WO2018024111A1 - 一种含金团簇的物质及其制备方法与应用 - Google Patents

一种含金团簇的物质及其制备方法与应用 Download PDF

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Publication number
WO2018024111A1
WO2018024111A1 PCT/CN2017/093671 CN2017093671W WO2018024111A1 WO 2018024111 A1 WO2018024111 A1 WO 2018024111A1 CN 2017093671 W CN2017093671 W CN 2017093671W WO 2018024111 A1 WO2018024111 A1 WO 2018024111A1
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Prior art keywords
gold
cysteine
cluster
gold cluster
ligand
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PCT/CN2017/093671
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English (en)
French (fr)
Inventor
孙涛垒
Original Assignee
北京深见投资基金管理有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
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Publication date
Priority to EP21218128.3A priority Critical patent/EP4000636A3/en
Application filed by 北京深见投资基金管理有限公司 filed Critical 北京深见投资基金管理有限公司
Priority to DK17836293.5T priority patent/DK3449945T3/da
Priority to JP2018569147A priority patent/JP2019533636A/ja
Priority to US16/084,553 priority patent/US10729718B2/en
Priority to AU2017306328A priority patent/AU2017306328B2/en
Priority to EP17836293.5A priority patent/EP3449945B1/en
Priority to ES17836293T priority patent/ES2908466T3/es
Publication of WO2018024111A1 publication Critical patent/WO2018024111A1/zh
Priority to US16/129,884 priority patent/US11058717B2/en
Priority to US16/396,727 priority patent/US11000543B2/en
Priority to US17/222,310 priority patent/US20210220395A1/en
Priority to US17/222,229 priority patent/US20210220394A1/en
Priority to AU2021211975A priority patent/AU2021211975B2/en

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    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
    • A61K9/1682—Processes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B22—CASTING; POWDER METALLURGY
    • B22F—WORKING METALLIC POWDER; MANUFACTURE OF ARTICLES FROM METALLIC POWDER; MAKING METALLIC POWDER; APPARATUS OR DEVICES SPECIALLY ADAPTED FOR METALLIC POWDER
    • B22F1/00—Metallic powder; Treatment of metallic powder, e.g. to facilitate working or to improve properties
    • B22F1/05—Metallic powder characterised by the size or surface area of the particles
    • B22F1/054—Nanosized particles
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B22—CASTING; POWDER METALLURGY
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    • B22F1/00—Metallic powder; Treatment of metallic powder, e.g. to facilitate working or to improve properties
    • B22F1/05—Metallic powder characterised by the size or surface area of the particles
    • B22F1/054—Nanosized particles
    • B22F1/0545—Dispersions or suspensions of nanosized particles
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B22—CASTING; POWDER METALLURGY
    • B22F—WORKING METALLIC POWDER; MANUFACTURE OF ARTICLES FROM METALLIC POWDER; MAKING METALLIC POWDER; APPARATUS OR DEVICES SPECIALLY ADAPTED FOR METALLIC POWDER
    • B22F1/00—Metallic powder; Treatment of metallic powder, e.g. to facilitate working or to improve properties
    • B22F1/14—Treatment of metallic powder
    • B22F1/145—Chemical treatment, e.g. passivation or decarburisation
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B22—CASTING; POWDER METALLURGY
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    • B22F1/00—Metallic powder; Treatment of metallic powder, e.g. to facilitate working or to improve properties
    • B22F1/14—Treatment of metallic powder
    • B22F1/148—Agglomerating
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B22—CASTING; POWDER METALLURGY
    • B22F—WORKING METALLIC POWDER; MANUFACTURE OF ARTICLES FROM METALLIC POWDER; MAKING METALLIC POWDER; APPARATUS OR DEVICES SPECIALLY ADAPTED FOR METALLIC POWDER
    • B22F2999/00—Aspects linked to processes or compositions used in powder metallurgy
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B22—CASTING; POWDER METALLURGY
    • B22F—WORKING METALLIC POWDER; MANUFACTURE OF ARTICLES FROM METALLIC POWDER; MAKING METALLIC POWDER; APPARATUS OR DEVICES SPECIALLY ADAPTED FOR METALLIC POWDER
    • B22F9/00—Making metallic powder or suspensions thereof
    • B22F9/16—Making metallic powder or suspensions thereof using chemical processes
    • B22F9/18—Making metallic powder or suspensions thereof using chemical processes with reduction of metal compounds
    • B22F9/24—Making metallic powder or suspensions thereof using chemical processes with reduction of metal compounds starting from liquid metal compounds, e.g. solutions
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
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    • C—CHEMISTRY; METALLURGY
    • C01—INORGANIC CHEMISTRY
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    • C01P2004/00—Particle morphology
    • C01P2004/90—Other morphology not specified above

Definitions

  • the invention relates to the field of nano drug technology, in particular to a substance containing gold clusters and a preparation method and application thereof.
  • AD Alzheimer's disease
  • PD Parkinson's disease
  • the clinical manifestations of AD are characterized by memory and cognitive dysfunction as well as personality and behavioral changes, while the clinical manifestations of PD include motor dysfunction such as resting tremor, bradykinesia, myotonia and posture gait disorder.
  • AD and PD mainly occur in the elderly, and the incidence increases with age. For example, AD, the incidence rate is 5% in people over 65 years old, and more than 30% in people over 80 years old.
  • AD has more than 40 million patients to date and is expected to reach 150 million by 2050.
  • the cost of caring for AD patients has exceeded $200 billion annually, twice as much as cancer, making it the most expensive disease in the world.
  • the number of PD patients worldwide is estimated to reach more than 10 million.
  • the etiology of these two types of diseases has not been known to date.
  • drugs have been approved by the US FDA for the treatment of mild and moderate AD or PD, these drugs are all neurotransmitter regulating drugs, which can only temporarily improve the patient's cognition or movement. Function, will rebound soon after stopping the drug, there is no drug to terminate or reverse its pathological process. Therefore, the development of new AD or PD therapeutic drugs is of great significance.
  • amyloid fibrosis protein in the brain of AD patients is mainly composed of ⁇ -amyloid (A ⁇ ) and Tau protein, and also contains a small amount of ⁇ -synuclein ( ⁇ -syn).
  • the hippocampus functioning as a memory learning and spatial positioning function in the brain.
  • the brain damage of PD patients begins with the midbrain substantia nigra responsible for somatic motor function.
  • the difference in the initial site of the disease determines the different symptoms of the patients in both diseases.
  • studies have shown that more than half of patients with AD develop motor dysfunction in the later stages, and most patients with PD also have symptoms of AD in the later stage, indicating an intrinsic correlation between the pathogenesis of the two diseases and the disease process.
  • a ⁇ is a polypeptide consisting of 36-43 amino acids, which is a hydrolysis product of fibrin precursor protein (APP), in which A ⁇ (1-40) accounts for more than 90% of the total amount of A ⁇ .
  • APP fibrin precursor protein
  • the pathological features of PD are mainly characterized by the progressive loss of dopaminergic (DA) neurons in the nigrostriatal system, accompanied by the production of Lewy bodies.
  • the Louise body is mainly composed of hollow radial amyloid fibers in which ⁇ -syn is denatured and aggregated. ⁇ -syn is in the presynaptic membrane of neurons, and its natural state is soluble and unfolded. Under the pathological conditions, it will fold incorrectly, produce ⁇ -sheet structure, and then aggregate fibrosis to form Lewy body lesions. structure. Studies have shown that amyloidosis of ⁇ -syn plays a key role in the pathological process of the disease.
  • MPTP 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine
  • MPP + 1-methyl-4-phenylpyridinium cation
  • MPP + can destroy DA neurons in the substantia nigra
  • MPP + can also interfere with an important substance in the respiratory chain, mitochondrial metabolism, NADH dehydrogenase, which leads 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 similar symptoms of PD. Therefore, MPTP and MPP + are widely used in the establishment of PD-related animal models and cell models, as well as drug development in PD.
  • Gold nanoparticles are gold particles of nanometer size (the gold nuclei used in the study are usually larger than 3 nm in diameter) because of their unique optical and electrical properties, good biocompatibility, and easy surface modification. Widely used in biological and medical related fields such as biosensors, medical imaging and tumor detection. Due to its chemical inertness and large specific surface area, as well as its ability to penetrate the blood-brain barrier at low concentrations, gold nanoparticles are also used as drug carriers for the study of drug-directed transport and controlled drug release.
  • the gold cluster is an ultrafine gold nanoparticle with a gold core diameter of less than 3 nm. It contains only a few to hundreds of gold atoms, causing the face-centered cubic stack structure of the gold atoms in the conventional gold nanoparticles to collapse and the energy level to split, thus showing a completely different color than the conventional gold nanoparticles above 3 nm.
  • the nature of the class of molecules On the one hand, due to energy level splitting, gold clusters do not have the surface plasmon effect and derived optics of conventional gold nanoparticles.
  • the plasmon resonance peak at 520 ⁇ 20 nm disappears in the ultraviolet-visible absorption spectrum of gold clusters, and one or more appears above 560 nm A new absorption peak, which is not observed in conventional gold nanoparticles, so the disappearance of the plasmon resonance absorption peak (520 ⁇ 20 nm) in the UV-visible absorption spectrum and the appearance of a new absorption peak above 560 nm are judged as gold groups. Whether clusters are important signs of success (HFQian, MZZhu, ZKWu, RC Jin, Accounts of Chemical Research 2012, 45, 1470). Gold clusters also have significantly different magnetic, electrical, catalytic, and photothermal effects from conventional gold nanoparticles, and thus have broad application prospects in the fields of single molecule photoelectric, molecular catalysis, and photothermal transition.
