffer solution or physiological saline.

[0076] The present invention provides the use of the ligand-bound gold clusters disclosed above for manufacturing for treating stroke in subjects, wherein stroke includes hemorrhagic stroke, ischemic stroke and transient ischemic attack (TIA).

[0077] This invention provides methods for treating stroke in subjects using the disclosed ligand-bound gold clusters, including hemorrhagic stroke, ischemic stroke, and transient ischemic attack (TIA). In some embodiments, the treatment method includes administering a pharmaceutically effective amount of the ligand-bound gold cluster to the subject. The pharmaceutically effective amount can be determined through routine in vivo studies. In some implementations, the pharmaceutically effective amount of the ligand-bound gold cluster is 0.001 mg / kg / day, 0.005 mg / kg / day, 0.01 mg / kg / day, 0.05 mg / kg / day, 0.1 mg / kg / day, 0.05 mg / kg / day, 1 mg / kg / day, 2 mg / kg / day, 3 mg / kg / day, 4 mg / kg / day, 5 mg / kg / day, 6 mg / kg / day, 7 mg / kg / day, 8 mg / kg / day, 9 mg / kg / day, 10 mg / kg / day, 15 mg / kg / day, 20 mg / kg / day, 30 mg / kg / day, 40 mg / kg / day, 50 mg / kg / day, 60 mg / kg / day, 70 mg / kg / day, 80 mg / kg / day, 90 mg / kg / day, or 100 mg / kg / day.

[0078] The following examples are provided only to illustrate the principles of the invention; they are by no means intended to limit the scope of the invention.

[0079] Example

[0080] Example 1: Preparation of ligand-bound gold clusters

[0081] 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;

[0082] 1.2 The ligand is dissolved in a solvent to obtain solution B, wherein the concentration of the ligand is 0.01–0.18 M; the ligand includes, but is not limited to, L-cysteine, D-cysteine ​​and other cysteine ​​derivatives, such as N-isobutyryl-L-cysteine ​​(L-NIBC), N-isobutyryl-D-cysteine ​​(D-NIBC), N-acetyl-L-cysteine ​​(L-NAC) and N-acetyl-D-cysteine ​​(D-NAC), cysteine-containing oligopeptides and their derivatives, including, but not limited to, dipeptides, tripeptides, tetrapeptides, pentapeptides and other cysteine-containing peptides, such as L(D)-cysteine-L(D)-arginine dipeptide (CR), L(D)-arginine-L(D)-cysteine ​​dipeptide (RC), L(D)-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), glycine-L(D)-cysteine-L(D)-serine-L(D)-arginine tetrapeptide (GCSR), cysteine-aspartic-glutamic-valine-aspartic-pentapeptide (CDEVD) and aspartic-glutamic-valine-aspartic-cysteine ​​pentapeptide (DEVDC), and other thiol-containing compounds, such as 1-[(2S)-2-methyl-3-thiol-1-oxopropyl]-L(D)- One or more of proline, mercaptoacetic acid, mercaptoethanol, thiophene, D-3-mercaptovaline, dodecyl mercaptan, 2-aminoethanethiol, 3-mercaptopropionic acid, and 4-mercaptobenzoic acid; the solvent is one or more of methanol, ethyl acetate, water, ethanol, n-propanol, pentane, formic acid, acetic acid, diethyl ether, acetone, anisole, 1-propanol, 2-propanol, 1-butanol, 2-butanol, pentanol, butyl acetate, tributyl methyl ether, isopropyl acetate, dimethyl sulfoxide, ethyl formate, isobutyl acetate, methyl acetate, 2-methyl-1-propanol, and propyl acetate;

[0083] 1.3 Mix solutions A and B to make the molar ratio of HAuCl4 to ligand 1:(0.01-100), stir in an ice bath for 0.1-48 h, add 0.025-0.8 M NaBH4 solution in water, ethanol, or methanol, and continue stirring in an ice-water bath for 0.1-12 h. The molar ratio of NaBH4 to ligand is 1:(0.01-100).

[0084] 1.4 After the reaction was completed, the reaction solution was centrifuged for 10-100 min at 8000-17500 r / min using an MWCO 3K-30K ultrafiltration tube to obtain ligand-bound gold clusters with different average particle sizes. The pore size of the filtration membrane of different MWCO ultrafiltration tubes directly determines the size of the ligand-bound gold clusters that can pass through the membrane.

[0085] This step can be omitted optionally;

[0086] 1.5 Dissolve the gold clusters with 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 1-7 days;

[0087] 1.6 After dialysis, the ligand-bound gold clusters were freeze-dried for 12-24 hours to obtain powdered or flocculant substances, i.e., ligand-bound gold clusters.

[0088] As detected, the particle size of the powdered or flocculant material obtained by the aforementioned method is less than 3 nm (typically distributed in the range of 0.5–2.6 nm). No significant absorption peak is observed at 520 nm. This confirms that the obtained powder or flocculant is a ligand-bound gold cluster.

[0089] Example 2: Preparation and identification of gold clusters bound to different ligands

[0090] 2.1 Preparation of L-NIBC-bound gold clusters, namely L-NIBC-AuCs

[0091] Taking ligand L-NIBC as an example, the preparation and identification of gold clusters bound by ligand L-NIBC are described in detail.

[0092] 2.1.1 Weigh 1.00 g of HAuCl4 and dissolve it in 100 mL of methanol to obtain a 0.03 M solution A;

[0093] 2.1.2 Weigh 0.57 g L-NIBC and dissolve it in 100 mL glacial acetic acid to obtain 0.03 M solution B;

[0094] 2.1.3 Take 1 mL of solution A and mix it with 0.5 mL, 1 mL, 2 mL, 3 mL, 4 mL, or 5 mL of solution B (i.e., the molar ratio between HAuCl4 and L-NIBC is 1:0.5, 1:1, 1:2, 1:3, 1:4, and 1:5, respectively). Stir the mixture in an ice bath for 2 h. When the solution changes from bright yellow to colorless, quickly add 1 mL of freshly prepared 0.03 M NaBH4 ethanol solution (weigh 11.3 mg NaBH4 and dissolve it in 10 mL of ethanol). After the solution turns dark brown, continue the reaction for 30 minutes and add 10 mL of acetone to terminate the reaction.

