HK1250489B - Substances containing gold clusters and preparation method and use thereof - Google Patents

Description

A substance containing gold clusters, its preparation method and application

Technical Field

This invention relates to the field of nanomedicine technology, and in particular to a gold-containing cluster substance, its preparation method, and its application.

Background Technology

Neurodegenerative diseases are a major threat to human health. Their common pathological features include abnormal protein tangles and amyloid fibrosis within nerve cells, along with related neuronal apoptosis and neurological dysfunction. Alzheimer's disease (AD) and Parkinson's disease (PD) are two of the most typical examples. AD is characterized by memory and cognitive impairment, as well as personality and behavioral changes, while PD primarily presents with motor dysfunction such as resting tremor, bradykinesia, rigidity, and postural and gait disturbances. Both AD and PD mainly affect the elderly, and their incidence increases with age. For example, the incidence of AD is 5% in people over 65 years of age, and over 30% in those over 80 years of age. Therefore, with increasing life expectancy and a growing aging population, the number of people suffering from these two diseases continues to rise. AD, in particular, has over 40 million patients to date, and this number is projected to reach 150 million by 2050. In the United States alone, the cost of caring for AD patients exceeds $200 billion annually, twice that of cancer, making it the most expensive disease in the world. Conservative estimates suggest that the number of PD patients worldwide exceeds ten million. However, the causes of these two diseases remain unknown. In terms of clinical treatment, although several drugs have been approved by the US FDA for the treatment of mild to moderate Alzheimer's disease (AD) or Parkinson's disease (PD), these drugs are all neurotransmitter modulators, which can only temporarily improve patients' cognitive or motor functions. The effects quickly rebound after discontinuation, and currently, no drug can stop or reverse the pathological progression. Therefore, developing novel AD or PD treatments is of great significance.

Studies have found that amyloid fibrillation proteins in the brains of Alzheimer's disease (AD) patients are mainly composed of β-amyloid (Aβ) and Tau protein, with a small amount of α-synuclein (α-syn). The initial site of lesion is the hippocampus, which is responsible for memory, learning, and spatial orientation. In contrast, brain damage in patients with progressive disease (PD) begins in the substantia nigra of the midbrain, which is responsible for motor function. This difference in the initial site of lesion determines the different symptoms of the two diseases. However, research shows that more than half of AD patients develop motor dysfunction in the later stages, while most PD patients also develop AD symptoms in the later stages, indicating an intrinsic correlation between the pathogenesis and disease progression of the two diseases.

The formation of senile plaques in the brain is one of the fundamental pathological features of Alzheimer's disease (AD). Aβ, a major component of senile plaques, is a polypeptide composed of 36-43 amino acids and is a hydrolysis product of fibrotic precursor protein (APP). Aβ(1-40) accounts for over 90% of the total Aβ content. Current research has clarified that although Aβ has normal physiological functions, regulating acetylcholinesterase catalytic activity to control acetylcholinesterase-mediated synaptic signal transmission, excessive accumulation and fibrosis of Aβ in the brain can cause synaptic dysfunction and subsequent secondary inflammatory responses, leading to neuronal loss and death. Therefore, developing substances that can inhibit Aβ accumulation and fibrosis and block its neurotoxicity is one of the important strategies in AD drug development.

The pathological features of Parkinson's disease (PD) are primarily characterized by the progressive loss of dopaminergic (DA) neurons in the substantia nigra-striatal system, accompanied by the formation of Lewy bodies. Lewy bodies are mainly composed of hollow, radially arranged amyloid fibers formed by the degeneration and aggregation of α-synuclein. α-synuclein is located at the presynaptic membrane terminals of neurons; in its natural state in vivo, it is soluble and unfolded. Under pathological conditions, it misfolds, producing β-sheet structures, which then aggregate and fibrose to form the pathological structure of Lewy bodies. Studies have shown that α-synuclein amyloidosis plays a crucial role in the pathological process of this disease. Therefore, inhibiting the aggregation and fibrosis of α-synuclein has become one of the strategies in the development of drugs for the prevention and treatment of PD. On the other hand, 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is a neurotoxin. While not toxic in itself, when it enters the brain, the 1-methyl-4-phenylpyridine cation (MPP ⁺ ) produced by its metabolism can destroy dorsal neuropathy (DA) neurons in the substantia nigra. Simultaneously, MPP ⁺ can interfere with NADH dehydrogenase, an important substance in the respiratory chain of mitochondrial metabolism, leading to cell death and the accumulation of free radicals. The resulting massive death of DA neurons severely affects the cerebral cortex's control of movement, leading to symptoms similar to Parkinson's disease (PD). Therefore, MPTP and MPP ⁺ are widely used in the establishment of PD-related animal and cell models, as well as in PD drug development.

Gold nanoparticles are nanoscale gold particles (the gold core diameter of the gold nanoparticles used in research is typically greater than 3 nm). Due to their unique optical and electrical properties, good biocompatibility, and ease of surface modification, they are widely used in biological and medical fields such as biosensors, medical imaging, and tumor detection. Because of their chemical inertness, large specific surface area, and ability to penetrate the blood-brain barrier at low concentrations, gold nanoparticles are also used as drug carriers in research on targeted drug delivery and controlled drug release. In recent years, studies have shown that combining gold nanoparticles with specific ligands (such as heteropoly acids and polypeptides with specific sequences) that inhibit the aggregation of fibrotic proteins has yielded certain results in in vitro experiments inhibiting protein fibrosis (Y.H. Liao, Y.J. Chang, Y. Yoshiike, Y.C. Chang, Y.R. Chen, Small 2012, 8, 3631; Y.D. Alvarez, J.A. Fauerbach, J.V. Pellegrotti, T.M. Jovin, E.A. Jares-Erijman, F.D. Stefani, Nano Letters 2013, 13). (See *Colloids and Surfaces B: Biointerfaces*, 2013, 112, 525). However, cell model results indicate that while gold nanoparticles (gold core size greater than 5 nm) have a synergistic effect on improving cell survival when used in combination with compounds that protect against fibrotic protein-damaged cells (N. Gao, H. Sun, K. Dong, J. Ren, X. Qu, *Chemistry-A European Journal*, 2015, 21, 829), their effect is not significant when used alone. No experiments at the AD animal model level have been reported. Furthermore, in these studies, gold nanoparticles were primarily used as drug carriers rather than as the active ingredient.

Gold clusters are ultra-micro gold nanoparticles with a gold core diameter of less than 3 nm. Containing only a few to hundreds of gold atoms, the face-centered cubic packing structure of gold atoms in conventional gold nanoparticles collapses, causing energy level splitting. This results in molecular-like properties that are completely different from those of conventional gold nanoparticles larger than 3 nm. On the one hand, due to energy level splitting, the gold clusters do not possess the surface plasmon effect and derived optical properties of conventional gold nanoparticles, but exhibit excellent fluorescence emission properties similar to semiconductor quantum dots. On the other hand, the plasmon resonance peak at 520±20 nm disappears in the UV-Vis absorption spectrum of the gold clusters, while one or more new absorption peaks appear above 560 nm. These absorption peaks are not observed in conventional gold nanoparticles. Therefore, the disappearance of the plasmon resonance absorption peak (520±20 nm) and the appearance of new absorption peaks above 560 nm in the UV-Vis absorption spectrum are important indicators for judging whether the gold clusters have been successfully prepared (H.F.Qian, M.Z.Zhu, Z.K.Wu, R.C.Jin, Accounts of Chemical Research 2012, 45, 1470). Gold clusters also possess magnetic, electrical, catalytic, and photothermal properties that are significantly different from conventional gold nanoparticles, thus showing broad application prospects in fields such as single-molecule photoelectric, molecular catalysis, and photothermal conversion.

Furthermore, gold clusters have also found applications in biological probes and medical imaging due to their excellent fluorescence emission properties. For example, Sandeep Verma's group used purine-modified gold clusters as green fluorescent probes for nuclear imaging (J.R. Wallbank, D. Ghazaryan, A. Misra, Y. Cao, J.S. Tu, B.A. Piot, M. Potemski, S. Wiedmann, U. Zeitler, T.L.M. Lane, S.V. Morozov, M.T. Greenaway, L. Evaes, A.K. Geim, V.I. Falko, K.S. Novoselov, A. Mishchenko, ACS Applied Materials & Interfaces 2014, 6, 2185). Such literature utilizes the fluorescence properties of gold clusters without addressing their medicinal activity.

Summary of the Invention

The purpose of this invention is to address the technical deficiencies in the prior art. Firstly, it provides a gold-containing cluster substance with pharmaceutical activity, comprising a gold cluster and an externally coated ligand Y, wherein the ligand Y is selected from L-cysteine-L-arginine dipeptide (CR), L-arginine-L-cysteine dipeptide (RC), L-cysteine-L-histidine dipeptide (CH), L-histidine-L-cysteine dipeptide (HC), L-lysine-L-cysteine-L-proline tripeptide... One or more of the following: peptide (KCP), L-proline-L-cysteine-L-arginine tripeptide (PCR), glycine-L-serine-L-cysteine-L-arginine tetrapeptide (GSCR), glycine-L-cysteine-L-serine-L-arginine tetrapeptide (GCSR), 1-[(2S)-2-methyl-3-mercapto-1-oxopropyl]-L-proline (Cap), D-3-mercaptovaline, and N-(2-mercaptopropionyl)-glycine.

The diameter of the gold core in the gold cluster is less than 3 nm, preferably 0.5-2.6 nm.

Secondly, the present invention provides a method for preparing the above-mentioned substance, comprising the following steps:

(1) Dissolve _HAuCl4 in one of methanol, water, ethanol, n-propanol, or ethyl acetate to prepare a solution A with a concentration of _HAuCl4 of 0.01–0.03 M;

(2) Dissolve ligand Y in a solvent to prepare a solution B with a concentration of 0.01-0.18 M;

(3) Mix solution A from step (1) and solution B from step (2), with a molar ratio of HAuCl ₄ to ligand Y of 1:(0.01-100) (preferably 1:(0.1-10), more preferably 1:(1-10)), stir in an ice bath for 0.1-48h (preferably 0.1-24h, more preferably 0.5-2h), add dropwise 0.025-0.8M NaBH ₄ solution (preferably an aqueous solution of NaBH ₄ , an ethanol solution of NaBH ₄ , or a methanol solution of NaBH ₄ ), and continue stirring in an ice-water bath for 0.1-12h (preferably 0.1-2h, more preferably 1-2h), with a molar ratio of NaBH 4 to ligand Y of 1:(0.01-100) (preferably 1:(0.1-8), more preferably 1:(1-8));

(4) Centrifuge the reaction solution of step (3) at 8000-17500 r/min for 10-100 min to obtain gold cluster precipitates with different average particle sizes; preferably, centrifuge the reaction solution of step (3) with an ultrafiltration tube with a molecular weight cutoff of 3K-30K at 8000-17500 r/min for 10-100 min to obtain gold clusters with different average particle sizes.

(5) Dissolve the gold cluster precipitates with different average particle sizes obtained in step (4) in water and put them into dialysis bags and dialyze them in water at room temperature for 1 to 7 days.

(6) Freeze-dry the gold cluster solution in the dialysis bag for 12-24 hours to obtain a substance containing gold clusters.

The solvent in step (2) is one or more of the following: 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, ethanol, butyl acetate, tributyl methyl ether, isopropyl acetate, dimethyl sulfoxide, ethyl acetate, ethyl formate, isobutyl acetate, methyl acetate, 2-methyl-1-propanol, and propyl acetate.

Thirdly, the present invention provides the application of the above-mentioned gold-containing substances in the preparation of near-infrared fluorescent probes in the fields of catalysts or molecular catalysis, chiral recognition, molecular detection, biomedical detection and imaging.

Fourthly, the present invention provides the use of the above-mentioned gold-containing clusters in the preparation of medicaments for diseases related to the aggregation and fibrosis of Aβ and/or diseases related to the aggregation and fibrosis of α-syn.

Fifthly, the present invention provides the use of the above-mentioned gold-containing clusters in the preparation of drugs for the prevention and treatment of Alzheimer's disease and/or Parkinson's disease.

In a sixth aspect, the present invention provides the use of gold clusters (gold core diameter less than 3 nm) modified by L-cysteine-L-arginine dipeptide (CR), L-arginine-L-cysteine dipeptide (RC) or 1-[(2S)-2-methyl-3-mercapto-1-oxopropyl]-L-proline (Cap) in the preparation of medicaments for diseases related to the aggregation and fibrosis of Aβ and/or diseases related to the aggregation and fibrosis of α-syn.

In a seventh aspect, the present invention provides the use of gold clusters (with a gold core diameter of less than 3 nm) modified by L-cysteine-L-arginine dipeptide (CR), L-arginine-L-cysteine dipeptide (RC) or 1-[(2S)-2-methyl-3-mercapto-1-oxopropyl]-L-proline (Cap) in the preparation of drugs for the prevention and treatment of Alzheimer's disease.

Eighthly, the present invention provides the use of gold clusters (gold core diameter less than 3 nm) modified by L-cysteine-L-arginine dipeptide (CR), L-arginine-L-cysteine dipeptide (RC) or 1-[(2S)-2-methyl-3-mercapto-1-oxopropyl]-L-proline (Cap) in the preparation of drugs for the prevention and treatment of Parkinson's disease.