  • gold clusters have also been used in bioprobe and medical imaging due to their excellent fluorescence emission properties.
  • the Sandeep Verma team used ⁇ modified gold clusters as green fluorescent probes for nuclear imaging (JRWallbank, D.Ghazaryan, A.Misra, Y.Cao, JSTu, BAPiot, M.Potemski,S .Wiedmann, U. Zeitler, TLMLane, SVMorozov, MT Greenaway, L. Evaes, AK Geim, VIFalko, KSNovoselov, A. Mishchenko, ACS Applied Materials & Interfaces 2014, 6, 2185), which uses gold The fluorescent properties of the cluster, but not its own pharmaceutically active activity.
  • a pharmaceutically active gold-containing cluster-containing material comprising a gold cluster and an externally coated ligand Y thereof.
  • the gold cluster has a gold core diameter of less than 3 nm, preferably 0.5 to 2.6 nm.
  • the ligand Y includes, but is not limited to, one or more of L(D)-cysteine and its derivatives, cysteine-containing oligopeptides and derivatives thereof, and other thiol-containing compounds.
  • the L(D)-cysteine and its derivative are preferably L(D)-cysteine, N-isobutyryl-L(D)-cysteine (L(D)-NIBC) or N-acetyl-L(D)-cysteine (L(D)-NAC) or the like.
  • the cysteine-containing oligopeptide and derivatives thereof are preferably a cysteine-containing dipeptide, a cysteine-containing tripeptide or a cysteine-containing tetrapeptide.
  • the cysteine-containing dipeptide is preferably L-cysteine-L-arginine dipeptide (CR), L-arginine-L-cysteine dipeptide (RC), L-histidine -L-cysteine dipeptide (HC) or L-cysteine-L-histidine dipeptide (CH).
  • CR L-cysteine-L-arginine dipeptide
  • RC L-arginine-L-cysteine dipeptide
  • HC L-histidine -L-cysteine dipeptide
  • CH L-cysteine-L-histidine dipeptide
  • the cysteine-containing tripeptide is preferably glycine-L-cysteine-L-arginine tripeptide (GCR), L-valine-L-cysteine-L-arginine III Peptide (PCR), L-lysine-L-cysteine-L-valine tripeptide (KCP) or L-glutathione (GSH).
  • GCR glycine-L-cysteine-L-arginine tripeptide
  • PCR L-valine-L-cysteine-L-arginine III Peptide
  • KCP L-lysine-L-cysteine-L-valine tripeptide
  • GSH L-glutathione
  • the cysteine-containing tetrapeptide is preferably glycine-L-serine-L-cysteine-L-arginine tetrapeptide (GSCR) or glycine-L-cysteine-L-serine-L- Arginine tetrapeptide (GCSR) and the like.
  • the other mercapto group-containing compound is preferably 1-[(2S)-2-methyl-3-indolyl-1-oxopropyl]-L-proline, mercaptoacetic acid, mercaptoethanol, thiophenol, D- 3-mercaptoproline, N-(2-mercaptopropionyl)-glycine or dodecanol or the like.
  • the substance is a powder or a floc.
  • the present invention provides a method of preparing the above gold-containing cluster-containing material, comprising the steps of:
  • the reaction solution of the step (3) is centrifuged at 8000 to 17500 r/min for 10 to 100 minutes to obtain gold cluster precipitates having different average particle diameters; preferably, the reaction liquid of the step (3) is cut off by molecular weight. 3K ⁇ 30K ultrafiltration tube centrifuged at 8000 ⁇ 17500r / min gradient for 10 ⁇ 100min, you can get gold clusters with different average particle size;
  • the gold clusters of different average particle diameters obtained in the step (4) are dissolved in water and placed in a dialysis bag and dialyzed in water for 1 to 7 days at room temperature;
  • the gold cluster solution in the dialysis bag is freeze-dried for 12 to 24 hours to obtain a gold-containing cluster.
  • the solvent in the step (2) is 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 One or more of methyl-1-propanol and propyl acetate.
  • the present invention provides the use of the above-described gold cluster-containing material in the preparation of a near-infrared fluorescent probe in the fields of catalyst or molecular catalysis, chiral recognition, molecular detection, biomedical detection and imaging.
  • the present invention provides the use of the above gold-containing cluster-containing substance in the preparation of a medicament for diseases associated with aggregation and fibrosis of A ⁇ and or diseases associated with aggregation and fibrosis of ⁇ -syn.
  • the present invention provides the use of the above gold-containing cluster material for the preparation of a medicament for preventing and treating Alzheimer's disease.
  • the present invention provides the use of the above gold-containing cluster material for the preparation of a medicament for preventing and treating Parkinson's disease.
  • the present invention provides the use of a gold cluster for the preparation of a medicament for diseases associated with aggregation and fibrosis of A ⁇ .
  • the disease associated with aggregation and fibrosis of A ⁇ is Alzheimer's disease.
  • the gold cluster is composed of L-glutathione (GSH), N-acetyl-L(D)-cysteine (L(D)-NAC), N-isobutyryl-L(D) -cysteine (L(D)-NIBC), L-cysteine-L-arginine dipeptide (CR), L-arginine-L-cysteine dipeptide (RC), 1-[(2S)-2-methyl-3-indolyl-1-oxopropyl]-L-proline (Cap) or L(D)-cysteine (L(D)-Cys) Etc.
  • GSH L-glutathione
  • L(D)-NAC N-acetyl-L(D)-cysteine
  • L(D)-NIBC N-isobutyryl-L(D) -cysteine
  • CR L-cysteine-L-arginine dipeptide
  • RC L-arginine-L-c
  • the present invention provides the use of a gold cluster for the preparation of a medicament for diseases associated with aggregation and fibrosis of ⁇ -syn.
  • the disease associated with aggregation and fibrosis of A ⁇ is Parkinson's disease.
  • the gold cluster is composed of L-glutathione (GSH), N-acetyl-L(D)-cysteine (L(D)-NAC), N-isobutyryl-L(D) -cysteine (L(D)-NIBC), L-cysteine-L-arginine dipeptide (CR), L-arginine-L-cysteine dipeptide (RC), 1-[(2S)-2-methyl-3-indolyl-1-oxopropyl]-L-proline (Cap) or L(D)-cysteine (L(D)-Cys) Etc.
  • GSH L-glutathione
  • L(D)-NAC N-acetyl-L(D)-cysteine
  • L(D)-NIBC N-isobutyryl-L(D) -cysteine
  • CR L-cysteine-L-arginine dipeptide
  • RC L-arginine-L-c
  • the gold-clustered substance provided by the present invention exhibits an excellent effect of inhibiting the aggregation of A ⁇ and ⁇ -syn in an in vitro experiment of inhibiting aggregation of A ⁇ and ⁇ -syn, in the A ⁇ -induced cell AD model and the MPP + induced cell PD model.
  • the experiment showed an excellent effect on improving cell survival rate.
  • the gold-containing cluster material can significantly improve the cognitive behavior of the diseased mice, and the A ⁇ (1-40) and A ⁇ (1-42) in the hippocampus and cerebral cortex of the mouse. )
  • the formation of plaques has a significant inhibitory effect.
  • the gold-containing cluster material can significantly improve and correct the motor behavior disorder of MPTP-injured model mice, improve the motor behavior of the diseased mice, and significantly inhibit MPTP-induced Specific apoptosis of mouse nigral and striatum DA neurons. It also has good biosafety at the cell level and animal level.
  • the above results indicate that the gold cluster-containing material of the present invention affects the aggregation and fibrosis of fibrotic proteins, and affects neurodegeneration at a deeper level such as energy metabolism of nerve cells and signal transduction function related to neurotransmitter metabolism. The progression of sexual diseases, therefore, the gold-clustered substances of the present invention are important for the development of new drugs for neurodegenerative diseases such as AD and/or PD.
  • 1 is an ultraviolet-visible spectrum, a transmission electron microscope photograph, and a particle size distribution diagram of a ligand L-NIBC modified gold nanoparticle having different particle diameters;
  • 2 is a UV-Vis spectrum, a transmission electron micrograph, and a particle size distribution diagram of a gold cluster modified with a different particle size of L-NIBC;
  • 3 is an infrared spectrum of a gold cluster of L-NIBC modified with different particle diameters
  • Figure 4 is a topographical view of AFM after incubation of A ⁇ (1-40) with gold nanoparticles or gold clusters modified with ligand L-NIBC;
  • Figure 5 is a graph showing the A ⁇ fibrillation kinetics of gold nanoparticles and gold clusters modified with ligand L-NIBC at different particle sizes and concentrations;
  • Figure 6 is a graph showing the effect of gold nanoparticles or gold clusters modified with ligand L-NIBC on cell viability of A ⁇ -induced AD cell model with different particle sizes and different concentrations;
  • Figure 7 is a UV, infrared, transmission electron microscope and particle size distribution diagram of ligand CR modified gold clusters (CR-AuNCs);
  • Figure 8 is a UV, infrared, transmission electron microscope and particle size distribution diagram of ligand RC modified gold clusters (RC-AuNCs);
  • Figure 9 is a gold cluster modified with the ligand 1-[(2S)-2-methyl-3-mercapto-1-oxopropyl]-L-proline (ie captopril (Cap)) ( Ultraviolet, infrared, transmission electron microscopy and particle size distribution of Cap-AuNCs);
  • Figure 10 is a diagram showing ultraviolet, infrared, transmission electron microscopy and particle size distribution of ligand GSH-modified gold clusters (GSH-AuNCs);
  • Figure 11 is a graph showing the ultraviolet, infrared, transmission electron microscopy and particle size distribution of ligand D-NIBC modified gold clusters (D-NIBC-AuNCs);
  • Figure 12 is a graph showing the inhibitory effect of different ligand-modified gold clusters on aggregation and fibrosis of A ⁇ (1-40);
  • Figure 13 is a schematic view of the water labyrinth experimental device in the fifth embodiment.