[0095] 2.1.4 After the reaction, the reaction solution was subjected to gradient centrifugation to obtain L-NIBC-AuCs powders of different particle sizes. Specific method: After the reaction was complete, the reaction solution was transferred to a 50 mL ultrafiltration tube with a MWCO of 30 K and centrifuged at 10,000 rpm for 20 min. The residue in the inner tube was dissolved in ultrapure water, yielding powder with a particle size of approximately 2.6 nm. Then, the mixed solution in the outer tube was transferred to a 50 mL ultrafiltration tube with a MWCO of 10 K and centrifuged at 13,000 rpm for 30 min. The residue in the inner tube was dissolved in ultrapure water, yielding powder with a particle size of approximately 1.8 nm. Then, the mixed solution in the outer tube was transferred to a 50 mL ultrafiltration tube with a MWCO of 3 K and centrifuged at 17,500 rpm for 40 min. The residue in the inner tube was dissolved in ultrapure water, yielding powder with a particle size of approximately 1.1 nm.

[0096] 2.1.5 The three different particle sizes of the precipitate obtained by gradient centrifugation were separated by removing the solvent, the crude product was dried by blowing with N2, dissolved in 5 mL of ultrapure water, placed in a dialysis bag (MWCO of 3 kDa), and placed in 2 L of ultrapure water. The water was changed every other day, and the dialysis was performed for 7 days. After freeze-drying, it was ready for use.

[0097] 2.2 Identification of L-NIBC-AuCs

[0098] Identification experiments were performed on the powder (L-NIBC-AuCs) obtained above. Meanwhile, gold nanoparticles modified with L-NIBC ligand (L-NIBC-AuNP) were used as a control. The preparation method of gold nanoparticles with L-NIBC ligand is described in the references (W. Yan, L. Xu, C. Xu, W. Ma, H. Kuang, L. Wang and NAKotov, Journal of the American Chemical Society 2012, 134, 15114; X. Yuan, B. Zhang, Z. Luo, Q. Yao, D. Leong, N. Yan and J. Xie, Angewandte Chemie International Edition 2014, 53, 4623).

[0099] 2.2.1 Morphological observation using transmission electron microscopy (TEM)

[0100] The test powders (L-NIBC-AuCs and L-NIBC-AuNPs samples) were dissolved in ultrapure water to a concentration of 2 mg / L to prepare the samples, which were then prepared using the hanging drop method. More specifically, 5 μL of the sample was dropped onto an ultrathin carbon film and allowed to evaporate naturally until the water droplet disappeared. The morphology of the sample was then observed using a JEM-2100F STEM / EDS field emission high-resolution TEM.

[0101] Four TEM images of L-NIBC-AuNP are shown Figure 1 In the B, E, H, and K images; three TEM images of L-NIBC-AuCs are shown Figure 2 Among the B, E and H images.

[0102] Figure 2 The images show that each L-NIBC-AuCs sample has a uniform particle size and good dispersibility, and the average diameters (referring to the diameter of the gold core) of L-NIBC-AuCs are 1.1 nm, 1.8 nm, and 2.6 nm, respectively. Figure 2 The results in the C, F, and I images are completely consistent. In contrast, the L-NIBC-AuNPs samples have larger particle sizes. Their average diameters (referring to the diameter of the gold core) are 3.6 nm, 6.0 nm, 10.1 nm, and 18.2 nm, respectively. Figure 1 The results for the C, F, I, and L amplitudes are in good agreement.

[0103] 2.2.2 Ultraviolet (UV)-Vis absorption spectrum

[0104] The test powders (L-NIBC-AuCs and L-NIBC-AuNPs samples) were dissolved in ultrapure water until the concentration reached 10 mg·L⁻¹, and the 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 path length of 1 cm, and the reference cell was filled with ultrapure water.

[0105] The UV-vis absorption spectra of four L-NIBC-AuNP samples of different sizes are shown in the figure. Figure 1 In images A, D, G, and J, the statistical distribution of granularity is shown in... Figure 1 The UV-vis absorption spectra of three L-NIBC-AuCs samples with different sizes are shown in the C, F, I, and L amplitudes. Figure 2 In images A, D, and G, the statistical distribution of granularity is shown in... Figure 2 In the C, F and I frames.

[0106] Figure 1 This indicates that L-NIBC-AuNP exhibits an absorption peak at approximately 520 nm due to surface plasmon resonance. The position of the absorption peak is related to particle size. When the particle size is 3.6 nm, the UV absorption peak appears at 516 nm; when the particle size is 6.0 nm, the UV absorption peak appears at 517 nm; when the particle size is 10.1 nm, the UV absorption peak appears at 520 nm; and when the particle size is 18.2 nm, the absorption peak appears at 523 nm. None of the four samples exhibited any absorption peak above 560 nm.

[0107] Figure 2 The results show that in the UV absorption spectra of L-NIBC-bound gold clusters with three different particle sizes, the surface plasmon resonance absorption peak at 520 nm disappears, while two distinct absorption peaks appear above 560 nm. The positions of these absorption peaks vary slightly with the particle size of the gold clusters. This is because the collapse of the face-centered cubic structure causes the gold clusters to exhibit molecular-like properties, leading to discontinuities in the density of states, energy level splitting, the disappearance of the plasmon resonance effect, and the appearance of new absorption peaks in the longer wavelength directions. It can be concluded that the three powder samples with different particle sizes obtained above are all ligand-bound gold clusters.

[0108] 2.2.3 Fourier Transform Infrared Spectroscopy

[0109] Infrared spectroscopy was performed on a Bruker VERTEX 80V Fourier transform infrared spectrometer using solid powder high-vacuum total reflectance mode, with a scanning range of 4000-400 cm⁻¹. -1The scan was performed 64 times. Taking L-NIBC-bound gold clusters as an example, the test samples consisted of three different sizes of L-NIBC-bound gold cluster dry powders, with pure L-NIBC powder as the control sample. Results are shown below. Figure 3 .

[0110] Figure 3 The image shows the infrared spectra of gold clusters bound to L-NIBCs of different sizes. Compared to pure L-NIBCs (bottom curve), the SH stretching vibrations in the 2500-2600 cm⁻¹ range completely disappear in the L-NIBC-bound gold clusters of different sizes, while other characteristic peaks of L-NIBCs are still observed. This demonstrates that L-NIBC molecules are successfully bound to the surface of the gold clusters via gold-sulfur bonds. The image also shows that the infrared spectra of ligand-bound gold clusters are independent of their size.