The gold-containing cluster substance provided by this invention exhibits excellent inhibitory effects on Aβ and α-syn aggregation in in vitro experiments, and demonstrates excellent effects on improving cell viability in Aβ-induced AD and MPP ⁺ -induced PD models. In transgenic mouse models of AD, this gold-containing cluster substance significantly improves the cognitive and behavioral abilities of affected mice, and significantly inhibits the formation of Aβ(1-40) and Aβ(1-42) plaques in the hippocampus and cerebral cortex. In MPTP-induced PD mouse models, this gold-containing cluster substance significantly improves and corrects motor behavioral disorders in MPTP-damaged model mice, enhances the motor behavioral abilities of affected mice, and significantly inhibits MPTP-induced specific apoptosis of dopaminergic neurons in the substantia nigra and striatum. Furthermore, it exhibits good biocompatibility at both the cellular and animal levels. The above results demonstrate that, in addition to affecting the aggregation and fibrillation of fibrin, the gold-containing clusters of this invention can also influence the progression of neurodegenerative diseases at a deeper level, such as the energy metabolism of nerve cells and the signal transduction function related to neurotransmitter metabolism. Therefore, the gold-containing clusters of this invention are of great significance for the development of new drugs for neurodegenerative diseases such as AD and/or PD.

On the other hand, since the ligand molecules themselves did not show an inhibitory effect on Aβ aggregation in in vitro kinetic experiments, and did not improve cell survival in Aβ-damaged AD cell models and MPP ⁺ -damaged PD cell models, this indicates that the efficacy against AD and PD comes from the gold clusters themselves, rather than the ligands. Based on the pharmaceutical activity of the gold clusters themselves, it is hoped that competitive new drugs can be developed.

Attached Figure Description

Figure 1 shows the UV-Vis spectra, transmission electron microscopy images, and particle size distribution of gold nanoparticles modified with ligand L-NIBC of different sizes.

Figure 2 shows the UV-Vis spectra, transmission electron microscopy images, and particle size distribution of gold clusters modified with L-NIBC ligands of different sizes.

Figure 3 shows the infrared spectra of gold clusters modified with L-NIBC ligands of different particle sizes;

Figure 4 shows the AFM morphology of Aβ(1-40) after co-incubation with gold nanoparticles or gold clusters modified with L-NIBC ligands for 48 h.

Figure 5 shows the Aβ fibrillation kinetics curves of gold nanoparticles and gold clusters modified with ligand L-NIBC at different particle sizes and concentrations.

Figure 6 shows the effect of gold nanoparticles or gold clusters with different particle sizes and concentrations, all modified with ligand L-NIBC, on the survival rate of Aβ-induced AD cell model cells.

Figure 7 shows the UV, IR, TEM and particle size distribution of CR-modified gold clusters (CR-AuNCs);

Figure 8 shows the UV, IR, TEM and particle size distribution of RC-modified gold clusters (RC-AuNCs);

Figure 9 shows the UV, IR, transmission electron microscopy and particle size distribution of gold clusters (Cap-AuNCs) modified with ligand 1-[(2S)-2-methyl-3-mercapto-1-oxopropyl]-L-proline (i.e. captopril (Cap)).

Figure 10 shows the UV, IR, TEM and particle size distribution of gold clusters (GSH-AuNCs) modified with ligand GSH.

Figure 11 shows the UV, IR, TEM and particle size distribution of gold clusters modified with ligand D-NIBC (D-NIBC-AuNCs).

Figure 12 shows the inhibitory effects of different ligand-modified gold clusters on Aβ(1-40) aggregation and fibrosis.

Figure 13 is a schematic diagram of the water maze experimental device in Example 5;

Figure 14 shows the effects of gold-containing clusters on cognitive behavior in the APP/PS1 double transgenic C57BL/6 mouse model (day 150 after drug administration).

Figure 15 shows the effect of gold-containing clusters on the expression of Aβ (1-40) in the hippocampus and cerebral cortex of APP/PS1 double transgenic C57BL/6 mouse model (100 days after administration);

Figure 16 shows the effect of gold-containing clusters on the expression of Aβ(1-42) in the hippocampus and cerebral cortex of APP/PS1 double transgenic C57BL/6 mouse model (100 days after drug administration);

Figure 17 shows the effect of gold-containing clusters on the expression of Aβ (1-40) in the hippocampus and cerebral cortex of APP/PS1 double transgenic C57BL/6 mouse model (150 days after drug administration);

Figure 18 shows the effect of gold-containing clusters on the expression of Aβ (1-42) in the hippocampus and cerebral cortex of APP/PS1 double transgenic C57BL/6 mouse model (150 days after drug administration);

Figure 19 shows the effect of gold-containing clusters on the kinetics of α-syn fibrillation;

Figure 20 shows the effect of gold-containing clusters on the survival rate of MPP ⁺ damaged PD cells (SH-sy5y) model cells.

Figure 21 shows the effect of gold-containing clusters on MPP ⁺ -induced apoptosis in a PD (PC12) cell model.

Figure 22 shows the effect of gold-containing clusters on spontaneous activity behavior in mice with MPTP injury model.

Figure 23 shows the effect of gold-containing clusters on the swimming ability of mice in the MPTP injury model.

Figure 24 shows the effect of gold-containing clusters on the rolling behavior of mice in the MPTP injury model.

Figure 25 shows the effect of gold-containing clusters on substantia nigra and striatal dopaminergic neurons in a mouse model of MPTP injury.

Figure 26 shows the effect of substances with different particle sizes and concentrations of gold-containing clusters on the survival rate of SH-sy5y neuroblastoma cells.

Detailed Implementation

In their research on the effect of gold nanoparticles with certain ligands on Aβ aggregation, the inventors discovered that as the diameter of the gold nanoparticle nucleus decreased, the aggregation of Aβ by gold nanoparticles modified with the same ligands on their surfaces changed from promoting to inhibiting. When the particle size was sufficiently small to form gold clusters, complete inhibition of Aβ aggregation could be achieved. Furthermore, it was found that gold clusters also had a complete inhibitory effect on α-syn. In this effect, the inhibitory effect was not exerted by the ligands, but by the gold clusters themselves.

Typically, the gold nanoparticles used in research have a gold core diameter greater than 3 nm. When the gold core diameter is less than 3 nm, it is called a gold cluster. The disappearance of the plasmon resonance absorption peak (520±20 nm) and the appearance of a new absorption peak above 560 nm in the UV-Vis absorption spectrum are indicators of whether the gold cluster has been successfully prepared. Gold clusters cannot exist stably in solution independently of ligands; they bind to thiol-containing ligands through Au-S bonds to form ligand-modified gold clusters (or simply gold clusters).

Existing literature discloses ligand-modified gold clusters such as L-glutathione (GSH), N-acetyl-L(D)-cysteine (L(D)-NAC), and N-isobutyryl-L(D)-cysteine (L(D)-NIBC), etc., and their preparation processes can be found in the literature (H.F. Qian, M.Z. Zhu, Z.K. Wu, R.C. Jin, Accounts of Chemical Research 2012, 45, 1470; C. Gautier, T. Bürgi, Journal of the American Chemical Society 2006, 128, 11079); their applications are mainly concentrated in catalysis, chiral recognition, molecular detection, biosensing, drug delivery, and bioimaging. G.Li,R.C.Jin,Accounts of Chemical Research 2013,46,1749;H.F.Qian,M.Z.Zhu,Z.K.Wu,R.C.Jin,Accounts of Chemical Research2012,45,1470;J.F.Parker,C. A.Fields-Zinna, R.W.Murray, Accounts of Chemical Research 2010,43,1289; S.H.Yau, O.Varnavski, T.Goodson, Accounts of Chemical Research 2013,46,1506).

This invention focuses on the effects of gold clusters on Alzheimer's disease (AD) and/or disease progression (PD), including at least the following: First, using gold clusters of different sizes with different ligands (ligands that do not inhibit Aβ aggregation) as the research object, the invention conducts research on three levels: in vitro experiments inhibiting Aβ aggregation and α-syn aggregation; Aβ-induced AD cell model and MPP ⁺ -induced PD cell model experiments; and AD transgenic mouse model and MPTP-induced PD mouse model experiments. Combined with experiments on the cytotoxicity, acute toxicity in mice, and in vivo distribution in mice, the invention provides ligand-modified gold clusters and discovers their application in the preparation of drugs for treating AD and PD. The results are compared with those of gold nanoparticles, clarifying that gold nanoparticles with a diameter greater than 3 nm are ineffective in this application and cannot be used to prepare drugs for treating AD or PD, while ligand-modified gold clusters can be used to prepare drugs for treating AD and/or PD.

The following specific embodiments illustrate the content of the present invention in more detail and further elaborate the present invention, but these embodiments are by no means intended to limit the present invention.

The purity of the raw materials used in the following examples only needs to reach chemical purity or above, and all of them can be purchased from the market.

Example 1: Preparation of ligand-modified gold clusters

This embodiment describes a method for preparing ligand-modified gold clusters, including the following steps:

(1) Dissolve _HAuCl4 in one of methanol, water, ethanol, n-propanol, or ethyl acetate to prepare solution A, wherein the concentration of _HAuCl4 is 0.01–0.03 M;

(2) Dissolve ligand Y in a solvent to prepare solution B, wherein the concentration of ligand Y is 0.01–0.18 M; ligand Y includes, but is not limited to, L(D)-cysteine and other cysteine derivatives, such as N-isobutyryl-L-cysteine (L-NIBC), N-isobutyryl-D-cysteine (D-NIBC), N-acetyl-L-cysteine, N-acetyl-D-cysteine, etc.; oligopeptides containing cysteine and their derivatives, including, but not limited to, dipeptides, tripeptides, tetrapeptides and other peptides containing cysteine, such as: L-cysteine-L-arginine dipeptide (CR), L-arginine-L-cysteine dipeptide (RC), L-cysteine-L-histidine (CH), glycine-L-cysteine-L-arginine tripeptide (GCR), L-proline-L-cysteine-L-arginine tripeptide (PCR). L-glutathione (GSH), glycine-L-serine-L-cysteine-L-arginine tetrapeptide (GSCR), glycine-L-cysteine-L-serine-L-arginine tetrapeptide (GCSR), etc.; and other thiol-containing compounds, such as 1-[(2S)-2-methyl-3-mercapto-1-oxopropyl]-L-proline, mercaptoacetic acid, mercaptoethanol, thiophenol, D-3-mercaptovaline, dodecanethiol, etc.; 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, ethanol, butyl acetate, tributyl methyl ether, isopropyl acetate, dimethyl sulfoxide, ethyl acetate, ethyl formate, isobutyl acetate, methyl acetate, 2-methyl-1-propanol, propyl acetate, etc.

(3) Mix solution A and solution B so that the molar ratio of HAuCl ₄ to ligand Y is 1:(0.01～100), stir the reaction in an ice bath for 0.1～48h, add 0.025～0.8M of NaBH ₄ solution in water, ethanol or methanol, and continue stirring the reaction in an ice-water bath for 0.1～12h, so that the molar ratio of NaBH ₄ to ligand Y is 1:(0.01～100);

(4) After the reaction is completed, the reaction solution is centrifuged at 8000-17500 r/min for 10-100 min using an ultrafiltration tube with a molecular weight cutoff of 3K-30K. This will yield gold cluster precipitates modified with ligands of different average particle sizes (the specific gradient centrifugation is as described in Example 2 (4). Since the pore size of the filter membrane of the ultrafiltration tube with different molecular weight cutoffs directly determines the size of the gold clusters that can pass through), this step can also be omitted. That is, after step (3), the process can proceed directly to step (5) to obtain gold clusters of different sizes.

(5) Dissolve the gold cluster precipitates with different average particle sizes obtained in step (4) in water and put them into dialysis bags and dialyze them in water at room temperature for 1 to 7 days.

(6) After dialysis, freeze-dry for 12-24 hours to obtain powder or flocculent material, which is the ligand-modified gold cluster.

Upon testing (see Example 2 for specific testing methods), the powder or flocculent material obtained by the above method has a particle size of less than 3 nm (generally distributed in the range of 0.5-2.6 nm), and its ultraviolet-visible absorption spectrum shows one or more absorption peaks above 560 nm, with no obvious absorption peak at 520 nm. Therefore, the obtained powder or flocculent material is identified as gold clusters.

Example 2: Preparation and confirmation of gold clusters modified with different ligands

Taking L-NIBC as an example, this paper details the preparation and confirmation of gold clusters modified with L-NIBC.

(1) Weigh 1.00g of HAuCl ₄ and dissolve it in 100mL of methanol to prepare a solution A with a concentration of 0.03M;

(2) Weigh 0.57g of L-NIBC and dissolve it in 100mL of glacial acetic acid to prepare a solution B with a concentration of 0.03M;

(3) Take 1 mL of solution A and mix it with 0.5 mL, 1 mL, 2 mL, 3 mL, 4 mL and 5 mL of solution B respectively (i.e., the molar ratio of HAuCl ₄ to L-NIBC is 1:0.5, 1:1, 1:2, 1:3, 1:4 and 1:5 respectively). React for 2 h under ice bath stirring. The solution color changes from bright yellow to colorless. Quickly add 1 mL of freshly prepared 0.03M NaBH ₄ aqueous solution (prepared by dissolving 11.3 mg NaBH ₄ in 10 mL of ethanol). After the solution color turns dark brown, continue to react for 30 min. Add 10 mL of acetone to terminate the reaction.