  • Figure 14 is a graph showing the effect of gold cluster-containing substances on cognitive behavior (day 150 of administration) of APP/PS1 double transgenic C57BL/6 mouse model;
  • Figure 15 is a graph showing the effect of gold-containing clusters on the hippocampus region of the APP/PS1 double transgenic C57BL/6 mouse model and the expression of A ⁇ (1-40) in the cerebral cortex (administered for 100 days);
  • Figure 16 is a graph showing the effect of gold cluster-containing substances on the hippocampus region of the APP/PS1 double transgenic C57BL/6 mouse model and the expression of A ⁇ (1-42) in the cerebral cortex (administered for 100 days);
  • Figure 17 is a graph showing the effect of gold-containing clusters on the hippocampus region of the APP/PS1 double transgenic C57BL/6 mouse model and the expression of A ⁇ (1-40) in the cerebral cortex (administered for 150 days);
  • Figure 18 is a graph showing the effect of gold-containing clusters on the hippocampus region of the APP/PS1 double transgenic C57BL/6 mouse model and the expression of A ⁇ (1-42) in the cerebral cortex (administered for 150 days);
  • Figure 19 is a graph showing the effect of a substance containing gold clusters on the kinetics of ⁇ -syn fiberization
  • Figure 20 is a graph showing the effect of gold cluster-containing substances on cell survival rate of MPP + injured PD cells (SH-sy5y) model;
  • Figure 21 is a graph showing the effect of gold cluster-containing substances on apoptosis of MPP + induced PD cells (PC12) model
  • Figure 22 is a graph showing the effect of gold-containing clusters on the spontaneous activity of mice with MPTP injury model
  • Figure 23 is a graph showing the effect of gold-containing clusters on the swimming activity of mice with MPTP injury model
  • Figure 24 is a graph showing the effect of gold cluster-containing substances on the roller behavior of mice in the MPTP injury model
  • Figure 25 is a graph showing the effect of gold-containing clusters on the substantia nigra and striatum DA neurons of MPTP-injured mice;
  • Figure 26 is a graph showing the effect of different particle size and concentration of gold-containing clusters on the survival rate of SH-sy5y neuroblastoma cells.
  • the gold nanoparticles used in the study have a gold core diameter of 3 nm or more, and when the gold core diameter is less than 3 nm, they are called gold clusters, and the disappearance of the plasmon resonance absorption peak (520 ⁇ 20 nm) in the ultraviolet visible absorption spectrum and 560 nm The appearance of the above new absorption peak is a sign to judge whether the gold cluster is successfully prepared.
  • the gold cluster cannot be stably separated from the ligand alone in the solution, and it binds to the ligand containing the thiol group through the Au-S bond to form a ligand-modified gold cluster (or gold cluster).
  • Ligand-modified gold clusters disclosed in the literature include L-glutathione (GSH), N-acetyl-L(D)-cysteine (L(D)-NAC), N-isodin Modified gold clusters such as acyl-L(D)-cysteine (L(D)-NIBC), etc., the preparation process of which is described in the literature (HFQian, MZZhu, ZKWu, RCJin, Accounts of Chemical Research 2012). , 45, 1470; C. Gautier, T. Bürgi, Journal of the American Chemical Society 2006, 128, 11079); its applications focus on catalysis, chiral recognition, molecular detection, biosensing, drug delivery, bioimaging, etc. (G.
  • the present invention studies the effects of gold clusters on AD and/or PD, and at least includes: firstly, by using gold clusters of different sizes with different ligands (ligands that do not inhibit aggregation of A ⁇ ), by inhibiting A ⁇ Aggregation and inhibition of ⁇ -syn aggregation in vitro, A ⁇ -induced AD cell model and MPP + induced PD cell model experiment, AD transgenic mouse model and MPTP-induced PD mouse model experiment, and combined with gold Cytotoxicity of clusters, acute toxicity test in mice, distribution experiments in mice, etc., provided ligand-modified gold clusters, and found its application in the preparation of drugs for the treatment of AD and PD, and experiments with gold nanoparticles.
  • the purity of the raw materials used in the following examples may be as long as it is more than chemically pure, and the sources are commercially available.
  • This example describes a method of preparing a ligand-modified gold cluster comprising the following steps:
  • Cystine derivatives such as N-isobutyryl-L-cysteine (L-NIBC), N-isobutyryl-D-cysteine (D-NIBC), N-acetyl-L- Cysteine, N-acetyl-D-cysteine, etc.; cysteine-containing oligopeptides and derivatives thereof, including but not limited to cysteine-containing dipeptides, tripeptides, tetrapeptides And other peptides, 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-valine-L-cysteine-L-arginine tripeptide (PCR) , L-glutathione (GSH), glycine-L-serine-L-cy
  • the reaction solution is centrifuged at 8000 to 17500 r/min for 10 to 100 minutes with an ultrafiltration tube having a molecular weight cut off of 3K to 30K to obtain a ligand-modified gold cluster precipitate with different average particle diameters.
  • Gradient centrifugation as described in (2) of Example 2 because the pore size of the filter membrane of the ultrafiltration tube with different molecular weight cutoff directly determines the size of the gold cluster that can pass, this step can also be omitted, ie in the step (3) Go directly to the step after the end Step (5), which is obtained by mixing gold clusters of different sizes;
  • the gold clusters of different average particle diameters obtained in the step (4) are dissolved in water and placed in a dialysis bag and dialyzed in water for 1 to 7 days at room temperature;
  • the obtained powdery or flocculent substance is a ligand-modified gold cluster.
  • the powder or flocculent material obtained by the above method has a particle diameter of less than 3 nm (generally distributed in 0.5-2.6 nm), and an ultraviolet-visible absorption spectrum appears above 560 nm. Or multiple absorption peaks, there is no obvious absorption peak at 520 nm, and it is determined that the obtained powder or floc is a gold cluster.
  • the reaction solution was subjected to gradient centrifugation to obtain L-NIBC modified gold cluster powder of different particle size.
  • the specific method after the reaction, the reaction solution was transferred to an ultrafiltration with a molecular weight of 30K and a volume of 50 mL. In the tube, centrifuge at 10000r/min for 20min, and the retentate in the inner tube is dissolved in ultrapure water to obtain a powder with a particle size of about 2.6nm, and then the mixed solution in the outer tube is transferred to a molecular weight of 10K.
  • the volume of the ultrafiltration tube is 50mL, centrifuged at 13000r/min for 30min, and the retentate in the inner tube is dissolved in ultrapure water to obtain a powder with a particle size of about 1.8nm, and then continue in the outer tube.
  • the mixed solution was transferred to an ultrafiltration tube with a volume of 50 mL and a molecular weight of 3K, and centrifuged at 17500 r/min for 40 min.
  • the retentate in the inner tube was dissolved in ultrapure water to obtain a particle size of about 1.1 nm. Powder.
  • the powder prepared above (the gold cluster of the ligand L-NIBC) was subjected to a characterization experiment, and gold nanoparticles having the same ligand as L-NIBC were used as a control.
  • Preparation method of gold nanoparticles with ligand L-NIBC reference literature W. Yan, L. Xu, C. Xu, W. Ma, H. Kuang, L. Wang and N. A. Kotov, Journal of The introduction 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).
  • the powder to be tested (the L-NIBC modified gold cluster sample prepared in Example 2 and the gold nanoparticle sample with the ligand of L-NIBC) was diluted with ultrapure water to 2 mg/L as a sample, and then suspended.
  • the method of dropping method is as follows: 5 ⁇ L of the sample is dropped onto the ultra-thin carbon film web, and naturally evaporated until the water droplets disappear, and then the morphology of the gold cluster is observed on a JEM-2100F STEM/EDS field emission high-resolution transmission electron microscope.
  • the photographs in Figure 2 show that the L-NIBC modified gold cluster samples have uniform particle size and good dispersion.
  • the average diameter of the gold clusters of L-NIBC modified (referred to as the gold core diameter) are 1.1 nm, 1.8 nm and 2.6 nm, respectively. It is consistent with the results of C, F and I in Figure 2.
  • the comparison of the ligands with L-NIBC gold nanoparticles has a larger particle size, and the average diameter (the diameter of the gold core) is 3.6 nm, 6.0 nm, 10.1 nm, and 18.2 nm, respectively, and the C-frame in Figure 1, The results of F, I and L are consistent.