[0111] Gold clusters bound to other ligands can be prepared using a similar method as described above, with slight adjustments to the solvent of solution B, the feed ratio of HAuCl4 to the ligand, the reaction time, and the amount of NaBH4 added. For example, when using L-cysteine, D-cysteine, N-isobutyryl-L-cysteine ​​(L-NIBC), or N-isobutyryl-D-cysteine ​​(D-NIBC) as ligands, acetic acid is chosen as the solvent; when using dipeptide CR, dipeptide RC, or 1-[(2S)-2-methyl-3-mercapto-1-oxopropyl]-L-proline as ligands, water is chosen as the solvent, and so on. Other steps are similar, so further details are not provided here.

[0112] This invention prepared and obtained a series of ligand-bound gold clusters using the above method. The parameters of the ligands and preparation method are shown in Table 1.

[0113] Table 1. Preparation parameters of gold clusters with different ligand bindings according to the present invention

[0114]

[0115]

[0116]

[0117] The samples listed in Table 1 were confirmed using the aforementioned methods. The characteristics of the 11 different ligand-bound gold clusters are shown in... Figure 4 (CR-AuCs), Figure 5 (RC-AuCs), Figure 6 (Cap-AuCs) (Cap represents 1-[(2S)-2-methyl-3-mercapto-1-oxopropyl]-L-proline), Figure 7 (GSH-AuCs), Figure 8 (D-NIBC-AuCs), Figure 9 (L-Cys-AuCs), Figure 10 (CSH-AuCs), Figure 11 (MPA-AuCs) Figure 12 (p-MBA-AuCs), Figure 13 (CDEVD-AuCs) Figure 14 (DEVDC-AuCs). Figures 4-12 Displays UV spectrum (A), infrared spectrum (B), TEM image (C), and particle size distribution (D). Figures 13-14 Displays UV spectrum (A), TEM image (B), and particle size distribution (C).

[0118] The results showed that the diameters of the gold clusters bound by the different ligands obtained in Table 1 were all less than 3 nm. The UV-Vis spectrum also showed the disappearance of the peak at 520 ± 20 nm, and the appearance of absorption peaks at other positions. The positions of these absorption peaks varied with the ligands, particle size, and structure. In some cases, no specific absorption peak appeared, mainly because a mixture of gold clusters of different sizes and structures was formed, or certain special gold clusters caused the absorption peaks to be located outside the range of conventional UV-Vis absorption spectroscopy. Simultaneously, the Fourier transform infrared spectroscopy also showed the disappearance of the thiol infrared absorption peak of the ligands. Figure 4-8 The peaks between the dashed lines in the middle B amplitude are visible, while other infrared characteristic peaks are retained, indicating that the ligand molecules have successfully bound to the gold atoms to form ligand-bound gold clusters, indicating that the present invention has successfully obtained gold clusters bound to the ligands listed in Table 1.

[0119] Example 3: Animal Model Experiment of Ischemic Stroke

[0120] 3.1 Test Samples

[0121] Gold clusters:

[0122] A1: L-NIBC ligand-bound gold clusters with a size of 0.5-3.0 nm;

[0123] A2: L-Cys ligand-bound gold clusters with a size of 0.5-3.0 nm;

[0124] A3: L-NAC ligand-bound gold clusters with a size of 0.5-3.0 nm;

[0125] A4: Gold clusters bound to DEVDC ligands, with a size of 0.5-3.0 nm.

[0126] Gold nanoparticles:

[0127] B1: L-NIBC modified gold nanoparticles, size 6.1±1.5 nm;

[0128] B2: L-NAC modified gold nanoparticles with a size of 9.0 ± 2.4 nm.

[0129] The preparation methods for all test samples were the same as those described above, with slight modifications; their quality was verified by the aforementioned methods.

[0130] 3.2 Test Methods

[0131] 3.2.1 Establishment of a rat model of middle cerebral artery infarction and administration of the test substance

[0132] Male SPF-grade Sprague Dawley (SD) rats (220-260g) were purchased from Shanghai Silex Laboratory Animal Co., Ltd. They were acclimatized to their environment for 7 days before the experiment. All rats were randomly divided into 14 groups (n=10 per group): sham-operated group, model control group, low-dose (2 mg / kg rat body weight) and high-dose (10 mg / kg rat body weight) groups of gold cluster drugs A1, A2, A3, and A4, and low-dose (2 mg / kg rat body weight) and high-dose (10 mg / kg rat body weight) groups of gold nanoparticles B1 and B2. On the day of the experiment, rats were anesthetized with 10% chloral hydrate (350 mg / kg body weight). The right common carotid artery, internal carotid artery, and external carotid artery were exposed through a midline cervical incision. An embolic suture was inserted through the external carotid artery (ECA) into the internal carotid artery (ICA) 18 mm ± 0.5 mm until the blood supply to the MCA region was blocked, inducing cerebral infarction. After 1.5 hours, the embolic suture was withdrawn from the ECA inlet for reperfusion. Preoperative baseline cerebral blood flow (CBF) and postoperative cerebral blood flow were measured using a flowmeter. Animals with a sustained decrease in CBF (rCBF ≥ 70%) were considered to have successfully established a rat model of middle cerebral artery occlusion (MCAO). After reperfusion, rats were administered the test drug or solvent (physiological saline) via intraperitoneal injection at 0h, 24h, 48h, and 72h. Neurological behavioral scores were assessed at 0h, 24h, 48h, 72h, and 96h. The experiment was terminated 96h post-operation. After euthanasia, the rats' brains were collected and stained with TTC, brain slices were photographed, and the percentage of cerebral infarction area was calculated.

[0133] 3.2.2 Neurobehavioral scoring

[0134] 0 points: No different from a normal mouse; 1 point: Right forepaw cannot be straightened, head turns to the opposite side; 2 points: Walks in open areas in discontinuous circles; 3 points: Walks in open areas in continuous circles; 4 points: Walks unconsciously and collapses to one side; 5 points: Death.