(4) After the reaction, the reaction solution was centrifuged using a gradient centrifugation method to obtain L-NIBC modified gold cluster powders with different particle sizes. The specific method was as follows: After the reaction, the reaction solution was transferred to an ultrafiltration tube with a molecular weight cutoff of 30K and a volume of 50mL. The tube was centrifuged at 10000r/min for 20min. The residue in the inner tube was dissolved in ultrapure water to obtain powder with a particle size of about 2.6nm. Then, the mixed solution in the outer tube was transferred to an ultrafiltration tube with a molecular weight cutoff of 10K and a volume of 50mL. The tube was centrifuged at 13000r/min for 30min. The residue in the inner tube was dissolved in ultrapure water to obtain powder with a particle size of about 1.8nm. Then, the mixed solution in the outer tube was transferred to an ultrafiltration tube with a molecular weight cutoff of 3K and a volume of 50mL. The tube was centrifuged at 17500r/min for 40min. The residue in the inner tube was dissolved in ultrapure water to obtain powder with a particle size of about 1.1nm.

(5) Remove the solvent from the three powder precipitates with different particle sizes obtained by gradient centrifugation, and after drying the crude product with _N2 , dissolve it in 5 mL of ultrapure water, put it into a dialysis bag (molecular weight cutoff of 3 kDa), place it in 2 L of ultrapure water, change the water every other day, dialyze for 7 days, and freeze dry for later use.

The powders (gold clusters with L-NIBC ligands) prepared above were characterized, with gold nanoparticles with the same L-NIBC ligand used as controls. The preparation method of gold nanoparticles with L-NIBC ligands was described in the literature (W. Yan, L. Xu, C. Xu, W. Ma, H. Kuang, L. Wang and N.A. Kotov, Journal of the American Chemical Society 2012, 134, 15114; X. Yuan, B. Zhang, Z. Luo, Q. Yao, D.T. Leong, N. Yan and J. Xie, Angewandte Chemie International Edition 2014, 53, 4623).

1. Morphological observation using transmission electron microscopy

The powder to be tested (the L-NIBC modified gold cluster sample and the gold nanoparticle sample with the same L-NIBC ligand prepared in Example 2) was diluted with ultrapure water to 2 mg/L as a sample. Then, the sample was prepared by the pendant drop method. The specific method was as follows: 5 μL of sample was dropped onto an ultrathin carbon film mesh and allowed to evaporate naturally until the water droplet disappeared. The morphology of the gold cluster was then observed on a JEM-2100F STEM/EDS field emission high-resolution transmission electron microscope.

Transmission electron microscopy (TEM) images of the four gold nanoparticle samples with L-NIBC ligands are shown in Figure 1 (images B, E, H, and K); TEM images of the three gold cluster samples with L-NIBC ligands are shown in Figure 2 (images B, E, and H).

The photographs in Figure 2 show that the L-NIBC-modified gold clusters have uniform particle size and good dispersibility. The average diameters (referring to the gold core diameter) of the L-NIBC-modified gold clusters are 1.1 nm, 1.8 nm, and 2.6 nm, respectively, which are consistent with the results in Figures C, F, and I. In contrast, the gold nanoparticles with L-NIBC as the ligand have larger particle sizes, with average diameters (referring to the gold core diameter) of 3.6 nm, 6.0 nm, 10.1 nm, and 18.2 nm, respectively, which are consistent with the results in Figures C, F, I, and L.

2. Ultraviolet-Visible Absorption Spectrum

The test powder was dissolved in ultrapure water to a concentration of 10 mg· ^L⁻¹ , and its ultraviolet-visible absorption spectrum was measured at room temperature. The scanning range was 190-1100 nm, the sample cell was a standard quartz cuvette with an optical path of 1 cm, and the reference cell contained ultrapure water.

The UV-Vis absorption spectra of the four gold nanoparticle samples with L-NIBC ligands are shown in Figure 1 (A, D, G, J), and the statistical distribution of particle size is shown in Figure 1 (C, F, I, L). The UV-Vis absorption spectra of the three gold cluster samples with L-NIBC ligands are shown in Figure 2 (A, D, G), and the statistical distribution of particle size is shown in Figure 2 (C, F, I).

As shown in Figure 1, due to the surface plasmon effect, gold nanoparticles with L-NIBC ligand exhibit an absorption peak at around 520 nm. The position of the absorption peak is related to the particle size. Specifically, the 3.6 nm UV absorption peak is at 516 nm, the 6.0 nm UV absorption peak is at 517 nm, the 10.1 nm UV absorption peak is at 520 nm, while the 18.2 nm absorption peak is redshifted to 523 nm. None of the four samples exhibit any absorption peaks above 560 nm.

As shown in Figure 2, the surface plasmon resonance absorption peak near 520 nm disappears in the UV absorption spectra of the three gold cluster samples with different particle sizes and L-NIBC ligands in Example 2, while two distinct absorption peaks appear above 560 nm. The positions of the absorption peaks vary slightly with the particle size of the gold clusters. This is because the gold clusters exhibit molecular-like properties due to the collapse of their face-centered cubic structure, resulting in a discontinuous density of states, energy level splitting, the disappearance of the plasmon resonance effect, and the appearance of new absorption peaks in the longer wavelength direction. Therefore, it can be determined that the three powder samples with different particle sizes obtained in Example 2 are all ligand-modified gold clusters.

3. Fourier transform infrared spectroscopy

Infrared spectroscopy was performed on a Bruker VERTEX 80V Fourier transform infrared spectrometer in high-vacuum total reflectance mode for solid powder, with a scanning range of 4000-400 ^cm⁻¹ and 64 scans. Taking the L-NIBC-modified gold cluster sample prepared in Example 2 as an example, the test samples were dried powders of three L-NIBC-modified gold clusters with different particle sizes, while the control sample was pure L-NIBC powder. The results are shown in Figure 3.

Figure 3 shows the infrared spectra of L-NIBC-modified gold clusters of different sizes. Compared with pure L-NIBC (the top curve), the SH stretching vibrations in the 2500-2600 ^cm⁻¹ range of L-NIBC-modified gold clusters of different sizes completely disappeared, while other characteristic peaks of L-NIBC were still observable. This proves that L-NIBC molecules were successfully anchored to the surface of gold clusters via gold-sulfur bonds. The figure also shows that the infrared spectra of ligand-modified gold clusters are independent of their size.

Other gold clusters modified with ligand Y can be prepared using a similar method, with slight adjustments to the solvent of solution B, the ratio of _HAuCl4 to ligand Y, the reaction time, and the amount of _NaBH4 added. For example, when L-cysteine, D-cysteine, N-isobutyryl-L-cysteine (L-NIBC), and N-isobutyryl-D-cysteine (D-NIBC) are used as ligand Y, acetic acid is chosen as the solvent; when dipeptide CR, dipeptide RC, and 1-[(2S)-2-methyl-3-mercapto-1-oxopropyl]-L-proline are used as ligand Y, water is chosen as the solvent, and so on. The remaining steps are similar and will not be described in detail.

The present invention prepared a series of ligand-modified gold clusters according to the above method. The ligands used and the parameters of the preparation process are shown in Table 1.

Table 1. Preparation parameters of gold clusters modified with different ligands in this invention

The samples of each example listed in Table 1 were confirmed using the same method described above. Figures 7-11 show the corresponding ultraviolet spectra (A in Figures 7-11), infrared spectra (B in Figures 7-11), transmission electron microscope images (C in Figures 7-11), and particle size distributions (D in Figures 7-11) of the gold clusters modified with ligands CR, RC, 1-[(2S)-2-methyl-3-mercapto-1-oxopropyl]-L-proline (abbreviation: Cap), GSH, and D-NIBC, respectively.

The results show that the diameters of the gold clusters modified with different ligands obtained in Table 1 are all below 3 nm. The UV spectra also show the disappearance of the peak at 520±20 nm, with absorption peaks appearing above 560 nm, although the position of these absorption peaks varies slightly depending on the ligand and particle size. Simultaneously, the Fourier transform infrared spectroscopy also shows the disappearance of the thiol infrared absorption peak of the ligands (located between the dashed lines in Figures 7-11, B), while other infrared characteristic peaks are retained. This indicates that each ligand molecule was successfully anchored to the surface of the gold clusters, demonstrating that the present invention successfully obtained the gold clusters modified with the ligands listed in Table 1.

Example 3: In vitro Aβ aggregation kinetics experiment

This embodiment verifies the function of ligand-modified gold clusters through in vitro Aβ aggregation kinetics experiments, and compares their effects with those of ligand-modified gold nanoparticles and ligand molecules alone on Aβ aggregation kinetics, demonstrating that the function originates from the gold clusters, not the ligands. The ThT fluorescence labeling method was used to characterize the Aβ(1-40) fibrillation aggregation kinetics.

Thioflavin T (ThT) is a dye specifically designed to stain amyloid fibers. When co-incubated with polypeptide or protein monomers, its fluorescence remains largely unchanged. However, when it encounters amyloid polypeptides or proteins with fibrous structures, it immediately couples with them, and its fluorescence intensity increases exponentially. Due to this characteristic, ThT is widely used as a marker for monitoring polypeptide or protein amyloidosis. The fibrillation process of Aβ(1-40) is also a nucleation-controlled polymerization process. Therefore, the growth curve of Aβ(1-40) fibers measured by ThT fluorescence labeling is mainly divided into three stages: the initiation stage, the growth stage, and the plateau stage. The initiation stage is mainly the stage where Aβ(1-40) undergoes a conformational change, forming misfolds and then aggregating to form nuclei. The growth stage is the stage where Aβ(1-40) monomers accumulate along the fiber axis onto the nucleus or oligomers to form fibers and grow rapidly. The plateau stage is the stage where all Aβ(1-40) molecules have formed mature long fibers, i.e., the fibers no longer grow. ThT fluorescent labeling can be used to conveniently monitor the kinetics of fibrillation and aggregation of Aβ(1-40) molecules.

1) Pretreatment of Aβ(1-40) monomers

Lyophilized amyloid peptide Aβ(1-40) powder (Invitrogen Corp.) was dissolved in hexafluoroisopropanol (HFIP) to obtain an Aβ(1-40) solution with a concentration of 1 g/L. After sealing, the solution was incubated at room temperature for 2-4 hours. Then, the hexafluoroisopropanol was dried in a fume hood using high-purity nitrogen ( _N₂ , 99.9%) at an appropriate gas flow rate (approximately 1 hour). Finally, the dried Aβ(1-40) was dissolved in 200 μL of dimethyl sulfoxide (DMSO), sealed, and stored at -20°C for up to one week. Before use, the DMSO solution of amyloid peptide was diluted with a large amount of phosphate-buffered saline (PBS, 10 mM, pH = 7.4) to a concentration of 20 μM for Aβ(1-40), obtaining an Aβ(1-40) PBS buffer solution. All Aβ(1-40) solutions used in experiments were freshly prepared and used immediately.

2) Sample preparation and testing

Ligand-modified gold clusters and gold nanoparticles were added to 20 μM Aβ(1-40) PBS buffer solution to form gold cluster samples with different concentrations and particle sizes, and corresponding gold nanoparticle samples with different ligand modifications. The ThT fluorescence labeling method was used, and the samples were continuously incubated in 96-well plates at 37°C, with fluorescence intensity monitored every 10 minutes using a microplate reader. The kinetics of Aβ(1-40) aggregation were characterized by changes in ThT fluorescence intensity.

The experimental group used three types of L-NIBC-modified gold clusters with particle sizes of 2.6 nm, 1.8 nm, and 1.1 nm, prepared in Example 2. The control group used four types of L-NIBC-modified gold nanoparticles with particle sizes of 18.2 nm, 10.1 nm, 6.0 nm, and 3.6 nm, as well as L-NIBC molecules not bound to gold clusters or gold nanoparticles. For each particle size of gold clusters or gold nanoparticles, six concentrations were used: 0 ppm (without gold clusters, gold nanoparticles, or L-NIBC, used as a control), 0.1 ppm, 1.0 ppm, 5.0 ppm, 10.0 ppm, and 20.0 ppm. When L-NIBC was used alone, two concentrations were used: 1.0 ppm and 10.0 ppm.

The results are shown in Figures 4 and 5.

Figure 4 shows the AFM morphology of Aβ(1-40) after co-incubation with each experimental group and control group for 48 h. Among them, A is the AFM morphology after Aβ(1-40) alone was incubated for 48 h, B is the AFM morphology after Aβ(1-40) was co-incubated with L-NIBC for 48 h, C and D are the AFM morphology after Aβ(1-40) was co-incubated with gold nanoparticles (modified with L-NIBC) with average particle sizes of 6.0 nm and 3.6 nm for 48 h, respectively, and E is the AFM morphology after Aβ(1-40) was co-incubated with gold clusters (modified with L-NIBC) with an average particle size of 1.8 nm for 48 h.

Figure 5 shows the Aβ(1-40) fibrosis kinetics curves in the presence of different concentrations of L-NIBC (Aβ1-40), Aβ(1-40) fibrosis kinetics curves in the presence of gold nanoparticles with particle sizes of 18.2 nm, 10.1 nm, 6.0 nm, and 3.6 nm at different concentrations, and Aβ(1-40) fibrosis kinetics curves in the presence of gold clusters with particle sizes of 2.6 nm, 1.8 nm, and 1.1 nm at different concentrations, respectively. In Figure 5, A-H, □ represents 0 ppm (i.e., no gold nanoparticles or gold clusters), ○ represents 0.1 ppm, △ represents 1 ppm, ▽ represents 5 ppm, ◇ represents 10 ppm, and ☆ represents 20 ppm of gold nanoparticles or gold clusters incubated with Aβ(1-40) for fibrosis kinetics.