  • the powder to be tested 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 is 190-1100 nm
  • the sample cell is a standard quartz cuvette with an optical path length of 1 cm
  • the reference cell contains ultrapure water.
  • the gold nanoparticles with ligand L-NIBC exhibit an absorption peak around 520 nm, and the position of the absorption peak is related to the particle size, wherein the ultraviolet absorption peak at 3.6 nm is at 516 nm.
  • the ultraviolet absorption peak at 6.0 nm is at 517 nm
  • the ultraviolet absorption peak at 10.1 nm is at 520 nm
  • the absorption peak at 18.2 nm is red-shifted to 523 nm.
  • the four samples have no absorption peak above 560 nm.
  • the surface plasmon absorption peak near 520 nm disappears in the ultraviolet absorption spectrum of the gold cluster sample of the three different particle diameter ligands of the L-NIBC of Example 2, and two of them appear above 560 nm.
  • the apparent absorption peak, the position of the absorption peak is slightly different depending on the particle size of the gold cluster. This is because the gold clusters exhibit the molecular-like nature due to the collapse of the face-centered cubic structure, resulting in the density of the gold clusters being discontinuous and producing energy levels. Splitting, the plasmon resonance effect disappears, and a new absorption peak appears in the long-wave direction. From this, it can be judged that the powder samples of the three different particle diameters obtained in Example 2 are all ligand-modified gold clusters.
  • Infrared spectra were measured on a VERTEX 80V Fourier transform infrared spectrometer manufactured by Bruker using a solid powder high vacuum total reflection mode with a scan range of 4000-400 cm -1 and 64 scans.
  • the test sample was a dry powder of three different particle diameter gold clusters modified by L-NIBC, and the control sample was a pure L-NIBC powder. The results are shown in Figure 3.
  • Figure 3 is an infrared spectrum of L-NIBC modified gold clusters of different particle sizes, compared with pure L-NIBC (top curve), L-NIBC modified gold clusters of different particle sizes at 2500-2600 cm -1
  • the SH stretching vibrations disappeared completely, while the characteristic peaks of other L-NIBCs were still observed, demonstrating that the L-NIBC molecules were successfully anchored to the gold cluster surface by gold-sulfur bonds.
  • the figure also shows that the infrared spectrum of the ligand-modified gold cluster is independent of its size.
  • ligand Y-modified gold clusters were prepared in a similar manner as described above except that the solvent of solution B, the ratio of HAuCl 4 to ligand Y, the reaction time and the amount of NaBH 4 added were slightly adjusted, such as: L-cysteine When acid, D-cysteine, N-isobutyryl-L-cysteine (L-NIBC), N-isobutyryl-D-cysteine (D-NIBC) are used as ligand Y, Select acetic acid as the solvent; when the dipeptide CR, the dipeptide RC, 1-[(2S)-2-methyl-3-mercapto-1-oxopropyl]-L-proline is used as the ligand Y, the water is selected. As a solvent, etc.; the remaining steps are similar and will not be repeated one by one.
  • L-cysteine When acid, D-cysteine, N-isobutyryl-L-cysteine (L-NIBC
  • the present invention prepares a series of ligand-modified gold clusters according to the above method, and the ligands used and the parameters of the preparation process are shown in Table 1.
  • the function of ligand-modified gold clusters was verified by in vitro A ⁇ aggregation kinetics experiments, and compared with the effects of ligand-modified gold nanoparticles and ligand molecules alone on A ⁇ aggregation kinetics. Its function comes from gold clusters, not from ligands.
  • the kinetics of A ⁇ (1-40) fibrosis was characterized by ThT fluorescent labeling.
  • ThT Thioflavin T
  • a polypeptide or a protein monomer When it is incubated with a polypeptide or a protein monomer, its fluorescence does not change substantially. When it encounters an amyloid polypeptide or protein with a fibrous structure, it immediately couples with an amyloid or protein, and its fluorescence intensity Rapidly increased exponentially. Because of this property, ThT is widely used to monitor markers of polypeptide or protein amyloidosis.
  • the fibrosis process of A ⁇ (1-40) is also a nucleation-controlled polymerization process. Therefore, the growth curve of A ⁇ (1-40) fiber measured by ThT fluorescent labeling is mainly divided into three stages: initial stage, Growth period and platform period.
  • the initial stage is mainly the stage where A ⁇ (1-40) undergoes conformational transformation to form misfolded and then aggregates into the nucleus; during the growth period, A ⁇ (1-40) monomer is added to the core or oligomer to form fibers along the axial direction of the fiber and The stage of rapid growth; the plateau is that the A ⁇ (1-40) molecules all form mature long fibers, that is, the stage in which the fibers no longer grow.
  • the ThT fluorescent labeling method can conveniently monitor the kinetics of fibrotic aggregation of A ⁇ (1-40) molecules.
  • the lyophilized amyloid polypeptide A ⁇ (1-40) powder (Invitrogen Corp.) was dissolved in hexafluoroisopropanol (HFIP) to obtain a solution of A ⁇ (1-40) at a concentration of 1 g/L, and sealed at room temperature. Incubate for 2-4 hours, then dry the hexafluoroisopropanol with high purity nitrogen (N 2 , 99.9%) at a suitable gas flow rate in a fume hood (about 1 hour), and finally dry it.
  • a ⁇ (1-40) was dissolved in 200 ⁇ L of dimethyl sulfoxide (DMSO), sealed and stored in a refrigerator at -20 °C for a period of not more than one week.
  • DMSO dimethyl sulfoxide
  • PBS phosphate buffer
  • the ligand-modified gold clusters and gold nanoparticles were separately added to 20 ⁇ M A ⁇ (1-40) in PBS buffer solution to form gold cluster samples with different ligands and different particle sizes modified and correspondingly different.
  • Ligand-modified gold nanoparticle samples The fluorescence intensity was monitored by a ThT fluorescent labeling method at 37 ° C in a 96-well plate and every 10 minutes with a microplate reader. Characterization of A ⁇ (1-40) aggregation by changes in fluorescence intensity of ThT Mechanical process.
  • the experimental group used three kinds of L-NIBC modified gold clusters prepared in Example 2 with particle diameters of 2.6 nm, 1.8 nm, and 1.1 nm, and the control group used four particles having particle diameters of 18.2 nm, 10.1 nm, 6.0 nm, and 3.6 nm.
  • L-NIBC modified gold nanoparticles, and L-NIBC molecules not bound to gold clusters or gold nanoparticles.
  • concentrations respectively: 0ppm (excluding gold clusters, gold nanoparticles or L-NIBC, for comparison), 0.1ppm, 1.0 Ppm, 5.0 ppm, 10.0 ppm, and 20.0 ppm.
  • L-NIBC was used alone, there were two concentrations: 1.0 ppm and 10.0 ppm, respectively.
  • Figure 4 shows the AFM topography after A ⁇ (1-40) was incubated with each experimental group and control group for 48 hours, in which A was the AFM topography after only 48 hours of A ⁇ (1-40) incubation.
  • B is the AFM topography after incubation with A ⁇ (1-40) and L-NIBC for 48h.
  • the C and D frames are A ⁇ (1-40) and gold nanoparticles with average particle size of 6.0nm and 3.6nm, respectively.
  • A is the A ⁇ (1-40) fibrillation kinetics curve in the presence of different concentrations of L-NIBC.
  • the B-E-frames are at different concentrations of 18.2 nm, 10.1 nm, 6.0 nm, and 3.6 nm, respectively.
  • a ⁇ (1-40) fibrillation kinetics curve in the presence of gold nanoparticles, F-H amplitude is A ⁇ (1) in the presence of gold clusters with particle sizes of 2.6 nm, 1.8 nm and 1.1 nm at different concentrations.
  • -40 Fibrillation kinetic curve.
  • A-H is represented by 0 ppm (i.e., gold-free nanoparticles and gold clusters), ⁇ represents 0.1 ppm, ⁇ represents 1 ppm, ⁇ represents 5 ppm, ⁇ represents 10 ppm, and ⁇ represents 20 ppm of gold nanoparticles or gold.
  • FIG. 4 It can be seen from Fig. 4 that the A-frame as a control is covered with A ⁇ fiber; the B-frame is also covered with A ⁇ fiber; although the fiber is reduced in the C-frame, the longer fiber can be seen in the D-frame. Although long fibers are not visible, a large amount of A ⁇ short fibers are still present. This indicates that L-NIBC has no significant effect on the formation of A ⁇ (1-40) fiber.
  • the addition of L-NIBC modified small-sized gold nanoparticles can delay the fibrillation process of A ⁇ (1-40), but it cannot be completely realized. Inhibition, because short fibers continue to grow and grow fibers after a longer period of time.
  • the E-frame of Figure 4 shows that there are neither long fibers nor short fibers, indicating that the L-NIBC-modified gold clusters can completely inhibit the fibrosis process of A ⁇ (1-40).
  • Figure 4 is a qualitative experiment
  • Figure 5 is a quantitative experiment
  • the results in Figure 5 show that the addition of L-NIBC has no significant effect on the fibrillation kinetics of A ⁇ (1-40) (Figure A, A);
  • Figure A, A When the particle diameter is greater than or equal to 10.1 nm, the addition of L-NIBC modified gold nanoparticles advances the growth phase and plateau time of A ⁇ aggregation kinetics (A ⁇ aggregation kinetics when the concentration of gold nanoparticles is 20 ppm)
  • the growth period is advanced to 12h, and the plateau time is advanced to 16h), indicating that L-NIBC modified gold nanoparticles can accelerate the aggregation of A ⁇ (Fig.