[0135] 3.2.3 Infarct area (TTC staining)

[0136] Rats were euthanized by carbon dioxide inhalation. Brains were harvested and placed in the sulcus for coronal sectioning (2 mm). The sections were stained with 2% TTC at room temperature in the dark, photographed, and the infarct area was analyzed using ImageJ. The percentage of infarct area (%) was calculated as: (contralateral hemisphere area - (ipsilateral hemisphere area - infarct area)) / contralateral hemisphere area × 100%.

[0137] 3.2.4 Statistical Analysis

[0138] Statistical analysis was performed using Graph Pad Prism software 7.0 (CA, US). Data are expressed as mean ± standard error. One-way ANOVA and Dunnett's test were used for statistical analysis. P < 0.05 was considered statistically significant.

[0139] 3.3 Experimental Results

[0140] 3.3.1 Cerebral blood flow in ischemic areas

[0141] A reduction of cerebral blood flow (rCBF ≥ 70%) in the ischemic area of ​​the rat brain was considered a successful MACO model. Except for the sham-operated group, the rCBF of all other groups was > 70%, with an average of around 80%, indicating that the model was successfully established in all groups.

[0142] 3.3.2 Effects of each test drug on the neurobehavioral behavior of rats

[0143] Figure 15The neurobehavioral scores of rats in each group are shown in the bar chart at each time point, from left to right: sham-operated group (blank), model control group, A1 low-dose group, A1 high-dose group, A2 low-dose group, A2 high-dose group, A3 low-dose group, A3 high-dose group, A4 low-dose group, A4 high-dose group, B1 low-dose group, B1 high-dose group, B2 low-dose group, and B2 high-dose group. Rats in the sham-operated group showed normal neurobehavioral behavior, with a behavioral score of 0. Rats in the model control group exhibited severe behavioral deficits at 0h, 24h, 48h, 72h, and 96h post-surgery (P<0.001 compared to the sham-operated group). Compared with the model control group, the neurobehavioral scores of the A1, A2, A3, and A4 low-dose and high-dose groups showed no significant improvement at 24 hours post-surgery. At 48 hours post-surgery, neurological behavioral scores in both the low-dose and high-dose groups of A1, A2, A3, and A4 began to decrease, but there was no statistically significant difference (P>0.05 compared to the model control group). At 72 hours post-surgery, neurological behavioral scores for all four drugs further decreased, with significant differences observed in the low-dose A1 group, the high-dose A1 group, and the high-dose A2 group (P<0.05, * compared to the model control group). At 96 hours post-surgery, significant differences were observed in both the low-dose and high-dose groups of all four drugs (P<0.05, * compared to the model control group). These results indicate that all four gold cluster drugs can significantly improve neurological behavioral deficits caused by ischemic stroke, and this effect shows a certain dose-dependent effect.

[0144] Compared with the model group, the administration of gold nanoparticles B1 and B2 in low and high dose groups did not significantly improve the neurological behavioral scores of MACO model rats at 24h, 48, 72h and 96h after surgery, indicating that gold nanoparticles could not significantly improve behavioral disorders caused by ischemic stroke.

[0145] 3.3.3 Effects of each test substance on the area of ​​cerebral infarction in MACO model rats

[0146] Figure 16The percentage of cerebral infarction area in each group of rats is shown in the bar chart (from left to right: sham-operated group (blank), model control group, A1 low-dose group, A1 high-dose group, A2 low-dose group, A2 high-dose group, A3 low-dose group, A3 high-dose group, A4 low-dose group, A4 high-dose group, B1 low-dose group, B1 high-dose group, B2 low-dose group, B2 high-dose group). The sham-operated group rats had normal brain tissue with no infarction, and the infarction area was 0%. The model control group had significant cerebral infarction, with an infarction area of ​​44.7% ± 4.5% (compared to the sham-operated group, P < 0.001, ###). Compared with the model control group, the percentage of cerebral infarction area in the A1, A2, A3, and A4 low-dose and high-dose groups all decreased significantly, but no significant difference was observed in the low-dose groups, while a significant difference was observed in the high-dose groups (compared to the model control group, P < 0.05, *). Taking A1 as an example, the infarct area in the low-dose group decreased from 44.7±4.5% to 36.0±4.0% (P>0.05 compared to the model control group), while that in the high-dose group decreased to 27.8±3.4% (P<0.05 compared to the model control group, *).

[0147] Figure 17 Taking drugs A1 and B1 as examples, representative images of TTC staining of MCAO rat brain tissue after administration of gold cluster drugs and gold nanoparticles are shown, where the white area represents the cerebral infarction area. Figure 17 Among them, (1) sham surgery group; (2) model control group; (3) low-dose A1 group; (4) high-dose A1 group; (5) low-dose B1 group; (6) high-dose B1 group. Figure 17 As can be seen, no cerebral infarction occurred in the sham-operated group rats, while the model control group experienced significant cerebral infarction (white area on the right). Low-dose administration of drug A1 reduced the cerebral infarction area (reduction in the white area on the right), while high-dose administration of drug A1 significantly reduced the cerebral infarction area (significant reduction in the white area on the right). Low-dose and high-dose administration of drug B1 had almost no effect on the cerebral infarction area (no reduction in the white area on the right). Drugs A2, A3, and A4 showed similar significant effects to drug A1 in reducing the infarction area, while drug B2, similar to drug B1, had no effect on reducing the infarction area.

[0148] Other ligand-bound gold clusters also have the same therapeutic effect on ischemic stroke, but their effects vary. They will not be described in detail here.

[0149] Example 4: Animal Model Experiment of Cerebral Hemorrhage Stroke

[0150] 4.1 Materials and Methods

[0151] 4.1.1 Test Drugs

[0152] Drug A: L-NIBC modified gold clusters (L-NIBC-AuNCs), with gold core diameters ranging from 0.5 to 3.0 nm;

[0153] Drug B: L-Cys-modified gold clusters (L-Cys-AuNCs) with gold core diameters ranging from 0.5 to 3.0 nm.

[0154] 4.1.2 Animals and Grouping

[0155] One hundred and forty SD female rats, eight weeks old and weighing 190-220g, were used. The rats were acclimatized for seven days in an SPF (Special Purpose Facility) environment and then randomly divided into seven groups.

[0156] (1) 20 normal control (NC) animals, with an average weight of 214.0±5.1g;

[0157] (2) The sham surgery group consisted of 20 animals with an average weight of 213.5 ± 6.5 g.