As shown in Figure 4, the control area A is filled with Aβ fibers; area B is also filled with Aβ fibers; although the number of fibers is reduced in area C, longer fibers are still visible; and although long fibers are not visible in area D, a large number of short Aβ fibers are still present. This indicates that L-NIBC has no significant effect on the formation of Aβ(1-40) fibers. Although the addition of small-sized gold nanoparticles modified with L-NIBC can delay the fiberization process of Aβ(1-40), it cannot completely inhibit it, because short fibers will continue to grow into long fibers after a longer period of time. Area E in Figure 4 shows neither long nor short fibers, indicating that the gold clusters modified with L-NIBC can completely inhibit the fiberization process of Aβ(1-40).

Figure 4 shows the qualitative experiment, and Figure 5 shows the quantitative experiment. The results in Figure 5 indicate that the addition of L-NIBC has no significant effect on the fibrosis kinetics of Aβ(1-40) (Figure 5, A). For gold nanoparticles, when the particle diameter is greater than or equal to 10.1 nm, the addition of L-NIBC-modified gold nanoparticles advances both the growth phase and plateau phase of Aβ aggregation kinetics (when the concentration of gold nanoparticles is 20 ppm, the growth phase of Aβ aggregation kinetics is advanced to 12 h, and the plateau phase is advanced to 16 h), indicating that L-NIBC-modified gold nanoparticles can accelerate Aβ aggregation at this time (Figure 5, B and C). However, when the particle size of gold nanoparticles is less than or equal to 6.0 nm (Figure 5, D and E), it can delay the time when Aβ begins to aggregate (when the concentration of L-NIBC-modified gold nanoparticles is 20 ppm, the growth phase of Aβ aggregation kinetics is delayed to 54 h), indicating that gold nanoparticles have a certain inhibitory effect on Aβ aggregation at this time. However, as shown in Figure 5, even at a high concentration (20.0 ppm), the addition of L-NIBC-modified gold nanoparticles could not achieve complete suppression (meaning no growth phase and a completely flat fluorescence curve). On the other hand, after the addition of L-NIBC-modified gold nanoparticles, since the fluorescence emission peak of ThT is located at 515 nm and the plasmon resonance absorption peak of L-NIBC-modified gold nanoparticles is located near 520 nm, the observed decrease in ThT fluorescence intensity is a partial quenching of ThT fluorescence by the plasmon resonance effect of gold nanoparticles, and cannot be attributed to the inhibitory effect of L-NIBC-modified gold nanoparticles on Aβ(1-40) aggregation.

Figure 5 (F-H sections) shows that all L-NIBC-modified gold clusters significantly inhibited Aβ aggregation (delaying the start of the growth phase; when the concentration of L-NIBC-modified gold clusters was 5 ppm, the start of the growth phase of the 20 μM Aβ aggregation kinetics was delayed to after 50 h). Furthermore, when the concentration of L-NIBC-modified gold clusters reached 10 ppm or higher, Aβ aggregation was completely inhibited (no growth phase occurred, and the fluorescence curve was completely flat). The minimum concentration of L-NIBC-modified gold clusters required for complete inhibition is related to the type of ligand and the diameter of the gold clusters. The minimum concentrations required for L-NIBC-modified gold clusters with particle sizes of 1.1 nm, 1.8 nm, and 2.6 nm were 5.0 ppm, 5.0 ppm, and 10.0 ppm, respectively. Furthermore, since the L-NIBC-modified gold clusters do not exhibit plasmon resonance, they do not quench the fluorescence of ThT. Therefore, the observed decrease in fluorescence intensity is entirely due to the inhibitory effect of the L-NIBC-modified gold clusters on Aβ(1-40) aggregation. The quantitative results in Figure 5 are in perfect agreement with the qualitative results in Figure 4.

This experiment shows that when the size of L-NIBC-modified gold nanoparticles is less than or equal to 6.0 nm, they have a certain inhibitory effect on Aβ aggregation and fibrosis, but the effect is limited. L-NIBC-modified gold clusters have the function of completely inhibiting Aβ aggregation and fibrosis. Since the L-NIBC molecule itself cannot affect Aβ aggregation and fibrosis (see Figure 4B and Figure 5A), this function comes from the gold clusters, not from L-NIBC as a ligand. This lays the foundation for the development of drugs for diseases related to Aβ aggregation and fibrosis, and can be classified as a substance containing gold clusters as defined in this invention.

This embodiment also verified the function of other gold clusters modified with different ligands listed in Table 1. For example, A-H in Figure 12 are the inhibitory effects of gold clusters modified with CR, N-acetyl-L-cysteine (L-NAC), GSH, 1-[(2S)-2-methyl-3-mercapto-1-oxopropyl]-L-proline (Cap), D-NIBC, RC, L-cysteine and D-cysteine (all at 10 ppm) on the aggregation and fibrosis of Aβ(1-40). Similar phenomena were observed with gold clusters modified with different ligands, leading to the same conclusion: these ligands themselves do not affect Aβ aggregation and fibrillation; ligand-modified gold nanoparticles larger than 3 nm have limited inhibitory effects on Aβ aggregation and fibrillation, and even larger gold nanoparticles may promote Aβ aggregation and fibrillation; however, ligand-modified gold clusters exhibit excellent inhibitory effects on Aβ aggregation and fibrillation, achieving complete inhibition at concentrations of 5-10 ppm or higher. The minimum concentration required for complete inhibition varies slightly depending on the ligand and the size of the gold cluster particles. These ligand-modified gold clusters are also classified as gold-clustered substances as defined in this invention.

Example 4: Experiment on an Aβ-induced AD cell model

This experiment used cell viability as an indicator, and the results detected by the CCK-8 assay reflected the effect of ligand-modified gold clusters or gold nanoparticles on the toxicity of Aβ(1-40), thus illustrating whether ligand-modified gold clusters or gold nanoparticles have a neuroprotective effect in the pathogenesis of amyloid protein misfolding. The cells used in the experiment were the SH-SY5Y neuroblastoma cell line. The construction of the Aβ-induced AD cell model was carried out according to the description in the literature (R. Liu, H. Barkhordarian, S. Emadi, C.B. Park, M.R. Sierks, Neurobiology of Disease 2005, 20, 74). The specific method was as follows:

1) Take SH-sy5y cells in logarithmic growth phase (cells passaged to the sixth generation), dilute with complete culture medium (MEM medium + 10% FBS + 1% penicillin-streptomycin) to a cell suspension density of 5 × ^10⁴ /mL, and seed 200 μL per well in a 96-well plate. Incubate at 37°C in a 5% _CO₂ incubator. After the cells adhere, add the sample.

2) Add 100 μL of ligand-modified gold cluster samples or ligand-modified gold nanoparticle samples with different particle sizes and concentrations of 0.04 ppm, 0.4 ppm, 4 ppm, 20 ppm, 40 ppm, and 80 ppm, prepared in maintenance medium (MEM medium + 2% FBS + 1% penicillin-streptomycin). After incubation in an incubator for 2 h, add 100 μL of Aβ(1-40) at a concentration of 80 μM and incubate for 24 h. Thus, the final concentrations of ligand-modified gold clusters or ligand-modified gold nanoparticles are 0.01 ppm, 0.1 ppm, 1 ppm, 5 ppm, 10 ppm, and 20 ppm, respectively, while the final concentration of Aβ(1-40) is 20 μM. Simultaneously, a blank control group without SH-sy5y cells, a negative control group containing SH-sy5y cells but without ligand-modified gold clusters or ligand-modified gold nanoparticles and Aβ(1-40), a cell model control group containing SH-sy5y cells and only Aβ(1-40) (final concentration 20 μM), and a ligand control group containing SH-sy5y cells, Aβ(1-40) (final concentration 20 μM), and L-NIBC (final concentration 20 ppm) were set up. After removing the culture medium, 100 μL of maintenance medium containing 10% CCK-8 was added to each well and incubated for 4 h. The absorbance of each well was measured at a wavelength of 450 nm to reflect the pre-protective and therapeutic effects of ligand-modified gold clusters on Aβ(1-40) damage.

Taking the L-NIBC-modified gold clusters of Example 2 as an example, and comparing them with L-NIBC-modified gold nanoparticles, the results are shown in Figure 6.

Figure 6 shows that A-C represent the effects of L-NIBC-modified gold clusters with particle sizes of 1.1 nm, 1.8 nm, and 2.6 nm at different concentrations on cell survival in the Aβ-induced AD cell model; and D-F represent the effects of L-NIBC-modified gold nanoparticles with particle sizes of 3.6 nm, 6.0 nm, and 10.1 nm at different concentrations on cell survival in the Aβ-induced AD cell model.

As shown in Figure 6, the addition of L-NIBC alone had no effect on improving cell survival. L-NIBC-modified gold clusters of different sizes (average sizes of 1.1, 1.8, and 2.6 nm, respectively) at very low concentrations (e.g., 0.1-1 ppm) increased the cell survival rate of the Aβ-induced AD cell model from nearly 60% to nearly 95% (P < 0.05, Figures A-C in Figure 6). L-NIBC-modified gold nanoparticles with an average diameter of 3.6 nm showed some improvement in cell survival rate of the AD cell model with increasing concentration (Figure 6, Figure ...

This embodiment also conducted experiments on gold clusters of different sizes modified with other ligands listed in Table 1, and the results showed that all of them significantly improved the cell survival rate of the Aβ-induced AD cell model. This indicates that, at least at the cell model level, gold clusters modified with different ligands have excellent therapeutic effects on Alzheimer's disease, and can be classified as substances containing gold clusters as defined in this invention, and used for the treatment of Alzheimer's disease.

Example 5: AD transgenic mouse model experiment

Experiment 1:

1) Weigh 1.0g of each of the ligand-modified gold clusters listed in Table 1, dissolve them in 100mL of water, and store the stock solution at 4℃ for later use. Before each use, take a small amount and dilute it with water.

2) One hundred and eighty transgenic mice of the B6/J-Tg(APPswe,PSEN1de9)85Dbo/MmNju strain (purchased from the Institute of Model Animals, Nanjing University) were randomly divided into three groups of 60 mice each: a control group, a low-dose treatment group, and a high-dose treatment group. From 100 days of age, the control group was fed normally every day, the low-dose treatment group was given 200 μL of a 0.5 g/L gold cluster aqueous solution orally once a day, and the high-dose treatment group was given 200 μL of a 2 g/L gold cluster aqueous solution orally once a day.

3) Mice in the control group, low-dose group, and high-dose group were randomly divided into 7 batches. At 140, 160, 180, 200, 230, 260, and 290 days of age, maze tests, open-field tests, and novel object recognition tests were used to study changes in the mice's learning and memory behaviors. The first four batches had 6 mice per group, and the last three batches had 6-8 mice per group (considering the mortality rate during mouse rearing, the same applies below).

4) After each batch of mice underwent behavioral studies, the Aβ content in their blood was measured: blood was collected from the orbital venous plexus, and the content of Aβ and Aβ aggregates was measured using the serum ELISA method.

5) After detecting the Aβ content in the blood of each batch of mice, the distribution of Aβ amyloid deposition in the hippocampus was detected: after blood was collected from the eyeballs and anesthetized, the brain was fixed by cardiac perfusion, the whole brain was taken, sucrose gradient sedimentation was performed, frozen sections were prepared, and the distribution of Aβ amyloid deposition in the hippocampus was detected by immunohistochemistry.

The results show that the ligand-modified gold clusters provided by this invention can significantly improve the cognitive behavior of AD transgenic mice, inhibit the formation of senile plaques in their brains, and inhibit the progression of the disease. They can be used as substances containing gold clusters to combat Alzheimer's disease.

Experiment 2:

1. Weigh 1.0g of each of the ligand-modified gold clusters listed in Table 1, dissolve them in 100mL of water, and store the stock solution at 4℃ for later use. Before each use, take a small amount of the stock solution and dilute it with water to form a gold cluster solution. Prepare the stock solution every two weeks.

2. Ninety transgenic mice of the B6/J-Tg(APPswe,PSEN1de9)85Dbo/MmNju strain (purchased from the Institute of Model Animals, Nanjing University) were randomly divided into three groups: a model control group, a low-dose administration group, and a high-dose administration group, with 30 mice in each group (considering that there is approximately a 30% mortality rate during the rearing of this strain of transgenic mice, the initial number of mice was greater than the number of mice used in later experiments to ensure sufficient mice for later experiments). From 100 days of age, the model control group was fed normally every day, while the low-dose administration group and the high-dose administration group were administered gold cluster solution via intraperitoneal intravenous injection at doses of 5 mg/kg body weight and 20 mg/kg body weight, respectively, every two days.