  • the fluorescence emission peak of the ThT is located at 515 nm, and the plasmon resonance absorption peak of the L-NIBC modified gold nanoparticles is located near 520 nm, so that it is observed here.
  • the 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 inhibition of A ⁇ (1-40) aggregation by L-NIBC modified gold nanoparticles.
  • the F-H panel of Figure 5 indicates that all of the L-NIBC-modified gold clusters can significantly inhibit the aggregation of A ⁇ (delaying the start of the growth phase, when the concentration of the L-NIBC-modified gold cluster is 5 ppm, The growth period of 20 ⁇ M A ⁇ aggregation kinetics can be delayed to 50 h later, and when the concentration of L-NIBC modified gold cluster reaches 10 ppm and above, the aggregation of A ⁇ can be completely inhibited (no growth period) The fluorescence curve is completely flat).
  • the minimum concentration of the L-NIBC modified gold cluster required for complete inhibition is related to the type of ligand and the diameter of the gold cluster, wherein the L-NIBC modified gold cluster having a particle size of 1.1 nm, 1.8 nm and 2.6 nm
  • the minimum concentrations required were 5.0 ppm, 5.0 ppm and 10.0 ppm, respectively.
  • the L-NIBC modified gold cluster does not have a plasmon resonance effect, there is no quenching effect on the fluorescence of ThT. Therefore, the decrease in fluorescence intensity observed here is entirely due to the L-NIBC modified gold cluster. Inhibition of A ⁇ (1-40) aggregation.
  • the quantitative results of Figure 5 are in complete agreement with the qualitative results of Figure 4.
  • the A-H frame of Figure 12 is CR, N-acetyl-L-cysteine, respectively.
  • L-NAC L-NAC
  • GSH 1-[(2S)-2-methyl-3-indolyl-1-oxopropyl]-L-proline (Cap)
  • D-NIBC D-NIBC
  • RC L-cysteine
  • the results of the CCK-8 assay were used to reflect the toxic effects of ligand-modified gold clusters or gold nanoparticle samples against A ⁇ (1-40). Whether the modified gold cluster or gold nanoparticle has a neuroprotective effect in the amyloid misfolding pathogenesis mechanism.
  • the cells used in the experiment were SH-SY5Y neuroblastoma cell lines, and the A ⁇ -induced AD cell model was constructed according to the literature (R. Liu, H. Barkhordarian, S. Emadi, CB Park, MRSierks, Neurobiology of Disease 2005, 20, 74). The description in ) is carried out. The specific method is:
  • the final concentrations of the ligand-modified gold clusters or ligand-modified gold nanoparticles were 0.01 ppm, 0.1 ppm, 1 ppm, 5 ppm, 10 ppm, and 20 ppm, respectively, and the final concentration of A ⁇ (1-40) was 20 ⁇ M.
  • a blank control group containing no SH-sy5y cells, a gold nanoparticle containing SH-sy5y cells without ligand modification or a ligand-modified gold nanoparticle, and a negative control group of A ⁇ (1-40), containing SH were provided.
  • -sy5y cells were supplemented with A ⁇ (1-40) (final concentration 20 ⁇ M) in a cell model control group, and contained SH-sy5y cells, A ⁇ (1-40) (final concentration 20 ⁇ M) and L-NIBC (final concentration) A ligand control group of 20 ppm).
  • the culture medium was removed, and 100 ⁇ L of 10% CCK-8 in maintenance medium was added to each well for 4 hours.
  • the absorbance of each well was measured at a wavelength of 450 nm to reflect the damage of the ligand-modified gold cluster to A ⁇ (1-40). Pre-protection and therapeutic effects.
  • the A-C panel in Figure 6 shows the effect of L-NIBC modified gold clusters with particle sizes of 1.1 nm, 1.8 nm, and 2.6 nm on cell viability in A ⁇ -induced AD cell model at different concentrations;
  • the -F web indicates the effect of L-NIBC modified gold nanoparticles having particle sizes of 3.6 nm, 6.0 nm, and 10.1 nm at different concentrations on cell viability in the A ⁇ -induced AD cell model, respectively.
  • L-NIBC modified gold clusters of different sizes are used at very low levels (eg 0.1-1 ppm)
  • the cell viability of the A ⁇ -induced AD cell model can be increased from nearly 60% to nearly 95% or more (P is less than 0.05, A-C panel in Fig. 6).
  • the L-NIBC modified gold nanoparticles with an average diameter of 3.6 nm increased the cell viability of the AD cell model with increasing concentrations (D panel in Figure 6), but none of them were significant (P > 0.05).
  • mice Take 180 B6/J-Tg (APPswe, PSEN1de9) 85Dbo/MmNju strain transgenic mice (purchased from the Institute of Model Animals, Nanjing University), randomly divided into three groups, 60 in each group: control group, low dose The drug group and the high dose administration group. From the 100th day of the mice, the control group was fed normally every day. The low-dose group was orally administered with 200 ⁇ L of 0.5g/L aqueous solution of gold clusters once a day, and the high-dose group was orally administered with 200 ⁇ L per day. It is a 2 g/L aqueous solution of gold clusters.
  • mice were randomly divided into 7 batches: 140 days, 160 days, 180 days, 200 days, 230 days, 260 days, and 290, respectively.
  • the changes in learning and memory behavior of mice were studied using labyrinth experiments, open field experiments, and new object recognition experiments. Among them, the first 4 batches of experiments were 6 in each group, and the last 3 batches of experiments were 6-8 in each group (considering a certain mortality rate during mouse feeding, the same below).
  • mice purchased from Nanjing University Model Animal Research Institute
  • model control group was fed normally every day.
  • the low-dose group and the high-dose group were given intraperitoneal injection according to the weight of the mice according to the body weight of 5mg/Kg body weight and 20mg/Kg body weight.
  • the cluster solution is administered once every two days.
  • the Morris water maze experiment is a kind of forced experimental animal swimming, learning to find the platform hidden in the water platform, mainly used to test the learning and memory ability of experimental animals to spatial position and direction perception, is widely used to evaluate the development of Alzheimer's disease drugs.
  • the behavior of the mice was tested using the Morris water maze test 150 days after administration of the model mice, and the experimental method was referred to the literature (C.V. Vorhees, M.T. Williams, Nature Protocols 2006, 1, 848). details as follows:
  • the Morris water maze test system consists of a circular pool and an automatic video recording and analysis system.
  • the camera is connected to the computer above the pool ( 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 liquid level is 0.5 cm above the platform and the water level is maintained at 22 ⁇ 0.5 °C.
  • the white pigment was used to dye the water to milky white.
  • the positioning navigation test was used to measure the learning and memory ability of mice in the water maze, which lasted 4 days. As shown in Fig. 13, the water maze is divided into four quadrants in the four directions of east (E) west (W) south (S) north (N).
  • the platform is placed in the middle of the SW quadrant, and the platform position is fixed throughout the experiment.
  • the mouse head is directed toward the wall of the pool from 1/2 radians of different quadrants, and is gently placed in the water near the outer wall.
  • the experiment was stopped by recording the time the mouse climbed onto the hidden platform (searching for latency) or reaching 60 s through the camera tracking system.
  • the mice were allowed to stay on the platform for 30 s after the platform. If the mouse did not find the platform within 60 s (when the latency was 60 s), the experimenter guided the mice to climb the platform and let them stay for 30 s.
  • Each mouse was removed after the test and lightly dried. Each animal was trained 4 times a day, with 15-20 minutes between training sessions and 4 days of continuous training.
  • a ⁇ (1-40) and A ⁇ (1-42) were used to detect the amyloid deposition distribution of A ⁇ (1-40) and A ⁇ (1-42) in hippocampus and cerebral cortex of rats.
  • the pathological deposition of A ⁇ in the cerebral cortex and hippocampus is a major pathological feature of AD.
  • a ⁇ (1-40) and A ⁇ (1-42) are important components of senile plaques in the brain, which are neurotoxic and can cause progressive cognitive dysfunction and memory loss.
  • the changes of A ⁇ (1-40) and A ⁇ (1-42) plaque formation in hippocampus and cerebral cortex were detected by immunohistochemistry.
  • mice after continuous administration of mice for 100 days and 150 days, 10-12 mice in each group were tested for hippocampus and cerebral cortex immunohistochemistry, and the mice after the 150th day of administration were subjected to water maze experiments.
  • Mouse The mice were anesthetized by intraperitoneal injection of 5% chloral hydrate (10 ⁇ L/g). The limbs were fixed on the bench and the chest was opened to fully expose the heart. Note that the liver cannot be cut during thoracotomy.
  • the left ventricle was first washed with 0.1 mL/L PBS buffer 50 mL for 5 min to remove blood, and then perfused with 0.1%/L PBS buffer containing 4% paraformaldehyde for 6 min.
  • the brain was removed after perfusion and placed in 4% paraformaldehyde at 4 ° C and fixed overnight.
  • the tissue was sequentially dehydrated with a 10%, 20%, and 30% sucrose solution gradient and stored at -80 °C until use.