[0158] (3) The model control (IVH) group consisted of 20 animals with an average weight of 214.7 ± 6.1 g;

[0159] (4) 20 rats were given low dose (4 mg / kg body weight) of drug A (AL), with an average body weight of 212.5 ± 7.3 g;

[0160] (5) 20 rats were given high dose (10 mg / kg body weight) of drug A (AH), with an average body weight of 212.1 ± 6.8 g;

[0161] (6) 20 rats were given low-dose (4 mg / kg body weight) drug B (BL) with an average body weight of 213.2 ± 6.3 g;

[0162] (7) 20 rats were given high dose (10 mg / kg body weight) of drug B (BH), with an average body weight of 211.2 ± 7.1 g.

[0163] There was no statistically significant difference in body weight among the groups.

[0164] 4.1.3 Modeling, Drug Administration, and Testing Methods:

[0165] A rat model of intraventricular hemorrhage (IVH) was established:

[0166] In the IVH group and each drug-treated group, rats were anesthetized by intraperitoneal injection of 2% barbiturate-free (50 mg / kg). After anesthesia, the rats' heads were prepared, and they were fixed in a prone position on a stereotaxic instrument. The stereotaxic instrument was adjusted to fix the rats' incisors to the incisor hooks so that the anterior and posterior fontanelles were on the same horizontal line. The ear rods were adjusted and fixed so that the rats' heads could not move. After disinfecting the skin on the rats' heads, the skin was cut in the midline, and the periosteum was eroded with 3% hydrogen peroxide to expose the anterior and posterior fontanelles and the coronal suture. A small hole with a diameter of about 1 mm was drilled 0.2 mm posterior to the anterior fontanelle and 3 mm lateral to the midline, without damaging the dura mater or brain tissue. The rat tails were heated and washed with 40°C warm water. After congestion, the tails were disinfected with ethanol. 50 μl of non-anticoagulated arterial blood was drawn using a 1 ml syringe and fixed on the stereotaxic instrument. The micro-syringe was inserted through the hole to a depth of 6 mm. The autologous arterial blood was injected twice. First, arterial blood was injected at a uniform rate into 10 μl of the needle. After injecting 40 μl of arterial blood, stop injecting for 2 minutes, then inject 40 μl of arterial blood evenly and slowly again, leave the needle in place for 4 minutes, withdraw the needle about 2.0 mm, stop injecting again for 4 minutes, and then slowly withdraw the syringe completely. Suture the incision and apply pressure with a sterile cotton ball to stop the bleeding. Observe the wound healing of the rats daily and check for any infection.

[0167] After the rats regained consciousness, behavioral observations were conducted, and the mNSS neurological function score was used to observe the rat model establishment. Postoperatively, the rats were administered the drug once daily via intraperitoneal injection on days 1, 2, 3, 4, 5, 6, and 7. The dosage for the AL and AH groups was 4 and 10 mg / kg body weight, respectively. The NC and SHAM groups received the same volume of physiological saline via intraperitoneal injection at the corresponding time points. Neurological function in all groups was assessed using the mNSS score at 12h, 24h, 3d, 5d, and 7d after intraperitoneal injection.

[0168] 4.1.4. Detection of blood-brain barrier permeability using the Evans blue staining method

[0169] Rats were treated and intravenously injected with Evans Blue Stain (0.5%), resulting in blue discoloration of their eyes and skin. After 0.5–1 hour, the rats were sacrificed, and brain tissue was collected. The brain tissue was placed in a 1.5 mL centrifuge tube, 1 mL of PBS was added, and the tissue was quickly homogenized using a tissue homogenizer and centrifuged. The supernatant was collected, and an equal volume of trichloroacetic acid was added. The mixture was incubated at 4°C for 15 min. The absorbance (OD) of the solution at 620 nm was measured using a spectrophotometer. Simultaneously, the OD values ​​of Evans Blue standards with different gradients were measured, and a standard curve was plotted. The Evans Blue content of the sample was calculated based on the standard curve.

[0170] 4.1.5 Brain tissue water content test

[0171] The wet-dry weight method was used. Rats subjected to ischemia-reperfusion for 24 hours were over-anesthetized, decapitated, and their brains were harvested. The olfactory bulb, cerebellum, and lower brainstem were removed, and the left and right cerebral hemispheres were separated. The wet weight was immediately measured, and the brain tissue was then dried in a 110℃ oven for 24 hours, followed by rapid weighing of the dry weight. Brain water content (%) was calculated as (wet weight - dry weight) / wet weight × 100%.

[0172] 4.1.6 H&E staining

[0173] Rat brain tissue was extracted and placed in an embedding cassette. The following steps were performed sequentially: dehydration, clearing, paraffin embedding, sectioning, and baking. Then, H&E staining was performed: sections were removed from the oven and treated twice with xylene for 15 minutes each time, followed by treatment with 100%, 95%, 80%, and 70% ethanol for 5 minutes each, and then treated twice with ultrapure water for 5 minutes each time to complete the dewaxing step. Hematoxylin was stained for 5 minutes, and the sections were rinsed three times with ultrapure water. After rinsing with tap water for 10 minutes until the cell nuclei turned blue, the sections were stained with 0.5% erythromycin for 5 minutes, rinsed five times with tap water, and then dried in a 60°C oven. Finally, the sections were mounted with neutral resin. Observation and image acquisition were performed using an optical microscope at 200x magnification.

[0174] 4.1.7 Immunofluorescence (IF) Detection

[0175] Rat brain tissue was extracted and placed in an embedding cassette. The tissue underwent dehydration, clearing, paraffin infiltration, embedding, paraffin sectioning, and baking. The sections were then removed from the oven and dewaxed. Following this, antigen retrieval, washing, permeabilization, iNOS primary antibody treatment, Goat anti-Rabbit secondary antibody treatment, and DAPI staining were performed. Finally, the sections were baked and mounted. Observation and image acquisition were conducted using a fluorescence microscope at 200x magnification.

[0176] 4.1.8 Western-Blot (WB) Detection

[0177] Protein sample pretreatment: Remove frozen tissue from the freezer, weigh an appropriate amount of tissue, add lysis buffer containing protease inhibitors at a ratio of 1:9, and shake at 4°C until the tissue block is completely broken. Lyse on ice for 20 min, centrifuge at 12000 rpm for 20 min at 4°C, and collect the supernatant. Protein concentration was determined using a BCA protein assay kit. Adjust the protein concentration according to the results to ensure consistency between different groups. After boiling at 95°C for 5 min, load the samples. Store the remaining samples at -80°C.