3. The Morris water maze test was used to assess the cognitive behavior of mice. The Morris water maze test is an experiment that forces laboratory animals to swim and learn to find platforms hidden in the water. It is mainly used to test the learning and memory abilities of laboratory animals in spatial location and orientation perception. It is widely used in the evaluation of Alzheimer's disease drug development and evaluation research. The shorter the latency to find the platform and the more times the mouse crosses the platform after removal, the longer the swimming distance and the longer the time spent in the target quadrant, the better the mouse's memory ability in spatial location and orientation. After 150 days of drug administration, the Morris water maze test was used to assess the behavior of the model mice. The experimental method was based on the literature (C.V. Vorhees, M.T. Williams, Nature Protocols 2006, 1, 848). Details are as follows:

(1) Orientation and Navigation Experiment: The Morris water maze testing system consists of a circular pool and an automatic video recording and analysis system. A camera is connected to a computer above the pool (as shown in Figure 13). The water maze consists of a circular pool with a diameter of 120 cm and a height of 60 cm, and a platform with a diameter of 9 cm. The water level is 0.5 cm above the platform, and the water temperature is maintained at 22 ± 0.5℃. The water is dyed milky white using white pigment. The orientation and navigation experiment was used to measure the learning and memory abilities of mice in the water maze and lasted for 4 days. As shown in Figure 13, the water maze was divided into 4 quadrants in a cross shape along the four directions of East (E), West (W), South (S), and North (N). The platform was placed in the middle of the SW quadrant, and its position remained fixed throughout the experiment. During training, the mice were gently placed in the water with their heads facing the pool wall from a point 1/2 arc in a different quadrant each day. The time it took for the mice to climb onto the hidden platform (platform-seeking latency) or 60 seconds was recorded by the camera tracking system, and the experiment was stopped when the latency reached the platform. After placing the mouse on the platform, allow it to remain there for 30 seconds. If the mouse does not find the platform within 60 seconds (the platform-finding latency is then counted as 60 seconds), the experimenter guides the mouse to climb onto the platform and allows it to remain there for another 30 seconds. After each experiment, remove the mouse and gently dry it. Each animal is trained four times a day, with 15-20 minute intervals between training sessions, for four consecutive days.

(2) Space exploration experiment: After training on the 4th day, the platform was removed on the 5th day. The mouse was gently placed into the water from the midpoint of the NE arc (the farthest point of the platform) facing the pool wall. The mouse's movement trajectory was recorded by a camera within 60 seconds. The software analyzed the number of times the mouse crossed the platform, the time spent in the target quadrant, and the swimming distance in the target quadrant.

4. Immunohistochemical assay to detect the distribution of Aβ(1-40) and Aβ(1-42) amyloid deposits in the hippocampus and cerebral cortex of rats. Pathological extraneuronal deposition of Aβ in the cerebral cortex and hippocampus is a major pathological feature of Alzheimer's disease (AD). Among them, Aβ(1-40) and Aβ(1-42) are important components of senile plaques in the brain, possessing neurotoxicity and leading to progressive cognitive impairment and memory loss. This experiment used immunohistochemistry to detect changes in the formation of Aβ(1-40) and Aβ(1-42) plaques in the hippocampus and cerebral cortex.

The specific methods are as follows: After 100 and 150 days of continuous drug administration, 10-12 mice from each group were used for immunohistochemical detection of the hippocampus and cerebral cortex. Mice administered on day 150 were those that had completed the water maze test. Mice were anesthetized by intraperitoneal injection of 5% chloral hydrate (10 μL/g), their limbs were fixed on the experimental table, and their chests were opened to fully expose the heart. Care was taken not to cut the liver during the chest opening. The left ventricle was first flushed with 50 mL of 0.1 mol/L PBS buffer for 5 min to remove blood, and then perfused and fixed with 0.1 mol/L PBS buffer containing 4% paraformaldehyde for 6 min. After perfusion and fixation, the brain was removed and fixed overnight in 4% paraformaldehyde at 4°C. The tissues were sequentially dehydrated with 10%, 20%, and 30% sucrose solutions and stored at -80°C for later use. Paraffin-embedded masses were prepared based on mouse brain atlases, and sections of the midbrain, hippocampus, and cerebral cortex (8 μm thickness) were prepared for immunohistochemical staining. The procedure is as follows: Frozen sections (8 μm) are incubated at room temperature for 30 min, then fixed with acetone at 4°C for 20 min. The sections are washed three times with PBS (5 min each time), followed by incubation with 3% hydrogen peroxide for 10 min to eliminate intrinsic peroxidase activity. After washing three times with PBS (5 min each time), sections are blocked with 10% normal goat serum at room temperature for 40 min (sections used for Aβ(1-42) immunohistochemistry are incubated with 10% formic acid for 10 min before blocking to restore antigen activity). The serum is discarded, and anti-Aβ(1-40) (ab20068, 1:20 dilution) or anti-Aβ(1-42) working solution (ab12267, 1:200 dilution) is added. The sections are incubated at room temperature for 2 h. The sections are washed three times with PBS (5 min each time). Horseradish enzyme-labeled streptavidin (PBS diluted) secondary antibody working solution is added, and the sections are incubated at room temperature for 1 h. After washing three times with PBS (5 min each time), the DAB blue reaction method was enhanced with nickel sulfate and developed for 10 min. When the positive product turned deep blue and the background was clear, the slides were rinsed three times with distilled water to stop the development. The slides were then counterstained with hematoxylin for 1 min, rinsed thoroughly with tap water, and air-dried in a ventilated area before mounting with neutral resin. The number of Aβ plaques in the entire hippocampus and cerebral cortex was observed and counted under a confocal microscope. Each sample was divided into left and right ventricles, and two slides were used in parallel experiments. The average values were used for statistical analysis. All data were processed using SPSS software (SPSS 21), and t-tests or one-way ANOVA were used. P < 0.05 was considered statistically significant.

Taking the L-NIBC-modified gold clusters with an average size of 1.8 nm from Example 2 as an example, the results of the water maze experiment after 150 days of drug administration are shown in Figure 14. The results showed that on days 1-2 of the positioning and navigation test training, there was no statistically significant difference in platform-finding latency between the model control group and the high- and low-dose drug groups (P>0.05, n=10⁻¹²/group) (Figure 14, section A). As the training time increased, the platform-finding latency of the high-dose drug group was significantly lower than that of the model group on days 3 and 4 (P<0.01 and P<0.05), while the platform-finding latency of the low-dose drug group was lower than that of the model group, but there was no statistically significant difference (P>0.05, see Figure 14, section A). After the mouse positioning and navigation experiment, the platform was removed for a spatial search experiment. The results showed that the number of platform crossings and the swimming distance in the target quadrant were significantly increased in the high-dose drug group compared to the model control group (P<0.05), and the time spent in the target quadrant was also significantly increased (P=0.05). While the number of platform crossings, swimming distance in the target quadrant, and time spent in the target quadrant were also improved in the low-dose administration group compared to the model control group, the results were not statistically significant (P>0.05) (Figures B-D in Figure 14). These results indicate that administration of the gold cluster for 150 days significantly improved the learning and memory abilities of APP/PS1 mice in terms of spatial location and orientation, and this effect was dose-dependent.

The results of immunohistochemical experiments to detect the distribution of amyloid deposits in the hippocampus and cerebral cortex of rats, specifically Aβ(1-40) and Aβ(1-42), are shown in Figures 15-18.

Figure 15 shows typical immunohistochemical sections of Aβ(1-40) plaques in the hippocampus and cerebral cortex of the high-dose group, low-dose group, and model control group after 100 days of drug administration, respectively. Figure 15 shows the statistical results. The experimental results indicate that, compared with the model control group, after 100 days of drug administration, high-dose administration significantly reduced the formation of Aβ(1-40) plaques in the hippocampus of model mice (44.6±12.2%, P<0.05), but had no significant effect on the formation of Aβ(1-40) plaques in the cerebral cortex (P>0.05). Low-dose administration had no significant effect on the formation of Aβ(1-40) plaques in either the hippocampus or cerebral cortex (P>0.05). Figure 16 shows the corresponding results for Aβ(1-42). The results indicate that high-dose administration significantly reduced the formation of Aβ(1-42) plaques in the cerebral cortex (reduced by 61.5 ± 11.4%, P < 0.05), but did not significantly reduce the formation of Aβ(1-42) plaques in the hippocampus (P > 0.05). Low-dose administration had no significant effect on the formation of Aβ(1-42) plaques in either the hippocampus or the cerebral cortex (P > 0.05). These results indicate that after 100 days of administration, the gold clusters showed a significant inhibitory effect on the formation of both Aβ(1-40) and Aβ(1-42) plaques, and this effect was clearly dose-dependent.

With prolonged administration time and increasing mouse age, compared to the model control mice administered for 100 days, the model control mice showed a significant increase in the formation of Aβ(1-40) and Aβ(1-42) plaques in the hippocampus and cerebral cortex after 150 days of administration. Specifically, Aβ(1-40) increased by 57.2±7.2% in the hippocampus (P<0.05) and 49.1±19.6% in the cerebral cortex (P<0.05), while Aβ(1-42) increased by 74.4±7.0% in the hippocampus (P<0.05) and 65±11.1% in the cerebral cortex (P<0.05). This indicates that the impact on memory and cognitive function in model mice may be greater with age. Figures 17A, B, and C show typical immunohistochemical sections of Aβ(1-40) in the hippocampus and cerebral cortex of the high-dose group, low-dose group, and model control group after 150 days of administration, respectively. Figure 17D shows the statistical results. The results showed that the high-dose administration significantly reduced Aβ(1-40) levels in both the hippocampus and cerebral cortex of mice (59.0±11.1% reduction in the hippocampus, P<0.05; 36.4±4.5% reduction in the cerebral cortex, P<0.05). In contrast, the low-dose administration had no significant effect on Aβ(1-40) plaque formation in the hippocampus (P>0.05), but significantly reduced the amount of Aβ(1-40) plaques in the cerebral cortex (26.9±2.1% reduction, P<0.05). This indicates that the gold clusters significantly inhibited the formation of Aβ(1-40) plaques at 150 days, and this effect was dose-dependent. Furthermore, SPSS analysis of the correlation between the number of Aβ(1-40) plaques and the number of times mice crossed platforms in the 150-day water maze experiment revealed a significant negative correlation between the amount of Aβ(1-40) plaques in the hippocampus and cortex and the number of platform crossings (hippocampus: R = -0.848, P < 0.01; cortex: R = -0.802, P < 0.05). This result further supports the correlation between the reduction of Aβ(1-40) plaques in the hippocampus and cortex induced by ginsenoside administration and the improvement in memory and learning ability in mice induced by ginsenoside administration.

Figure 18 shows the results of Aβ(1-42) treatment for 150 days. The results indicate that high-dose administration of the gold cluster significantly inhibited the formation of Aβ(1-42) plaques in the hippocampus and cerebral cortex of mice (hippocampus decreased by 51.1±6.7%, P<0.05; cerebral cortex decreased by 62.8±4.6%, P<0.05). Low-dose administration had no significant effect on the formation of Aβ(1-42) plaques in the hippocampus and cerebral cortex of mice (P>0.05). This suggests that the gold cluster has a significant inhibitory effect on the formation of Aβ(1-42) plaques at 150 days, and this effect is dose-dependent. SPSS correlation analysis revealed a significant negative correlation between the number of Aβ(1-42) plaques in the hippocampus and cortex and the number of times mice crossed platforms (hippocampus: R＝-0.794, P<0.05; cerebral cortex: R＝-0.802, P<0.05). This further supports the correlation between the reduction of Aβ(1-42) plaques in the hippocampus and cerebral cortex induced by gold cluster administration and the improvement of memory and learning ability in mice by gold cluster administration.

In summary, gold clusters significantly improve the cognitive and behavioral abilities of AD model mice and significantly inhibit the formation of Aβ(1-40) and Aβ(1-42) plaques in the hippocampus and cerebral cortex of mice, thereby inhibiting the progression of the disease in affected mice. They can be used as substances containing gold clusters for the prevention and treatment of AD.

Other gold clusters modified with different ligands listed in Table 1 also have similar effects, which will not be elaborated here.

Example 6: In vitro α-syn aggregation kinetics experiment

This embodiment verifies the function of ligand-modified gold clusters through in vitro α-syn aggregation kinetics experiments and compares the effect with that of using ligand molecules alone on α-syn aggregation kinetics, proving that the function comes from the gold clusters rather than from the ligands.

Thioflavin T (ThT) is a dye specifically used to stain amyloid fibers. When co-incubated with peptides or protein monomers, its fluorescence remains largely unchanged. However, when it encounters amyloid peptides or proteins with fibrous structures, it immediately couples with them, resulting in an exponential increase in fluorescence intensity. Therefore, it is widely used as a marker for monitoring peptide or protein amyloid degeneration. This example uses ThT fluorescence labeling to monitor the kinetics of α-syn fibrillation and aggregation in the presence of gold clusters. The specific experimental method is as follows:

Pretreatment of α-syn monomer: Lyophilized α-syn powder (Bachem Corp.) was dissolved in hexafluoroisopropanol (HFIP) to obtain an α-syn solution with a concentration of 1 g/L. After sealing, the solution was incubated at room temperature for 2-4 hours. The hexafluoroisopropanol was then dried under high-purity nitrogen in a fume hood. The dried α-syn was dissolved in 200 μL of dimethyl sulfoxide (DMSO), sealed, and stored at -20°C for up to one week. Before use, the DMSO solution of α-syn was diluted with a large amount of phosphate-buffered saline (PBS, 10 mM, pH = 7.4) to a concentration of 20 μM to obtain an α-syn PBS buffer solution. All α-syn PBS buffer solutions used in the experiments were prepared fresh each time.

Sample preparation and detection: Gold clusters modified with ligands listed in Table 1 at different concentrations were added to 35 μM α-syn PBS buffer solution. The samples were continuously incubated at 37°C in 96-well plates using the ThT fluorescence labeling method. Fluorescence intensity was monitored every 10 minutes using a microplate reader, and the kinetics of α-syn aggregation were characterized by changes in ThT fluorescence intensity. The experimental group used L-NIBC-modified gold clusters with a particle size of 1.8 nm prepared in Example 2 as an example. The ligand control group used L-NIBC molecules that were not bound to the gold clusters. Four concentrations of gold clusters were used: 0 ppm (containing only α-syn, without gold clusters or L-NIBC, serving as a model control group), 1.0 ppm, 5.0 ppm, and 10.0 ppm. Two concentrations of L-NIBC were used when used alone: 1.0 ppm and 10.0 ppm.