  • the tissue was subjected to a paraffin-embedded reference mouse brain map, and the midbrain hippocampus and cortical slices (thickness 8 ⁇ m) were used for immunohistochemical staining.
  • the procedure was as follows: frozen section 8 ⁇ m, left at room temperature for 30 min, fixed in acetone at 4 ° C for 20 min, washed three times with PBS (5 min each), and then incubated with 3% hydrogen peroxide for 10 min to eliminate the intrinsic peroxidase activity.
  • the nickel sulfate amine enhanced DAB blue reaction method was used for color development for 10 min.
  • the positive product was dark blue and the background was clear, it was washed with distilled water for 3 times to terminate the color development.
  • the number of A ⁇ plaques in the hippocampus and cerebral cortex was observed and counted. Each sample was divided into left and right ventricles, and two sections were paralleled. The average was taken for statistical analysis. All data were processed by SPSS software (SPSS 21) using t test or one-way analysis of variance. P ⁇ 0.05 indicated that the difference was statistically significant.
  • the A, B and C panels of Figure 15 are typical immunohistochemistry of the hippocampus and cerebral cortex of A ⁇ (1-40) in the high-dose, low-dose, and model control groups at 100 days of administration, respectively.
  • the slice result, the D picture of Fig. 15 is the statistical result.
  • the experimental results showed that compared with the model control group, high dose administration significantly decreased A ⁇ (1-40) plaque formation in the hippocampus of model mice (44.6 ⁇ 12.2%, P ⁇ 0.05). There was no significant effect of A ⁇ (1-40) plaque formation in the cerebral cortex (P>0.05). Low dose administration had no significant effect on A ⁇ (1-40) plaque formation in hippocampus and cerebral cortex (P>0.05).
  • Figure 16 is the result of the corresponding A ⁇ (1-42).
  • the age of the mice increased, compared with the model control mice administered for 100 days, A ⁇ (1-40) and A ⁇ (1) in the hippocampus and cerebral cortex of the model control group at 150 days of administration. -42)
  • the formation of plaques increased significantly, with A ⁇ (1-40) hippocampus increased by 57.2 ⁇ 7.2% (P ⁇ 0.05), cerebral cortex increased by 49.1 ⁇ 19.6% (P ⁇ 0.05), A ⁇ (1-42) Hippocampus increased by 74.4 ⁇ 7.0% (P ⁇ 0.05) and cerebral cortex increased by 65 ⁇ 11.1% (P ⁇ 0.05), indicating that the memory and cognitive function of model mice may be affected with age.
  • the A, B and C panels of Figure 17 are typical immunohistochemistry of the hippocampus and cerebral cortex of A ⁇ (1-40) in the high-dose, low-dose, and model control groups at 150 days of administration, respectively.
  • the slice result, the D picture of Fig. 17 is a statistical result.
  • the results showed that the A ⁇ (1-40) in the hippocampus and cerebral cortex of the high-dose group was significantly decreased (the hippocampus decreased by 59.0 ⁇ 11.1%, P ⁇ 0.05; the cerebral cortex decreased by 36.4 ⁇ 4.5%, P ⁇ 0.05).
  • Figure 18 is the result of the corresponding administration of A ⁇ (1-42) for 150 days.
  • the results showed that high dose of gold clusters significantly inhibited the formation of A ⁇ (1-42) plaques in hippocampus and cerebral cortex (51.1 ⁇ 6.7% in hippocampus, P ⁇ 0.05; 62.8 ⁇ 4.6 in cerebral cortex). %, P ⁇ 0.05).
  • Low dose administration had no significant effect on A ⁇ (1-42) plaque formation in hippocampus and cerebral cortex of mice (P>0.05). This indicates that gold clusters have a significant inhibitory effect on the formation of A ⁇ (1-42) plaques at 150 days, and this effect is also dose dependent.
  • gold clusters significantly improved the cognitive behavior of AD model mice, and significantly formed the formation of A ⁇ (1-40) and A ⁇ (1-42) plaques in hippocampus and cerebral cortex of mice. Inhibition, thereby inhibiting the development of diseased mice, can be used as a gold cluster-containing substance for the prevention and treatment of AD.
  • ThT Thioflavin T
  • a polypeptide or a protein monomer When it is incubated with a polypeptide or a protein monomer, its fluorescence does not change substantially. When it encounters an amyloid polypeptide or protein with a fibrous structure, it immediately couples with an amyloid or protein, and its fluorescence intensity The index is enhanced and is therefore widely used to monitor markers of polypeptide or protein amyloidosis.
  • This example uses ThT fluorescent labeling to monitor the kinetics of fibrotic aggregation of ⁇ -syn in the presence of gold clusters.
  • the specific experimental methods are as follows:
  • ⁇ -syn monomer Pretreatment of ⁇ -syn monomer: lyophilized ⁇ -syn powder (Bachem Corp.) was dissolved in hexafluoroisopropanol (HFIP) to obtain ⁇ -syn solution at a concentration of 1 g/L, and sealed at room temperature. Incubate for 2-4 hours, dry hexafluoroisopropanol with high purity nitrogen in a fume hood, dissolve the dried ⁇ -syn in 200 ⁇ L of dimethyl sulfoxide (DMSO), seal and place in a refrigerator at -20 ° C. Save the backup in the middle, the storage time should not exceed one week.
  • DMSO dimethyl sulfoxide
  • PBS phosphate buffer
  • the ⁇ -syn PBS buffer solution in all experiments was now available.
  • the gold clusters used four concentrations, respectively: 0 ppm (only ⁇ -syn, no gold clusters or L-NIBC, as model control), 1.0 ppm, 5.0 ppm, and 10.0 ppm, L-NIBC When used alone, there are two concentrations used, namely: 1.0 ppm and 10.0 ppm.
  • the fluorescence intensity of ThT label was significantly lower than that of the model control group and the ligand control group without added gold clusters at lower concentrations (such as 1.0 ppm and 5.0 ppm). And the onset time was significantly delayed (Fig. 19B), indicating that the addition of gold clusters can significantly inhibit the aggregation and fibrosis of ⁇ -syn.
  • the fluorescence intensity of the ThT mark remained near the baseline during the experimental time of 168 hours without any increase (Fig. 19B), indicating that when the gold cluster concentration is sufficient, Aggregation and fibrosis of ⁇ -syn are completely inhibited.
  • Example 7 MPP + induced PD cell (SH-sy5y) model experiment
  • the first group is added with 100 ⁇ L of gold cluster or gold nanoparticle solution modified with ligands of different particle sizes and different concentrations listed in Table 1 to achieve final concentrations of 0.01 ppm, 0.1 ppm, and 1 ppm, respectively. , 5 ppm, 10 ppm, and 20 ppm, as a drug-administered group; after pretreatment of ligand-modified gold clusters or gold nanoparticles for 2 hours, MPP + (final concentration of 1 mM) was added to the administration group and the cell model control group, respectively.
  • Gold clusters and gold nanoparticles modified with different ligands were tested in the same procedure.
  • the results indicate that the ligand-modified gold cluster provided by the present invention has a protective effect on nerves in Parkinson's neurodegenerative diseases, and this effect is also derived from the gold cluster itself, not the ligand, and can be used as a substance containing gold clusters. Used to fight Parkinson's disease.
  • the first group is added with 100 ⁇ L of gold cluster or gold nanoparticle solution modified with ligands of different particle sizes and different concentrations listed in Table 1 to achieve final concentrations of 0.01 ppm, 0.1 ppm, and 1 ppm, respectively. 5 ppm, 10 ppm, and 20 ppm, as a drug-administered group; after pretreatment with ligand-modified gold clusters or gold nanoparticles for 2 hours, MPP + (final concentration of 1 mM) was added to the administration group and the cell model control group, respectively.
  • the cell viability of the model control group supplemented with 1 mM MPP + but no gold clusters was reduced to 65.1 ⁇ 4.0% (P ⁇ 0.01 for the blank control group), and the cell survival rate of the ligand control group was 61.5 ⁇ 3.8% (right
  • the blank control group (P ⁇ 0.01) indicated that there was no improvement in the survival rate of the MPP + injured cell model when the ligand was used alone.
  • the cell viability of the administration group with 1ppm, 5ppm, 10ppm and 40ppm gold clusters increased to 97.9 ⁇ 2.8% (P ⁇ 0.01 for the model control group) and 99.7 ⁇ 4.0% (P ⁇ 0.001 for the model control group).
  • the ligand-modified gold cluster provided by the present invention is in the Parkinson's neurodegenerative disease Nerve cells have a protective effect, which is also derived from the gold cluster itself, not the ligand.
  • the gold nanoparticles of the corresponding ligands had no effect on the survival rate of the model cells at the three experimental concentrations, indicating that the gold nanoparticles could not be used as a drug for the prevention and treatment of PD.
  • Example 8 MPP + induced PD cell (PC12) model experiment
  • MPP + (100 mM) was used to induce apoptosis of PC12 cells, and cell flow technique was used to observe the protective effect of gold clusters on MPP + cell damage and apoptosis.
  • DETAILED Experimental method Experimental set MPP + was not added and the blank control group of gold clusters, MPP + only add the MPP + model group, the control group added only gold clusters and clusters of gold and gold clusters added to MPP + test group.