[0178] Then perform the WB experiment as follows:

[0179] Preparation of electrophoresis gel: After washing and drying the glass plate, fix it on the gel casting apparatus. Begin preparing the separating gel by pouring it into the gaps in the glass plate to an appropriate height. Cover the separating gel with anhydrous ethanol until the gel is completely polymerized. Pour out the anhydrous ethanol, gently rinse with double-distilled water, and then blot dry with filter paper. Next, add the stacking gel to an appropriate height and insert the comb teeth. After the stacking gel has completely polymerized, remove the comb teeth.

[0180] Sample loading and electrophoresis: Load the processed sample at a total volume of 40 μg per well. Perform electrophoresis with an 80V stacking gel and a 120V separating gel per gel, using a constant voltage power supply. Determine the position of the target protein based on the relative position of the pre-stained marker and the molecular weight of the target protein. Stop electrophoresis when the target protein is in the optimal resolvable position in the lower 1 / 3 of the separating gel surface.

[0181] Transfer: Immerse the cut PVDF membrane in methanol for 10 seconds, then place it in fresh transfer buffer. Remove the gel and cut the target band according to the marker. Rinse with distilled water. Cut a PVDF membrane and filter paper to the same size as the PAGE gel. Immerse the PVDF membrane and filter paper together in the electrotransfer buffer. Arrange the plates in the following order: black plate - fiber pad - filter paper - gel - PVDF membrane - filter paper - fiber pad - white plate. Clamp the plates and place them in the transfer apparatus, with the black plate facing the black negative electrode. Fill the transfer tank with transfer buffer and begin the transfer. The transfer process is performed at 4°C (ice bath) under the following conditions: 200 mA, 90 min (0.45 μm membrane) or 200 mA, 60 min (0.2 μm membrane).

[0182] Blocking: The membrane was washed three times with PBST, and then blocked with TBST blocking solution containing 5% milk for 2 hours on a shaker at room temperature.

[0183] Primary antibody: Dilute the corresponding primary antibody with TBST containing 3% BSA, immerse the PVDF membrane in the primary antibody incubation solution, and incubate overnight at 4°C. MMP9 was diluted 1:1000, and iNOS was diluted 1:2000. After incubation, wash the PVDF membrane thoroughly with TBST three times for 10 min each time to remove excess primary antibody.

[0184] Secondary antibody: Dilute HRP-labeled secondary antibody (1:5000) with blocking buffer, immerse the PVDF membrane in the secondary antibody incubation solution, and incubate on a shaker at room temperature for 1 hour. After incubation, wash the PVDF membrane thoroughly with TBST 3 times for 10 minutes each time to remove excess secondary antibody.

[0185] Color development and exposure: The enhancement solution in the ECL kit was mixed with the stable peroxidase solution at a ratio of 1:1 to prepare the working solution. An appropriate amount of the working solution was dropped onto the PVDF membrane and exposed using a fully automated chemiluminescence image analysis system.

[0186] Membrane elution and regeneration: After exposure, thoroughly wash the PVDF membrane three times with TBST, 5 min each time. Add an appropriate amount of membrane regeneration solution, immerse the PVDF membrane in the membrane regeneration solution, and elute on a shaker at room temperature for 20 min. After elution, thoroughly wash the PVDF membrane three times with TBST, 5 min each time, to remove excess membrane regeneration solution.

[0187] The process involved blocking again, incubation with internal control, binding with secondary antibody, and exposure, following the same procedure as described above. Both β-tubulin and GAPDH were diluted 1:5000.

[0188] The exposure results were analyzed using ImageJ software to determine the grayscale values.

[0189] 4.1.9 Detection of lipid peroxidation

[0190] Sample processing: Add the corresponding volume of PBS (mass-volume ratio of 1:9) to the tissue, homogenize thoroughly, incubate on ice for 10 min, centrifuge at 4000 rpm for 10 min, and collect the supernatant for testing.

[0191] Malondialdehyde (MDA) was tested using a reagent kit. Blank tubes, standard tubes, assay tubes, and control tubes were prepared according to the instructions. The absorbance (OD) value at 532 nm was measured. The MDA content in the supernatant of the tissue was calculated using the following formula: MDA content (nmol / mL) = (Assessed OD value - Control OD value) / (Standard OD value - Blank OD value) × Standard concentration (10 nmol / mL).

[0192] 4.1.10 Detection of Superoxide Dismutase (SOD) Levels

[0193] Sample processing: Add the corresponding volume of PBS (mass-volume ratio of 1:9) to the tissue, homogenize thoroughly, incubate on ice for 10 min, centrifuge at 4000 rpm for 10 min, and collect the supernatant for testing.

[0194] The SOD kit was used for testing according to the instructions. The following formulas were used to calculate the SOD inhibition rate (%): SOD inhibition rate (%) = ((OD value of control wells - OD value of control blank wells) - (OD value of assay wells - OD value of assay blank wells)) / (OD value of control wells - OD value of control blank wells) × 100%; SOD activity (U / mgprot) = SOD inhibition rate ÷ 50% × reaction system dilution factor ÷ protein concentration of the sample to be tested (mgprot / mL).

[0195] 4.1.11 Electron Microscopy Detection

[0196] A tissue block of approximately 1 cubic millimeter was taken. After fixation, dehydration, embedding, and solidification, it was sectioned using an ultramicrotome to a thickness of 70 nm. The section was then double-stained with 2% uranium acetate and lead citrate. Observation and imaging were performed using a transmission electron microscope.

[0197] 4.2 Test Results

[0198] Figure 18 The results of the mNSS neurological function scores at different time points after modeling were presented, including the normal control group (NC); the sham operation group (SHAM); the model control group (IVH); the low-dose drug A group (AL); the high-dose drug A group (AH); the low-dose drug B group (BL); and the high-dose drug B group (BH).

[0199] like Figure 18 As shown in Figure A, the results showed that 12 hours after modeling, the average mNSS neurological function scores were as follows: Normal control (NC) group (0.0±0.0), Sham operation (SHAM) group (0.0±0.0), Model control (IVH) group (14.7±1.2), Low-dose drug A (AL) group (14.7±1.0), High-dose drug A (AH) group (14.4±1.1), Low-dose drug B (BL) group (14.7±1.3), High-dose drug B (BH) group (14.7±1.1). The NC and SHAM groups showed significant statistical differences compared with the IVH group (P<0.001, ***), while the other groups showed no significant statistical differences compared with the IVH group.