The results are shown in Figure 19. The results show that during incubation at 37°C with 35 μM α-syn, the fluorescence intensity of the ThT-labeled α-syn increased rapidly starting from the 48th hour, indicating that α-syn aggregated and fibrillated. This is consistent with the results reported in the literature (V.N. Uversky, J. Li, P. Souillac, I.S. Millett, S. Doniach, R. Jakes, M. Geodert, A.L. Fink, Journal of Biological Chemistry 2002, 277, 11970). The results of the ligand control group showed that L-NIBC alone had no significant effect on the aggregation kinetics of α-syn (Figure 19A). In the experimental group with added gold clusters, at lower concentrations (e.g., 1.0 ppm and 5.0 ppm), the fluorescence intensity of ThT-labeled cells was significantly lower than that of the model control group and ligand control group without gold clusters, and the onset time was significantly delayed (Figure 19B). This indicates that the addition of gold clusters can significantly inhibit the aggregation and fibrillation of α-syn. When the gold cluster concentration reached 10 ppm, the fluorescence intensity of ThT-labeled cells remained near the baseline for 168 hours during the experiment without any increase (Figure 19B), indicating that when the gold cluster concentration is sufficient, it can completely inhibit the aggregation and fibrillation of α-syn.

This embodiment also conducted the experiment on other gold clusters modified with different ligands listed in Table 1. For example, Figures C-J in Figure 19 show the inhibitory effects of gold clusters modified with D-NIBC, CR, RC, 1-[(2S)-2-methyl-3-mercapto-1-oxopropyl]-L-proline (Cap), GSH, N-acetyl-L-cysteine (L-NAC), L-cysteine (L-Cys), and D-cysteine (D-Cys) (all at 10 ppm) on the aggregation and fibrillation of α-syn. Similar phenomena were observed for gold clusters modified with different ligands, and the same conclusion could be drawn: these ligands themselves do not affect the aggregation and fibrillation of α-syn, while the ligand-modified gold clusters have excellent inhibitory effects on the aggregation and fibrillation of α-syn, and complete inhibition can be achieved when the concentration reaches 10 ppm. The minimum concentration required to achieve complete inhibition varies slightly depending on the ligand used. These ligand-modified gold clusters are also classified as gold-containing substances as defined in this invention. Similar effects are observed with other gold clusters listed in Table 1, except that the concentration of gold clusters required to completely inhibit α-syn aggregation and fibrillation differs, and will not be elaborated upon here.

Example 7: MPP ⁺ -induced PD cell (SH-sy5y) model experiment

Experiment 1:

This experiment used cell viability as an indicator, and the results detected by the CCK-8 assay, reflected the toxicity of ligand-modified gold clusters or gold nanoparticles against the commonly used neurotoxin MPP ⁺ -induced SH-sy5y neural cell model in Parkinson's disease, thus illustrating its neuroprotective role in Parkinson's neurodegenerative disease. The MPP ⁺ -induced PD cell model was constructed according to the description in the literature (Cassarino, DS; Fall, CP; Swerdlow, RH; Smith, TS; Halvorsen, EM; Miller, SW; Parks, JP; Parker, WD Jr; Bennett, JP Jr. Elevated reactive oxygen species and antioxidant enzyme activities in animal and cellular models of Parkinson's disease. Biochimica et biophysica acta. 1997. 1362. 77-86). The specific methods were as follows:

1) Take SH-sy5y cells in the logarithmic growth phase, dilute them with complete culture medium to a cell suspension density of 5× ^10⁴ /mL, and seed 200 μL per well in a 96-well plate. Incubate at 37℃ in a 5% _CO₂ incubator. After the cells adhere, add the sample.

2) The first group was treated with 100 μL of ligand-modified gold clusters or gold nanoparticles of different particle sizes and concentrations prepared from maintenance medium as listed in Table 1, with final concentrations of 0.01 ppm, 0.1 ppm, 1 ppm, 5 ppm, 10 ppm, and 20 ppm, respectively. This group was used as the drug administration group. After 2 h of pretreatment with ligand-modified gold clusters or gold nanoparticles, MPP ⁺ (final concentration of 1 mM) was added to both the drug administration group and the cell model control group. Simultaneously, a blank control group without SH-sy5y cells, a negative control group containing SH-sy5y cells but without the addition of gold clusters or gold nanoparticles and MPP ⁺ , a cell control group containing SH-sy5y cells treated with only 1 mM MPP ⁺ , and a cell control group containing SH-sy5y cells treated with 1 mM MPP+ were also established. ^{Simultaneously} with the treatment, a ligand control group containing the corresponding ligand molecules (final concentration of 20 ppm) was added and incubated at 37°C for 24 h. After centrifugation to remove the culture medium, 100 μL of maintenance medium containing 10% CCK-8 was added to each well and incubated for 4 h. The absorbance value of each well was measured at a wavelength of 450 nm to reflect the pre-protective and therapeutic effects of ligand-modified gold clusters on MPP ⁺ damage.

The same experimental procedures were followed for gold clusters and gold nanoparticles modified with different ligands. The results showed that the ligand-modified gold clusters provided by this invention have a neuroprotective effect on Parkinson's disease. This effect is derived from the gold clusters themselves, rather than the ligands, and they can be used as gold cluster-containing substances to combat Parkinson's disease.

Experiment 2:

This experiment used cell viability as an indicator, and the results detected by the CCK-8 assay reflected the toxicity of ligand-modified gold clusters or gold nanoparticles against the commonly used neurotoxin MPP ⁺ -induced SH-sy5y neural cell model in Parkinson's disease, thus illustrating its neuroprotective role in Parkinson's neurodegenerative disease. The MPP ⁺ -induced PD cell model was constructed according to the description in the literature (DSCassarino, CPFall, RHSwerdlow, TSSmith, EMHalvorsen, SWMiller, JPParks, WDJr. Parker, JPJr. Bennett, Biochimica et Biophysica Acta 1997, 1362, 77). The specific methods were as follows:

1) Take SH-sy5y cells in the logarithmic growth phase, dilute them with complete culture medium to a cell suspension density of 5× ^10⁴ /mL, and seed 200 μL per well in a 96-well plate. Incubate at 37℃ in a 5% _CO₂ incubator. After the cells adhere, add the sample.

2) The first group was treated with 100 μL of ligand-modified gold clusters or gold nanoparticles of different particle sizes and concentrations prepared from maintenance medium as listed in Table 1, with final concentrations of 0.01 ppm, 0.1 ppm, 1 ppm, 5 ppm, 10 ppm, and 20 ppm, respectively. This group was used as the drug administration group. After 2 h of pretreatment with ligand-modified gold clusters or gold nanoparticles, MPP ⁺ (final concentration of 1 mM) was added to both the drug administration group and the cell model control group. Simultaneously, a blank control group without SH-sy5y cells, a negative control group containing SH-sy5y cells but without the addition of gold clusters or gold nanoparticles and MPP ⁺ , a cell control group containing SH-sy5y cells treated with only 1 mM MPP ⁺ , and a cell control group containing SH-sy5y cells treated with 1 mM MPP+ were also established. ^{Simultaneously} with the treatment, a ligand control group containing the corresponding ligand molecules (final concentration of 20 ppm) was added and incubated at 37°C for 24 h. After centrifugation to remove the culture medium, 100 μL of maintenance medium containing 10% CCK-8 was added to each well and incubated for 4 h. The absorbance value of each well was measured at a wavelength of 450 nm to reflect the pre-protective and therapeutic effects of ligand-modified gold clusters on MPP ⁺ damage.

The experimental results using L-NIBC-modified gold clusters or gold nanoparticles are shown in Figure 20. The results show that after 24 hours of culture, the cell survival rate of the control group (containing 100 mM gold clusters but without MPP ⁺ treatment) increased to 108.5 ± 7.1% compared to the blank control group (set as 100%) (P < 0.01), indicating that the gold clusters are non-toxic. The cell survival rate of the model control group (containing 1 mM MPP ⁺ but without gold clusters) decreased to 65.1 ± 4.0% (P < 0.01 compared to the blank control group), while the cell survival rate of the ligand control group was 61.5 ± 3.8% (P < 0.01 compared to the blank control group), indicating that the ligand alone did not improve the survival rate of MPP ⁺ -damaged cell models. The cell survival rates of the treatment groups supplemented with 1 ppm, 5 ppm, 10 ppm, and 40 ppm gold clusters increased to 97.9 ± 2.8% (P < 0.01 compared to the model control group), 99.7 ± 4.0% (P < 0.001 compared to the model control group), 95.3 ± 1.7% (P < 0.01 compared to the model control group), and 93.2 ± 0.4% (P < 0.01 compared to the model control group), respectively. This indicates that the ligand-modified gold clusters provided by this invention have a protective effect on nerve cells in Parkinson's disease, and this effect originates from the gold clusters themselves, not from the ligands. On the other hand, the gold nanoparticles with the corresponding ligands had no effect on the survival rate of model cells at any of the three experimental concentrations, indicating that gold nanoparticles cannot be used as drugs for the prevention and treatment of PD.

The gold clusters modified with different ligands listed in Table 1 were subjected to the same experimental procedures and exhibited similar effects, which will not be elaborated upon here. (Note: Add new experimental results here)

Example 8: MPP ⁺ -induced PD cell (PC12) model experiment

This experiment used an MPP ⁺ (100mM)-induced apoptosis model in PC12 cells and combined it with flow cytometry to observe the protective effect of gold clusters against MPP ⁺ -induced cell damage and apoptosis. The specific experimental methods were as follows: a blank control group without MPP ⁺ and gold clusters, an MPP ⁺ model group with only MPP ⁺ , a gold cluster control group with only gold clusters, and an experimental group with both MPP ⁺ and gold clusters. In the experimental groups, PC12 cell suspensions were pre-filled with a solution of L-NIBC-modified gold clusters (final concentration 20ppm) with an average particle size of 1.8nm for half an hour, followed by incubation with MPP ⁺ for 24 hours. Cell growth viability and apoptosis were detected using the Annexin V-FITC/PI apoptosis detection kit (purchased from Roch) and a FACSCalibur flow cytometer. Data were acquired and analyzed using CellQuest Pro.

The experimental results are shown in Figure 21. The results showed that after 24 hours of MPP ⁺ treatment, flow cytometry analysis revealed that the percentage of apoptotic cells in the blank control group (without MPP ⁺⁾ was 23.5±2.8%, and the percentage of apoptotic cells in PC12 cells incubated with 20 ppm gold clusters alone was 28.47±3.2%, showing no significant difference compared to the blank control group, indicating that the gold clusters had no significant cytotoxic effect. However, the percentage of apoptotic cells in the MPP ⁺ model group was 49.5±10.1%, showing a significant increase in apoptotic cells (P<0.001 compared to the blank control group). After PC12 cells were pre-incubated with gold clusters for half an hour before being incubated with MPP ⁺ for 24 hours, the percentage of apoptotic cells decreased to 35.9±2.2%, significantly lower than the MPP ⁺ model group (P<0.05).

The gold clusters with different ligand modifications listed in Table 1 were tested using the same steps and had similar effects, which will not be elaborated here.

The results of Examples 7 and 8 together show that the gold clusters significantly improved the cell survival rate of the MPP ⁺ -induced PD cell model and significantly inhibited cell apoptosis.

Example 9: MPTP-induced PD mouse model experiment

Experiment 1:

Experimental animals: 80 male C57bl/6 mice, 8 weeks old, weighing 25-30g; 3 mice per cage, all kept in an environment with a room temperature of 22-27℃, with a 12h diurnal rhythm, free access to food and water, for 7 days to acclimatize.

MPTP nerve injury mouse model: Mice were randomly divided into four groups of 20 mice each: blank control group, gold cluster normal control group, MPTP model group, and gold cluster treatment group. The MPTP model group and gold cluster treatment group received subcutaneous injections of 20 mg/kg (free base) of MPTP every 2 hours for four times. The normal solvent control group received subcutaneous injections of 20 mg/kg of physiological saline every 2 hours for four times. Eight hours after the last injection, the normal solvent control group and MPTP model group received daily tail vein injections of 10 μL of physiological saline, while the normal solvent control group and gold cluster treatment group received daily intraperitoneal vein injections of 10 μL of physiological saline solution containing ligand-modified gold clusters (concentration of gold clusters 10 g/L) listed in Table 1 for 7 consecutive days. Animals were placed in enclosures with clean bedding and allowed free access to water and food.

Behavioral testing: The roller test requires animals to maintain balance and move continuously on the roller. It is a widely used test for testing motor coordination. The roller diameter is 6cm and the rotation speed is 20rpm. After five adaptations, the interval between each test is 1min. The time it takes for the animal to fall off the roller is recorded. The average value is taken after 5 consecutive tests.

Neurotransmitter assay: After the behavioral experiment, the animals were euthanized, and striatal tissue from mice was collected and stored at -80°C. For the assay, the striatum was treated with a homogenate (0.1M perchloric acid, 0.1mM EDTA-2Na) at 10 μL/mg (striatum), followed by ultrasonic lysis in an ice bath for 30 minutes. After lysis, the tissue was centrifuged at 10000 rpm for 10 minutes, and the supernatant was extracted. The supernatant was filtered through a 0.25 μm filter and injected into an HPLC column. The levels of dopamine (DA) and its metabolites 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) in the striatum were detected using a laboratory-established high-performance liquid chromatography system. Before each assay, the column was maintained with freshly prepared mobile phase for 2 hours. HPLC conditions: flow rate: 1 mL/min; column temperature: 30°C; excitation and absorption wavelengths of the fluorescence detector were 280 nm and 330 nm, respectively.