  • L-NIBC modified gold cluster solution with an average particle diameter of 1.8 nm (final concentration of 20 ppm) was added to the PC12 cell suspension of PC12 cells for half an hour, and added with MPP + for 24 hours, using Annexin V-
  • the FITC/PI apoptosis assay kit (purchased from Roch) was used to detect cell growth viability and apoptosis by FACSCalibur flow cytometry. CellQuest Pro obtained data and analyzed it.
  • Example 7 The results of Example 7 and Example 8 together showed that the gold cluster significantly improved the cell survival rate of the MPP + induced PD cell model and significantly inhibited cell apoptosis.
  • mice 80 male C57bl/6 mice, 8 weeks old, weighing 25-30 g; 3 mice per cage, all in the environment at room temperature 22-27 ° C, 12 h circadian rhythm, free to eat and drink, Adapt to 7 days.
  • mice were randomly divided into four groups, 20 in each group, divided into blank control group, normal control group of gold cluster, MPTP model group and gold cluster treatment group.
  • the MPTP model group and the gold cluster treatment group were injected subcutaneously with 20 mg/kg (free base) MPTP every 2 hours for four injections.
  • the saline normal solvent control group was injected subcutaneously with 20 mg/kg normal saline every 2 hours, and injected four times.
  • the normal saline control group and the MPTP model group were injected with 10 ⁇ L of normal saline per day, and the gold cluster normal control group and the gold cluster treatment group were intraperitoneally injected with 10 ⁇ L of the ligands listed in Table 1 daily.
  • the physiological saline solution of the modified gold cluster (concentration of gold clusters of 10 g/L) was continuously injected for 7 days, and the animals were placed in a feeding box with a clean litter, freely drinking water, and fed.
  • Neurotransmitter measurement After the behavioral experiment, the animals were sacrificed, and the mouse striatum was taken and frozen at -80 °C. The striatum was treated with homogenate (0.1 M perchloric acid, 0.1 mM EDTA-2Na) 10 ⁇ L/mg (striatum), ultrasonically lysed under ice bath, lysed for 30 minutes, and placed in a cryogenic centrifuge 10000 r/ After centrifugation for 10 min, the supernatant was extracted, filtered through a 0.25 ⁇ m filter, and injected into a HPLC liquid chromatography column to detect dopamine (DA) transmitters and their metabolism in the striatum using a laboratory-established high-performance liquid phase system.
  • homogenate 0.1 M perchloric acid, 0.1 mM EDTA-2Na
  • HPLC conditions flow rate: 1 mL/min; column temperature 30 ° C; fluorescence detector excitation and absorption wavelengths were 280 and 330 nm, respectively.
  • tyrosine hydroxylase brain tissue was removed and fixed in 4 wt% PFA + 2 wt% sucrose for 4-6 h, then dip After entering the 30 wt% sucrose solution, the brain tissue was immersed in the bottom, embedded in OCT, and subjected to continuous coronary patching by a frozen slicer. ABC (Avidibiotin-peroxidase complex) staining was performed, and frozen tissue sections of the substantia nigra were taken for TH staining. , diphenylamine color development, microscopic observation.
  • mice 80 male C57bl/6 mice, 8 weeks old, weighing 25-30 g; 3 mice per cage, all in the environment at room temperature 22-27 ° C, 12 h circadian rhythm, free to eat and drink, Adapt to 7 days.
  • MPTP nerve injury mouse model mice were randomly divided into four groups, 20 mice in each group, divided into blank control group, gold cluster control group (divided into low dose group and high dose group according to the amount of gold clusters), MPTP model Group, gold cluster experimental group (divided into low dose group and high dose group according to the amount of gold clusters).
  • the MPTP model group and the gold cluster experimental group were intraperitoneally injected with 30 mg/kg (free base) MPTP every day for seven consecutive days.
  • the blank control group was subcutaneously injected with 30 mg/kg physiological saline for 7 consecutive days.
  • the low-dose group in the gold cluster control group and the gold cluster experimental group were intraperitoneally injected with 100 ⁇ L of 1 g/L L-NIBC modified gold cluster physiological saline solution with an average particle diameter of 1.8 nm, while the high dose was administered.
  • the group was intraperitoneally injected with 100 ⁇ L of 4 g/L L-NIBC modified gold cluster physiological saline solution with an average particle diameter of 1.8 nm for 7 days, and the animals were placed in a feeding box with clean litter. Free drinking water and eating.
  • roller test Animals need to maintain balance and continuous movement on the roller. It is widely used to test the coordination of motion. The diameter of the roller is 6cm, the rotation speed is 20rpm. After five times of adaptation, the interval between each detection is 1min. The time it was dropped from the drum was averaged five times.
  • the tissue was subjected to paraffin-coated reference mouse brain map, and the midbrain substantia nigra and striatum coronal sections were sliced to a thickness of 3 ⁇ m/sheet. Brain slices were used for immunofluorescence, hypersensitivity two-step immunohistochemistry and other experiments.
  • the immunohistochemical staining procedure was as follows: 0.3% hydrogen peroxide in methanol (30% hydrogen peroxide 1 mL + methanol 80 mL + PBS 19 mL) for 30 min, 0.3% Triton X-100 in PBS for 30 min, immersed in mouse anti-tyrosine hydroxylase ( TH) Monoclonal antibody (1:200) or IBa1 (diluted 1:250) was incubated for 48 h (4 ° C), immersed in biotinylated rabbit anti-mouse secondary antibody (1:500) for 2 h (room temperature), rinsed rapidly with distilled water After the nickel sulfate amine enhanced DAB blue reaction method for color development for 20 to 30 minutes, when the positive product is dark blue and the background is clear, the distilled water is washed 3 times to terminate the color development.
  • TH mouse anti-tyrosine hydroxylase
  • Striatum protein immunoblotting (WB) assay Tyrosine hydroxylase (TH) is a key enzyme in the dopamine (DA) biosynthesis pathway, and TH immunohistochemistry can show DA in the substantia nigra and striatum Neuronal changes (D. Luo, J. Zhao, Y. Cheng, SMLee, J. Rong, Molecular Neurobiology 2017, DOI: 10.107/s 12035-017-0486-6).
  • 5 rats in each group were tested for WB of striatum.
  • the brain part was taken out on ice, RIPA lysate was lysed, homogenized at 4 ° C for 12000 g, centrifuged for 30 min, protein was extracted, and samples were prepared.
  • mice showed tremor, decreased movement, arched back, hind limb opening, gait instability, vertical tail, vertical hair, etc., and individual epileptic seizures occurred. After 30 to 60 minutes, the above symptoms occurred. Gradually reduced, after 24h, it basically returned to normal, but with the increase of the number of doses, the acute reaction performance was reduced, but after 24h, the performance of exercise decreased, gait instability, and slow response became more and more obvious. Seven days after continuous MPTP injection, the spontaneous movement and movement speed of the mice were significantly lower than that of the blank control group (P ⁇ 0.01), showing symptoms of bradykinesia.
  • the gold cluster (high dose administration) and the MPTP concomitant administration group increased the swimming distance (P ⁇ 0.05), and the swimming time also increased significantly (P ⁇ 0.05) (B and D of Fig. 23). Amplification), indicating that gold clusters have a significant improvement effect on MPTP-induced swimming behavior disorder in mice.
  • Fig. 25 The immunohistochemical examination of the substantia nigra and striatum and the WB test results of the striatum are shown in Fig. 25.
  • the MPTP model group showed a significant decrease in the number of TH-positive immunogens (ie, DA-energy neurons), residual neuronal shrinkage, decreased or disappeared protuberances, and striatum TH immunopositive cells and nerve fiber density.
  • TH-positive immunogens ie, DA-energy neurons
  • residual neuronal shrinkage decreased or disappeared protuberances
  • striatum TH immunopositive cells and nerve fiber density Decreased, WB analysis showed that the striatum DA neurons were reduced to 55.8 ⁇ 5.6% (100% in the blank control group) (P ⁇ 0.01 for the blank control group, see Figure C, C).
  • Gold clumps alone had no significant effect on TH and striatum TH immunopositive cells and nerve fiber density ( Figures A and B).
  • the combination of gold clusters and MPTP significantly inhibited MPTP down-regulation of nigrostria and striatum cells and nerve fiber TH immunopositive expression.
  • the ligand-modified gold cluster provided by the present invention can significantly improve the spontaneous activity, exercise ability and body coordination ability of MPTP-induced PD model mice, and is specific to DA neurons in the substantia nigra and striatum.
  • Sexual loss has a significant protective effect, indicating that substances containing gold clusters can be used against PD.
  • the specific method is as follows: collect the SH-sy5y cells in which the cells are propagated in the log phase (the cells are passed to the sixth generation), adjust the concentration of the cell suspension, add 100 ⁇ L per well, and plate the cells to adjust the density to 1000-10000 wells.
  • Cell culture plates (96 well flat bottom edge wells filled with cell culture medium) were placed in a cell incubator and incubated for 24 h in a 5% CO 2 , 37 ° C environment to allow cells to adhere.
  • the 96-well plate was taken out, alcohol-sterilized, placed in a biosafety cabinet, and the original cell culture medium was aspirated, and the ligands listed in Table 1 diluted with the cell culture solution to 1 ppm, 10 ppm, 50 ppm, 100 ppm, 200 ppm, and 500 ppm were respectively added.
  • the gold cluster solution, the control group (no gold cluster) was added with the same amount of fresh cell culture medium, and then placed in the cell incubator for 48 hours, and the experimental group and the control group each set 6 replicate wells.