[0200] like Figure 18 As shown in Figure B, the results showed that one day after modeling, the average mNSS neurological function scores were: NC group (0.0±0.0), SHAM group (0.0±0.0), IVH group (14.3±1.3), AL group (14.6±1.1), AH group (14.3±1.0), BL group (14.9±0.9), and BH group (14.3±1.0). The NC and SHAM groups showed significant statistical differences compared with the IVH group (P<0.001, ***), while the other groups showed no significant statistical differences compared with the IVH group.

[0201] like Figure 18As shown in Figure C, the results indicated that two days after modeling, the average mNSS neurological function scores were as follows: NC group (0.0±0.0), SHAM group (0.0±0.0), IVH group (11.0±0.9), AL group (8.6±1.5), AH group (7.7±1.7), BL group (9.9±1.8), and BH group (10.2±1.9). The NC, SHAM, AL, and AH groups showed statistically significant differences compared to the IVH group (P<0.001, ***). While the average scores of the BL and BH groups were slightly lower than those of the IVH group, no statistically significant difference was observed.

[0202] like Figure 18 As shown in Figure D, the results showed that 4 days after modeling, the average mNSS neurological function scores were: NC group (0.0±0.0), SHAM group (0.0±0.0), IVH group (8.0±1.5), AL group (6.0±1.8), AH group (6.1±1.7), BL group (7.2±1.8), and BH group (7.0±1.8). There were no statistically significant differences between the BL and BH groups and the IVH group, while the remaining groups showed statistically significant differences compared with the model group (P<0.001, ***).

[0203] like Figure 18 As shown in Figure E, the results indicated that 7 days after modeling, the average mNSS neurological function scores were: NC group (0.0±0.0), SHAM group (0.0±0.0), IVH group (5.4±1.7), AL group (2.9±0.9), AH group (2.8±1.0), BL group (4.5±1.8), and BH group (4.1±1.6). There were no statistically significant differences between the BL and BH groups and the IVH group, while all other groups showed statistically significant differences compared to the model group (P<0.001, ***). Figure 1 As shown in E.

[0204] The results above indicate that different doses of drug A significantly improved the mNSS neurological function scores of IVH-induced rats from day 2 after modeling, demonstrating the excellent therapeutic effect of drug A on IVH-induced rats. However, while drug B showed a slight decrease in mNSS scores compared to the IVH group from day 2, the differences were not statistically significant (P>0.05), indicating that drug B was less effective in treating IVH-induced rats.

[0205] To further investigate the therapeutic effect of drug A on IVH-induced rats, we conducted research from multiple perspectives.

[0206] Figure 19These are the results of an Evans blue staining assay to detect blood-brain barrier (BBB) ​​permeability. As can be seen, after Evans blue staining, the Evans blue content in the brain tissue of the IVH group rats was significantly higher (P<0.001, ***) than that in the NC and SHAM groups. The Evans blue content in the brain tissue of rats in the low-dose (AL) and high-dose (AH) drug A groups was significantly lower than that in the IVH group rats (P<0.05, *; P<0.01, **). This result indicates that IVH modeling leads to a significant increase in blood-brain barrier permeability in rats, and both low-dose and high-dose drug A administration can significantly improve the increased blood-brain barrier permeability caused by IVH modeling in rats.

[0207] Cerebral edema induced by IVH modeling is a significant cause of increased intracranial pressure, brain herniation, and death in animals. The wet-dry weight method can be used to assess the degree of cerebral edema in rat brain tissue. Figure 20 These are the results of an experiment measuring the water content of rat brain tissue using the wet-dry weight method. Analysis revealed that the brain tissue water content of rats in the NC group and SHAM group was significantly lower than that of rats in the IVH model (P<0.001, ***). The brain tissue water content of rats in the low-dose (AL) and high-dose (AH) drug A groups was also significantly lower than that of the IVH group (P<0.05, *; P<0.01, **). These results indicate that both low-dose and high-dose administration of drug A can significantly improve cerebral edema induced by IVH modeling, thereby improving the animals' prognosis.

[0208] Figure 21 These are representative images from the H&E staining assay. The results show that in the NC group, some gaps were visible around the blood vessels and cells. Figure 21 (A portion of the image), the SHAM group showed mild damage (A portion of the image). Figure 21 In the B-mode (IVH) group, significantly enlarged spaces around blood vessels and cells in the brain tissue were observed, along with cell degeneration and vacuolation, indicating severe brain damage. Figure 21 The C-range), while the low-dose administration of drug A (AL) ( Figure 21 D-amplitude) and high-dose administration (AH) Figure 21 Both the E-amplitude and the E-amplitude of the brain significantly reduced brain damage in IVH-induced rats.

[0209] Brain injury leads to the expression of damage-inducible nitric oxide synthase (iNOS). Figure 22 This is a typical image of iNOS immunofluorescence staining of rat brain tissue. It can be seen that the IVH group ( Figure 22 The C group showed higher iNOS positive expression, while the other groups, including the NC group, showed lower expression. Figure 22 A-frame), SHAM group ( Figure 22 (B group), AL group ( Figure 22 (D-group) and AH-group ( Figure 22 The expression of iNOS in the brain tissue of rats was lower than that in the IVH group to varying degrees, indicating that IVH modeling increased iNOS expression in rat brain tissue, while both low and high concentrations of drug A could reduce iNOS expression and have a protective effect against brain injury.

[0210] Gelatinase B (MMP9) is not expressed or is expressed at low levels in normal brain tissue, but after brain damage caused by IVH modeling, MMP9 plays an important role in vasogenic cerebral edema and other brain injury-related processes by disrupting the blood-brain barrier. Figure 23 The expression of MMP9 protein in the brain tissue of rats in each group, among which Figure 23 Image A represents the MMP9 protein bands from six parallel samples obtained by Western blotting. Figure 23 The B-square represents the relative protein expression level of MMP9 (with GAPDH as a reference). It can be seen that the relative expression levels of MMP9 in the brain tissue of rats in the NC, SHAM, AL, and AH groups were significantly lower than those in the IVH group, and the differences were statistically significant (P<0.05, * relative to the IVH group). These results indicate that both low and high doses of drug A can significantly reduce the increase in MMP9 protein expression caused by brain injury in model rats, suggesting that drug A has a protective effect against brain injury.