Assay of tyrosine hydroxylase: After brain tissue was removed, it was fixed in 4wt% PFA + 2wt% sucrose for 4-6 hours, then immersed in 30wt% sucrose solution. After the brain tissue settled, it was embedded in OCT and serially mounted on a cryostat. The tissue was stained using the ABC (Avidibiotin-peroxidase complex) method. Frozen tissue sections of the substantia nigra were removed, TH stained, and developed with bisphenylamine. The tissue was then observed and photographed under a microscope.

The results show that the ligand-modified gold clusters provided by this invention can significantly improve the motor behavior of MPTP-induced PD model mice, increase the number of dopaminergic neurons, and improve the brain level of dopamine neurotransmitter. They can be used as substances containing gold clusters to combat Parkinson's disease.

Experiment 2:

Experimental animals: 80 male C57bl/6 mice, 8 weeks old, weighing 25-30g; 3 mice per cage, all kept in an environment with a room temperature of 22-27℃, with a 12h diurnal rhythm, free access to food and water, for 7 days to acclimatize.

MPTP nerve injury mouse model: Mice were randomly divided into four groups of 20 mice each: a blank control group, a gold cluster control group (divided into low-dose and high-dose groups based on the gold cluster dosage), an MPTP model group, and a gold cluster experimental group (divided into low-dose and high-dose groups based on the gold cluster dosage). The MPTP model group and the gold cluster experimental group received intraperitoneal intravenous injections of 30 mg/kg (free base) MPTP daily for seven consecutive days. The blank control group received subcutaneous injections of 30 mg/kg physiological saline daily for seven consecutive days. The low-dose groups in both the gold cluster control and experimental groups received intraperitoneal intravenous injections of 100 μL of a 1 g/L L-NIBC-modified gold cluster physiological saline solution with an average particle size of 1.8 nm, while the high-dose groups received intraperitoneal intravenous injections of 100 μL of a 4 g/L L-NIBC-modified gold cluster physiological saline solution with an average particle size of 1.8 nm daily for seven consecutive days. Animals were placed in enclosures with clean bedding and had free access to water and food.

1. Behavioral testing:

(1) Spontaneous activity counting experiment: The animal was transferred from the feeding cage to the spontaneous activity detection device. After the animal adapted to the new environment for 5 minutes, its spontaneous activities and changes within 5 minutes were recorded. The strength of its activity ability was measured by the animal's activity distance and movement speed within 5 minutes.

(2) Swimming experiment: Following Donnan's test method (G.A. Donnan, G.L. Willis, S.J. Kaczmarezyk, P. Rowe, Journal of the Neurological Science 1987, 77, 185), the test mice were placed in a Morris water tank with a water depth of 60 cm and a water temperature of 22℃. The distance the animals swam within 10 minutes was recorded, and the swimming time was used to measure their activity level.

(3) Roller test: The animal needs to maintain balance and move continuously on the roller. It is a widely used test to detect motor coordination. The roller diameter is 6cm and the rotation speed is 20rpm. After five adaptations, the interval between each test is 1min. The time it takes to fall off the roller is recorded. The average value is taken after 5 consecutive tests.

2. Immunohistochemical detection of the substantia nigra and striatum: After behavioral testing, 5 mice from each group underwent immunohistochemical detection of the substantia nigra and striatum. After intraperitoneal anesthesia with 1 mL of 0.5% sodium pentobarbital, the blood was flushed with 15 mL of 0.9% saline via the aorta after thoracotomy. The tissue was then perfused and fixed for 1 hour with 100 mL of 0.1 mol/L phosphate-buffered saline (PBS, pH 7.2) containing 4% paraformaldehyde, initially rapidly then slowly. After perfusion and fixation, the brain was harvested and placed in 4% paraformaldehyde. Paraffin-embedded sections were prepared using a reference mouse brain atlas, and coronal sections of the substantia nigra and striatum were prepared. Brain slices were 3 μm thick per slice. The prepared brain slices were used for immunofluorescence, ultrasensitive two-step immunohistochemistry, and other experiments. The immunohistochemical staining procedure is as follows: Incubate with 0.3% hydrogen peroxide methanol solution (1 mL 30% hydrogen peroxide + 80 mL methanol + 19 mL PBS) for 30 min, followed by 0.3% Triton X-100 in PBS for 30 min. Incubate with mouse anti-tyrosine hydroxylase (TH) monoclonal antibody (1:200) or IBa1 (1:250 dilution) for 48 h (4℃). Incubate with biotinylated rabbit anti-mouse secondary antibody (1:500) for 2 h (room temperature). After rapid rinsing with distilled water, develop the DAB blue reaction using nickel sulfate-enhanced ammonium sulfate for 20–30 min. When the positive product turns deep blue and the background is clear, rinse three times with distilled water to terminate the staining. Each step requires three washes with 0.01 mol/L PBS for 10 min each. The primary antibody is diluted with PBS containing 1% bovine serum and 0.3% Triton X-100, while the secondary antibody and ABC complex are diluted with PBS. Patch, dehydrated, clear, neutral resin sealing.

3. Western Blot (WB) Detection of Striatal Proteins: Tyrosine hydroxylase (TH) is a key enzyme in the dopamine (DA) biosynthesis pathway. TH immunohistochemistry can reveal changes in dopaminergic neurons in the substantia nigra and striatum (D. Luo, J. Zhao, Y. Cheng, S.M. Lee, J. Rong, Molecular Neurobiology 2017, DOI:10.1007/s12035-017-0486-6). After behavioral testing, five animals from each group underwent striatal WB detection. The desired brain regions were removed on ice, lysed with RIPA lysis buffer, homogenized, and centrifuged at 12000g for 30 min at 4℃. Proteins were extracted, samples were prepared, and SDS-polyacrylamide gel electrophoresis was performed at 55V-60V for 4.5 h. Semi-dry constant current transfer was then performed at 60mA for approximately 1.5 h. Block with 5% skim milk powder at room temperature for 1 hour; add rabbit anti-TH diluted with TBST (1:300 dilution) and incubate overnight at 4°C; recover the antibody, wash 3 times with TBST for 10 minutes each time; add IRDye R 680RD Goat anti-Rabbit diluted with TBST (1:3000 dilution); wash 3 times with TBST for 10 minutes each time; scan protein signals using a dual-color infrared laser imaging system.

The results of the spontaneous activity counting experiment in mice are shown in Figure 22. Three to five minutes after MPTP administration, mice exhibited changes such as tremors, reduced movement, arched back, hind limbs extended, unsteady gait, tail erection, and hair tufting. Some mice even experienced epileptic-like seizures. These symptoms gradually subsided after approximately 30 to 60 minutes, and mice largely returned to normal after 24 hours. However, with increasing administration frequency, the acute reaction symptoms lessened, but after 24 hours, the reduced movement, unsteady gait, and slowed reaction became increasingly pronounced. After seven consecutive days of MPTP injection, the spontaneous activity distance and movement speed of mice were significantly reduced compared to the control group (P<0.01), exhibiting symptoms of bradykinesia. Gold clusters alone had no significant effect on the spontaneous activity distance and movement speed of normal mice (Figures A and C of Figure 22). In MPTP model mice, combined administration of gold clusters (high dose) significantly increased spontaneous activity distance and movement speed (Figures B and D in Figure 22), indicating that gold clusters significantly improved spontaneous activity in MPTP model mice, and the difference was statistically significant compared with the MPTP model group (spontaneous activity distance: P<0.05; movement speed: P<0.01).

The results of the mouse behavioral swimming experiment are shown in Figure 23. After seven consecutive days of MPTP injection, mice were placed in a water tank for swimming ability testing. The longer the swimming time and the farther the distance, the better the limb coordination. The blank control group and the group treated with ginsenoside alone had no significant effect on swimming time and distance (Figures A and C of Figure 23). Compared with the blank control group, the MPTP model group showed a significantly shorter swimming distance within 10 minutes (P<0.05) and a significantly reduced time spent in the water tank (P<0.05), indicating that MPTP significantly reduces the swimming ability of mice. Compared with the MPTP model group, the group treated with ginsenoside (high-dose administration) in conjunction with MPTP showed an increased swimming distance (P<0.05) and a significantly increased swimming time (P<0.05) (Figures B and D of Figure 23), indicating that ginsenoside has a significant ameliorative effect on MPTP-induced swimming behavioral disorders in mice.

The results of the mouse rolling behavior test are shown in Figure 24. Mice underwent rolling behavior testing 7 days after continuous MPTP injection. In the normal saline control group, the drop latency and drop percentage were 12.1±4.6 min and 33.3±1.5%, respectively (Figures A and C of Figure 24). Compared with the blank control group, the drop latency in the MPTP model group was significantly shortened to 5.5±3.7 min, and the drop percentage was significantly increased to 83.3±3.4%. This indicates that MPTP administration led to decreased motor coordination in mice, unstable grip, and increased drop rate (Figures B and D of Figure 24). Gold clusters alone had no significant effect on the drop latency in mice (Figure A of Figure 24), but under prolonged rolling conditions, the drop percentage was significantly increased (P<0.001 compared to the blank control group), indicating that gold clusters themselves had a certain effect on the rolling behavior of mice (Figure C of Figure 24). However, compared with the MPTP model group, the group receiving both MPTP and gold clusters showed a significantly prolonged drop latency (low-dose administration: P<0.01; high-dose administration: P<0.05) and a significantly decreased drop percentage (both high-dose and low-dose administration: P<0.001), as shown in Figures B and D of Figure 24. This indicates that gold clusters have a role in improving MPTP-induced motor coordination dysfunction.

The results of immunohistochemical staining of the substantia nigra and striatum, and Western blot analysis of the striatum are shown in Figure 25. Compared with the blank control group, the MPTP model group showed a significant reduction in the number of TH immunopositive neurons (i.e., dopaminergic neurons), with residual neurons showing shrinkage, reduced or absent processes, and decreased density of TH immunopositive cells and nerve fibers in the striatum. Western blot analysis showed that the number of striatal dopaminergic neurons decreased to 55.8 ± 5.6% (with the blank control group as 100%) (P < 0.01 for the blank control group, see Figure 25, section C). Gold-containing compounds alone had no significant effect on the density of TH immunopositive cells and nerve fibers in the substantia nigra and striatum (Figure 25, sections A and B). When gold clusters were administered in combination with MPTP, the effect of MPTP in downregulating the expression of TH in substantia nigra and striatal cells and nerve fibers was significantly inhibited. Western blot analysis showed that the proportion of striatal dopaminergic neurons was 65.6±6.3% of the control group when gold clusters were used at low doses (P<0.01 for the MPTP model group, see Figure 25, section C). When gold clusters were used at high doses, the proportion of striatal dopaminergic neurons reached 84.7±4.5% of the control group (P<0.001 for the MPTP model group). The results indicate that gold clusters have a significant anti-MPTP cytotoxic effect and a significant protective effect against the specific loss of dopaminergic neurons in substantia nigra and striatum.

The gold clusters with different ligand modifications listed in Table 1 were tested using the same method and showed similar effects, which will not be elaborated here.

The above results indicate that the ligand-modified gold clusters provided by this invention can significantly improve the spontaneous activity, motor ability and body coordination of MPTP-induced PD model mice, and have a significant protective effect against the specific loss of dopaminergic neurons in the substantia nigra and striatum, suggesting that substances containing gold clusters can be used to combat PD.

Example 10: Biosafety Evaluation

1. The SH-sy5y cell line was used to evaluate the biosafety of gold-containing clusters at the cellular level.

The specific method is as follows: SH-sy5y cells in the logarithmic growth phase (cells passaged to the sixth generation) were collected. The cell suspension concentration was adjusted, and 100 μL was added to each well. The cells were seeded to adjust the density to 1000-10000 wells. The cell culture plate (a 96-well flat-bottomed plate with the edge wells filled with cell culture medium) was placed in a cell incubator and incubated at 37°C with 5% _CO2 for 24 hours to allow the cells to adhere. The 96-well plate was removed, sterilized with alcohol, and placed in a biosafety cabinet. The original cell culture medium was aspirated, and gold cluster solutions with ligands modified as listed in Table 1 were added, diluted with cell culture medium to 1 ppm, 10 ppm, 50 ppm, 100 ppm, 200 ppm, and 500 ppm, respectively. The control group (without gold clusters) was treated with an equal volume of fresh cell culture medium. The plates were then placed in a cell incubator and incubated for another 48 hours. Each experimental and control group had 6 replicates. After 48 hours of incubation, centrifuge to remove the culture medium, wash 2-3 times with PBS, add 100 μL of fresh culture medium and 20 μL of thiazolyl blue (MTT) solution (5 mg/ml, i.e., 0.5% MTT) to each well, continue incubation for another 4 hours, then terminate the culture. Remove the 96-well plate, centrifuge (1000 rpm) for 10 minutes, aspirate the supernatant, add 200 μL of DMSO to each well, and shake on a shaker at low speed for 10 minutes until the color in the wells is uniform and the crystals are fully dissolved; measure the absorbance of each well at 490 nm using a microplate reader. All the above operations must be performed in a sterile environment. All steps except the detection are completed in a biosafety cabinet. Experimental supplies must be sterilized in a high-temperature steam sterilizer before use.