[0211] Malondialdehyde (MDA) is the end product of the peroxidation reaction between free radicals and unsaturated fatty acids in cell membranes. It is an important biomarker of lipid oxidative damage and can indirectly reflect the degree of tissue peroxidative damage. Its level is closely related to the severity of clinical symptoms in the pathogenesis of stroke and has important clinical significance in the diagnosis, treatment and prognosis of stroke. Figure 24 The expression levels of MDA in the brain tissue of rats in each group were shown. It can be seen that the MDA expression in the NC, SHAM, AL, and AH groups was significantly lower than that in the IVH group, indicating that brain injury caused by IVH modeling led to a significant increase in MDA expression in the brain (NC and SHAM groups vs. IVH group, P < 0.001, ***). Both low and high concentrations of drug A significantly reduced brain injury-related MDA expression (vs. IVH group, P < 0.01, ** and 0.001, ***, respectively).

[0212] Superoxide dismutase (SOD) is the most effective free radical scavenging enzyme in the body, and the amount of SOD produced in the body can reflect the status of free radicals. Studies have shown that the expression and activity of SOD in brain tissue of hemorrhagic stroke patients are significantly lower than those in normal brain tissue. Figure 25The expression levels of superoxide dismutase (SOD) in the brain tissue of rats in each group were analyzed. The results showed that the SOD expression levels in the NC, SHAM, AL, and AH groups were significantly higher than those in the IVH group, indicating that brain damage caused by IVH modeling led to a significant decrease in SOD expression in the brain (NC and SHAM groups vs. IVH group, P < 0.001, ***). Both low-dose and high-dose administration of drug A significantly increased SOD expression in the brain (vs. IVH group, P < 0.001, ***).

[0213] Figure 26 The results of transmission electron microscopy examination of ultrathin sections of brain tissue are shown.

[0214] In the NC group, glial cells had normal or slightly irregular morphology, with irregularly elliptical nuclei (N); mitochondria (M) in the cytoplasm were short and round or short rod-shaped, structurally intact, with slightly blurred internal crests, and the endoplasmic reticulum (ER) was slightly dilated. Figure 26 (Image A).

[0215] In the SHAM group, glial cells exhibited slightly irregular morphology, with oval nuclei (N); mitochondria (M) in the cytoplasm were dumbbell-shaped with blurred inner crests, and the endoplasmic reticulum (ER) was slightly dilated. Figure 26 (B version).

[0216] In the IVH group, glial cells exhibited irregular morphology, with irregularly shaped nuclei (N); intracytoplasmic mitochondria (M) were round and significantly swollen, the endoplasmic reticulum (ER) was markedly expanded, and autophagosomes (AP) were visible. Figure 26 (C-axis). This indicates that IVH modeling causes severe damage to brain tissue cells.

[0217] In the AL group, glial cells showed slightly irregular morphology, with oval nuclei (N); mitochondria (M) in the cytoplasm were round or oval, slightly swollen, with some internal crura broken; the endoplasmic reticulum (ER) was slightly dilated, and a small amount of lipofuscin (L) was visible. Figure 26 (D amplitude). This indicates that low-dose administration of drug A can effectively improve or repair cell damage in brain tissue caused by IVH modeling.

[0218] In the AH group, glial cells were generally regular or slightly irregular in morphology, with oval nuclei (N); mitochondria (M) in the cytoplasm were oval or rod-shaped, with clear internal cristae and intact structure; the endoplasmic reticulum (ER) was slightly dilated, and some showed a small amount of lipofuscin (L). Figure 26 (E amplitude). This indicates that high-dose administration of drug A can effectively improve or repair cellular damage to brain tissue caused by IVH modeling, with almost no difference in brain tissue cells compared to the NC group.

[0219] The above results demonstrate that gold clusters with L-NIBC as ligands have unexpected therapeutic effects on hemorrhagic stroke and can be used in the development of drugs for the treatment of hemorrhagic stroke.

[0220] Industrial applicability

[0221] Ligand-bound gold clusters can be used to treat stroke. They are also suitable for industrial applications.

[0222] References

[0223] Amani H, Mostafavi E, Mahmoud Reza Alebouyeh MR, Arzaghi H, Akbarzadeh A, Pazoki-ToroudiH, Webster TJ. Would Colloidal Gold Nanocarriers Present An Effective Diagnosis Or Treatment For Ischemic Stroke? Int J Nanomedicine.2019Oct 7;14:8013-8031.

[0224] Zheng Y, Wu Y, Liu Y, Guo Z, Bai T, Zhou P, Wu J, Yang Q, Liu Z, LuX. Intrinsic Effects of Gold Nanoparticles on Oxygen-Glucose Deprivation / Reperfusion Injury in Rat Cortical Neurons. Neurochem Res. 2019 Jul;44(7):1549-156.

Claims

1. Use of ligand-bound gold clusters for the preparation of a medicament for the treatment of a stroke in a patient, characterized in that, The ligand-bound gold cluster comprises: a gold core; and a ligand bound to the gold core; the ligand is one selected from the group consisting of L-cysteine, N-isobutyryl-D-cysteine (D-NIBC), L(D)-cysteine-L(D)-arginine dipeptide (CR), L(D)-arginine-L(D)-cysteine dipeptide (RC), cysteine-aspartate-glutamate-valine-aspartate (CDEVD), aspartate-glutamate-valine-aspartate-cysteine (DEVDC), N-isobutyryl-L-cysteine (L-NIBC), N-acetyl-L-cysteine (L-NAC), L(D)-glutathione (GSH), 1-[[(2S)-2-methyl-3-thiol-1-oxopropyl]-L(D)proline, 2-aminoethanethiol, 3-mercaptopropionic acid and 4-mercaptobenzoic acid; the diameter of the gold core is 0.5-3 nm; wherein the stroke includes a cerebral hemorrhagic stroke or a cerebral ischemic stroke.

2. The use for the production according to claim 1, characterized in that, the diameter of the gold core is 0.5-2.6 nm. wherein the stroke includes a cerebral hemorrhagic stroke or a cerebral ischemic stroke.

Images