Taking the L-NIBC-modified gold clusters from Example 2 as an example, the results are shown in Figure 26. A-C represent the effects of gold clusters with particle sizes of 2.6 nm, 1.8 nm, and 1.1 nm, and final concentrations of 1 ppm, 10 ppm, and 50 ppm, and 100 ppm, 200 ppm, and 500 ppm, respectively, on the survival rate of SH-sy5y cells. It can be seen that at relatively high concentrations (e.g., 100 ppm), the addition of L-NIBC-modified gold clusters has almost no effect on cell survival. At even higher concentrations (e.g., 200 and 500 ppm), the addition of L-NIBC-modified gold clusters causes a small degree of cell damage (cell death rate less than 20%). Since 100 ppm is much higher than the effective concentration of the drug (0.1–1 ppm or lower), it can be considered that L-NIBC-modified gold clusters have high safety at the cellular level.

Other ligand-modified gold clusters of different sizes listed in Table 1 also have similar effects, which will not be elaborated here.

2. The acute toxicity of substances containing gold clusters was evaluated using an acute toxicity test in mice.

The specific method is as follows: For the gold clusters with different ligand modifications listed in Table 1 (taking the L-NIBC modified gold cluster with an average diameter of 1.8 nm in Example 2 as an example), 60 adult mice were taken and divided into four groups of 15 mice each, namely the control group and three experimental groups. The control group was fed normally, while the three experimental groups were fed the gold clusters orally by gavage at doses of 0.1 g/kg body weight, 0.3 g/kg body weight, and 1 g/kg body weight per day, respectively, for one week. After stopping the feeding of gold clusters, normal feeding was resumed for 30 days. Abnormal reactions of the mice were observed.

In mouse experiments, the intake of three different concentrations of gold clusters of different sizes had no effect on the survival and activity of the mice. Even with a high dose of 1 g/kg body weight, the mice remained healthy.

Similar results were obtained for other gold clusters modified with different ligands listed in Table 1, which will not be elaborated here. From the above results, we can conclude that gold clusters are very safe.

Example 11: Tissue and metabolic distribution of gold cluster-containing substances in mice

Experiment 1:

Procedure: Eighty mice were randomly divided into four groups of 20 each. They were orally administered gold clusters modified with the ligands listed in Table 1 via gavage at doses of 100 mg/kg, 20 mg/kg, 5 mg/kg, and 1 mg/kg, respectively. After administration, each group of 20 mice was randomly divided into four subgroups of 5 mice each. Mice were sacrificed at 2 h, 6 h, 24 h, and 48 h post-administration. Heart, liver, spleen, lung, kidney, and brain tissues were isolated. Each tissue was weighed, and 2 mL of water was added for homogenization. After homogenization, 2 mL of aqua regia was added and vortexed. The mixture was then shaken for 72 h. Finally, 2 wt% dilute nitric acid solution was added to bring the volume to 10 mL, and the mixture was centrifuged at 15000 rpm for 15 min. 4 mL of the supernatant was collected, and the gold content in the tissue fluid was determined by atomic absorption spectrometry.

The results show that the gold clusters can cross the blood-brain barrier and reach the brain, and can be excreted from the body over time, thus without significant accumulation in the body. Therefore, the gold cluster-containing substance provided by this invention has good prospects for application in the treatment of AD or PD.

Experiment 2:

Procedure: Eighty mice were randomly divided into four groups of 20 mice each. The mice were administered gold clusters modified with the ligands listed in Table 1 via intraperitoneal intravenous injection. The dosage of gold clusters in each group (using L-NIBC-modified gold clusters with an average diameter of 1.8 nm as an example) relative to the mouse body weight was 100 ppm, 20 mg ppm, 5 ppm, and 1 ppm, respectively. After injection, the 20 mice in each group were randomly divided into four subgroups of 5 mice each. Mice were sacrificed at 2 h, 6 h, 24 h, and 48 h after feeding, and heart, liver, spleen, lung, kidney, and brain tissues were isolated. Each tissue was weighed, and 2 mL of water was added for tissue homogenization. After homogenization, 2 mL of aqua regia was added and vortexed. The mixture was then shaken on a shaker for 24 h. Finally, 2 wt% dilute nitric acid solution was added to bring the volume to 5 mL, and the mixture was centrifuged at 15000 rpm for 15 min. 1 mL of the supernatant was collected, and the gold content in the tissue fluid was determined by graphite furnace atomic absorption spectrometry.

The above experimental steps were used to conduct experiments on the other gold clusters with different ligands listed in Table 1.

The results showed that after 2 hours, the gold content in the brain reached 1%-10% of the initial administered concentration. After 6 hours, the brain content remained at a similar level. After 24 hours, the brain content decreased significantly, and after 48 hours, except for the sample with a dose of 100 ppm, the content in all samples decreased to near or below the detection limit. These results indicate that substances containing gold clusters also have good biocompatibility in animals, can cross the blood-brain barrier, and do not accumulate significantly in vivo.

In summary, the experimental results above demonstrate the following points (the terms "gold nanoparticles" and "gold clusters" mentioned below refer to those with ligand modification):

(1) In the in vitro experiment on inhibiting Aβ aggregation (Example 3), it was found that the effect of gold nanoparticles on Aβ aggregation kinetics was size-dependent. When the particle diameter was greater than or equal to 10.1 nm, the addition of gold nanoparticles accelerated Aβ aggregation, while when the particle size was less than or equal to 6.0 nm, it inhibited Aβ aggregation but could not completely inhibit it. However, when gold clusters (average diameter less than 3 nm) were used, all gold clusters could significantly inhibit Aβ aggregation in vitro, and this effect was related to the concentration of gold clusters. When the concentration of gold clusters reached 5-10 ppm, Aβ aggregation could be completely inhibited. The minimum concentration required for complete inhibition was related to the type of ligand and the diameter of the gold cluster. In the in vitro experiment on inhibiting α-syn aggregation (Example 6), it was also found that gold clusters had the same complete inhibitory effect on α-syn aggregation and fibrosis.

(2) In the Aβ-induced AD and MPP ⁺ -induced PD cell models (Examples 4, 7, and 8), it was found that using small-sized gold nanoparticles (such as gold nanoparticles with an average diameter of 3.6 nm or 6.0 nm) had no significant effect on improving the cell survival rate of both Aβ-induced AD and MPP ⁺ -induced PD cell models. This indicates that gold nanoparticles do not exhibit significant pharmacological effects on AD and PD at the cellular level and therefore cannot be directly used as an active ingredient to prepare drugs for treating AD or PD. However, for gold clusters of different sizes modified with different ligands (with an average diameter of less than 3 nm) used in this invention, it was found that even with very low amounts of gold clusters (such as 0.1-1 ppm), the cell survival rate of both models could be increased from 50%-65% to over 95%. This indicates that gold clusters have significant pharmacological effects at the cellular level. Since the ligands used had no effect on Aβ aggregation or on either of the two cell models (Examples 4, 7, and 8), it can be concluded that the efficacy of the gold clusters comes from the clusters themselves, which provides a new approach to the application of gold clusters.

(3) Furthermore, the present invention uses a transgenic mouse model of AD and a PD mouse model induced by MPTP (Example 5, Example 9) to further verify the efficacy of the gold clusters, indicating that the gold clusters used have significant effects on improving the cognitive and motor abilities of mice, inhibiting the formation of senile plaques in the brain and inhibiting the specific apoptosis of MPTP-induced substantia nigra and striatal dopaminergic neurons, and can be used as a drug for the prevention or treatment of related diseases.

(4) In further experiments evaluating biosafety (Example 10), the gold clusters had no significant effect on cell survival when co-cultured with nerve cells at a concentration of 100 ppm by weight. However, cell survival decreased slightly at concentrations exceeding 100 ppm (far greater than the effective concentration). Since the effective concentration of the gold clusters (0.1-1 ppm) is far below 100 ppm, it can be considered that the gold clusters have excellent biosafety at the cellular level. In the acute toxicity experiment in mice, mice administered 1 g/kg body weight (equivalent to 1000 ppm) once daily for seven consecutive days showed no adverse reactions. In the in vivo distribution and pharmacokinetic experiment in mice (Example 11), after 2 hours, the gold content in the brain reached 1%-10% of the initial administered concentration. After 6 hours, the brain content remained at a similar level. After 24 hours, the brain content decreased significantly, and after 48 hours, except for the sample administered at 100 ppm, all samples decreased to below the detection limit. The above results demonstrate that substances containing gold clusters exhibit good biocompatibility in animals, can cross the blood-brain barrier, and do not accumulate significantly in vivo. Therefore, these substances show promising potential for application in the preparation of drugs for treating Alzheimer's disease (AD) or Parkinson's disease (PD).

(5) Compared to existing technologies, the ligands used in this invention are not specifically designed for the aggregation behavior of Aβ and α-syn, and comparative experiments show that the ligands used have no significant effect on the aggregation of Aβ and α-syn (Example 3). However, since the size of the gold clusters is smaller than that of the protein itself, the aggregation of Aβ and α-syn can be significantly inhibited through the binding of the scale effect and weak intermolecular interactions. The excellent results in Aβ-induced AD cell models and transgenic animal models further verify the feasibility of using gold cluster-containing substances in the preparation of drugs for treating AD. Furthermore, the excellent effects of gold-containing clusters in MPP ⁺ -induced PD cell models and MPTP-induced PD animal models indicate that gold-containing clusters also have broad application prospects in the preparation of drugs for treating other neurodegenerative diseases. Moreover, since MPP ⁺ -induced PD cell models and MPTP-induced PD animal models do not involve protein fibrosis, but rather act on deeper mechanisms such as energy metabolism and neurotransmitter metabolism-related signal transduction functions of nerve cells, it can be inferred that gold-containing clusters, in addition to affecting protein fibrosis, can also influence the progression of neurodegenerative diseases at a deeper level, which will be of great significance for the development of new drugs for neurodegenerative diseases.

The above description is only a preferred embodiment of the present invention. It should be noted that, for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims (12)

1. A substance containing gold clusters, characterized in that it comprises gold clusters and an externally coated ligand Y, wherein the ligand Y is L-cysteine-L-arginine dipeptide (CR), L-arginine-L-cysteine dipeptide (RC), L-cysteine-L-histidine dipeptide (CH), L-histidine-L-cysteine dipeptide (HC), L-lysine-L-cysteine-L-proline tripeptide (KCP), L-proline-L-cysteine- One or more of the following: L-arginine tripeptide (PCR), glycine-L-serine-L-cysteine-L-arginine tetrapeptide (GSCR), glycine-L-cysteine-L-serine-L-arginine tetrapeptide (GCSR), 1-[(2S)-2-methyl-3-mercapto-1-oxopropyl]-L-proline (Cap), and N-(2-mercaptopropionyl)-glycine, wherein the diameter of the gold core of the gold cluster is 0.5-2.6 nm. 2. A method for preparing the substance of claim 1, characterized in that it comprises the following steps: (1) Dissolve _HAuCl4 in one of methanol, water, ethanol, n-propanol, or ethyl acetate to prepare a solution A with a concentration of _HAuCl4 of 0.01–0.03 M; (2) Dissolve ligand Y in a solvent to prepare a solution B with a concentration of 0.01–0.18 M; (3) Mix solution A from step (1) and solution B from step (2), with a molar ratio of HAuCl ₄ to ligand Y of 1: (0.01-100), stir in an ice bath for 0.1-48 h, add 0.025-0.8 M NaBH ₄ solution dropwise, and continue stirring in an ice-water bath for 0.1-12 h, with a molar ratio of NaBH ₄ to ligand Y of 1: (0.01-100). (4) Centrifuge the reaction solution from step (3) at 8000-17500 r/min for 10-100 min to obtain gold cluster precipitates with different average particle sizes; (5) Dissolve the gold cluster precipitates with different average particle sizes obtained in step (4) in water and put them into dialysis bags and dialyze them in water at room temperature for 1 to 7 days; (6) Freeze-dry the gold cluster solution in the dialysis bag for 12-24 h to obtain a substance containing gold clusters. 3. The method according to claim 2, wherein the solvent in step (2) 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. 4. The method according to claim 2, wherein in step (3), the molar ratio of HAuCl ₄ to ligand Y is 1:(0.1-10). 5. The method according to claim 4, wherein in step (3), the molar ratio of HAuCl ₄ to ligand Y is 1:(1-10). 6. The method according to claim 2, wherein the _NaBH4 solution in step (3) is an aqueous solution of _NaBH4 , an ethanol solution of _NaBH4 , or a methanol solution of _NaBH4 . 7. The method according to claim 2, wherein in step (3), the molar ratio of _NaBH4 to ligand Y is 1:(1-8). 8. The method according to claim 2, wherein step (4) specifically comprises: centrifuging the reaction solution of step (3) with an ultrafiltration tube having a molecular weight cutoff of 3K to 30K at a gradient of 8000 to 17500 r/min for 10 to 100 min to obtain gold clusters with different average particle sizes. 9. The use of the gold-containing cluster material of claim 1 in the preparation of catalysts. 10. The application according to claim 9, wherein the catalyst is a molecular catalyst. 11. The application of the gold-containing cluster material of claim 1 in the preparation of chiral recognition and molecular detection reagents. 12. The application of the gold-containing cluster material of claim 1 in the preparation of near-infrared fluorescent probes in the field of biomedical detection and imaging.