  • the culture solution was removed by centrifugation, washed with PBS for 2-3 times, and 100 ⁇ L of fresh medium and 20 ⁇ L of thiazolyl blue (MTT) solution (5 mg/ml, 0.5% MTT) were added to each well, and the culture was stopped for 4 hours.
  • Culture take out 96-well plate, centrifuge (1000r/min) for 10min, aspirate the supernatant, add 200 ⁇ L DMSO to each well, shake it on a shaker for 10min at low speed until the color in the well is evenly dissolved to dissolve the crystal; use the microplate reader to measure The absorbance of each well at 490 nm. All of the above operations must be performed in a sterile environment. All procedures are completed in the biosafety cabinet except for testing. The laboratory supplies must be sterilized by a high temperature steam sterilizer before use.
  • Example 2 Taking the L-NIBC modified gold cluster of Example 2 as an example, the results are shown in Fig. 26, wherein the A-C-webs are 2.6 nm, 1.8 nm, and 1.1 nm, respectively, and the final concentrations are 1 ppm, 10 ppm, and 50 ppm, respectively.
  • L-NIBC modified gold clusters has little effect on cell viability, at higher concentrations (eg, 200 and 500 ppm), L-NIBC modified The addition of gold clusters results in a small degree of cellular damage (cell mortality is less than 20%). Since 100 ppm is much larger than the effective concentration of the drug (0.1 to 1 ppm or less), it can be considered that the L-NIBC-modified gold cluster is highly safe at the cell level.
  • the gold clusters of different sizes modified by other ligands listed in Table 1 have similar effects, and are not described here.
  • the specific method is as follows: For the different ligand-modified gold clusters listed in Table 1 (for example, the gold cluster having an average diameter of 1.8 nm modified by L-NIBC in Example 2), 60 adult mice were taken. Divided into four groups of 15 each, which were the control group and three experimental groups. The control group was fed normally, while the three experimental groups were fed with gold clusters by oral gavage at a daily dose of 0.1 g/kg body weight, 0.3 g/kg body weight and 1 g/kg body weight. One week. Stop feeding the gold clusters and continue normal feeding for 30 days. Observe the abnormal response of the mice.
  • mice In the mouse experiment, three different concentrations of gold clusters of different sizes had no effect on the survival and activity of the mice. Even at high doses of 1 g/Kg body weight, the mice remained healthy.
  • Example 11 Tissue distribution and metabolic distribution of gold-containing clusters in mice
  • mice were randomly divided into four groups, 20 in each group.
  • the ligand-modified gold clusters listed in Table 1 were fed by oral gavage.
  • the amount of gold clusters fed in each group was 100 mg/kg. 20 mg/kg, 5 mg/Kg and 1 mg/kg.
  • 20 mice in each group were randomly divided into 4 groups, 5 rats in each group.
  • the mice were sacrificed at 2h, 6h, 24h and 48h after feeding, and the heart, liver, spleen and lung were separated. Kidney and brain tissue. The tissues were weighed, then 2 mL of water was added for tissue homogenization.
  • the results show that gold clusters can reach the brain through the blood-brain barrier and can be excreted over time and thus have no obvious accumulation in the body. Therefore, the gold cluster-containing substances provided by the present invention are used in the preparation of AD or PD. Have good prospects.
  • mice were randomly divided into four groups, 20 in each group.
  • the mice were given the ligand-modified gold clusters listed in Table 1 by intraperitoneal injection.
  • Each group of gold clusters (with L-NIBC)
  • the amount of modified gold cluster having an average diameter of 1.8 nm is 100 ppm, 20 mg ppm, 5 ppm, and 1 ppm, respectively, relative to the body weight of the mouse.
  • 20 mice in each group were randomly divided into 4 groups, 5 rats in each group. The mice were sacrificed at 2h, 6h, 24h and 48h after feeding, and the heart, liver, spleen and lung were separated. Kidney and brain tissue.
  • gold clusters of different sizes modified by various ligands used in the present invention are found to be capable of two types in the case where the amount of gold clusters is very low (e.g., 0.1-1 ppm).
  • Model cell viability increased from 50%-65% to over 95%. It shows that the cell layer has a significant effect on the gold cluster. Since the ligands used have no effect on the aggregation of A ⁇ and both cell models (Example 4, Example 7 and Example 8), the efficacy of gold clusters can be derived from its own conclusions. This puts forward new ideas for the application of gold clusters.
  • the present invention uses the transgenic mouse model of AD and the MPTP-induced PD mouse model (Example 5, Example 9) to further verify the efficacy of the gold cluster, indicating that the gold cluster used is improved.
  • Mice have cognitive effects on cognitive behavior, motor behavior, inhibition of senile plaque formation in the brain, and inhibition of MPTP-induced nigral and striatum-specific neuronal apoptosis. They can be used as prevention of related diseases. medicine.
  • the gold cluster had no significant effect on the cell survival rate when co-cultured with nerve cells at a concentration of 100 ppm by weight, exceeding 100 ppm (much greater than the effect of the drug) At the concentration), the cell survival rate decreased slightly. Since the effective concentration of gold clusters (0.1-1 ppm) is much lower than 100 ppm, it is considered that gold clusters have excellent biosafety at the cell level.
  • the administration of 1 g/kg body weight (equivalent to 1000 ppm) once a day was continued for seven days, and the mice showed no adverse reactions.
  • the content of gold in the brain reached 1%-10% of the initial concentration. After 6 hours, the content in the brain could be maintained similarly. At the level of 24 hours, the content in the brain decreased significantly. At 48 hours, except for the sample with 100 ppm dose, it decreased below the detection limit.
  • the above results indicate that the gold-clustered material also has good biosafety at the animal level, can penetrate the blood-brain barrier, and has no obvious accumulation in the body.
  • the gold-containing clusters are at the animal level. It also has good biosafety, can penetrate the blood-brain barrier, and has no obvious accumulation in the body, so it has a good prospect in the preparation of drugs for the treatment of AD or PD.
  • the ligand used in the present invention is not specifically designed for the aggregation behavior of A ⁇ and ⁇ -syn, and comparative experiments show that the ligand used has no significant effect on the aggregation of A ⁇ and ⁇ -syn (implementation) Example 3), but since the size of the gold cluster is smaller than the size of the protein itself, the aggregation of A ⁇ and ⁇ -syn can be greatly suppressed by the combination of the scale effect and the weak intermolecular interaction.
  • the superior effects of the A ⁇ -induced AD cell model and the transgenic animal model further demonstrate the feasibility of using gold-clustered materials for the preparation of drugs for the treatment of AD.
  • MPP + induced PD cell model and MPTP induced PD animal model indicate that gold cluster-containing substances also have broad application prospects in the preparation of drugs for the treatment of other neurodegenerative diseases.
  • MPP + induced PD cell model and the MPTP-induced PD animal model do not involve protein fibrosis, but a deeper mechanism for energy metabolism and neurotransmitter metabolism-related signaling functions of nerve cells, It can be speculated that in addition to affecting protein fibrosis, substances containing gold clusters can affect 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 gold cluster-containing material provided by the invention can improve cognitive behavior, motor behavior and inhibition of senile plaque formation in the brain in the transgenic mouse model of AD and the MPTP-induced PD mouse model, and at the animal level. It also has good biosafety and is suitable for industrial applications.

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Abstract

一种金团簇的医药用途以及一种含金团簇的物质及其制备方法与应用。金团簇及含金团簇的物质能够抑制Aβ、α-syn的聚集，并且在细胞模型和动物模型层面也具有优异的效果，可用于制备预防和治疗阿兹海默病和/或帕金森的药物。

Description

一种含金团簇的物质及其制备方法与应用 技术领域
本发明涉及纳米药物技术领域，特别是涉及一种含金团簇的物质及其制备方法与应用。
背景技术
神经退行性疾病是人类健康的重大威胁之一，其共通的病理特征是神经细胞内存在蛋白质异常缠结及淀粉样纤维化变性，以及与之相关的神经细胞凋亡及神经功能损伤。阿兹海默病(AD)和帕金森症(PD)是其中最典型的两种。AD的临床表现以记忆与认知功能障碍以及人格和行为改变为特征，而PD的临床表现主要包括静止性震颤、运动迟缓、肌强直和姿势步态障碍等运动功能障碍。AD与PD均主要发病于老年人，且发病率随着年龄的增大而增大，如AD，65岁以上人群发病率为5％，而80岁以上人群则高达30％以上。因此，随着人类寿命的延长和人口老龄化的加剧，这两种疾病的患病人数持续增加。特别是AD，迄今已有超过四千万患者，预计2050年将达到1.5亿。仅美国，照顾AD病人的费用每年就已超过2000亿美元，为癌症的2倍，使其成为世界上最昂贵的疾病。全世界PD患者数目据保守估计也达到一千万以上。然而，这两类疾病病因迄今未知。在临床治疗方面，虽然已有几种药物被美国FDA批准用于轻度和中度AD或PD的治疗，但这些药物都属于神经递质调节类药物，仅能暂时改善患者的认知或运动功能，停药之后会很快反弹，目前尚无任何药物能终止或逆转其病理进程。因此，开发新型AD或PD治疗药物具有重大意义。
研究发现：AD患者脑部的淀粉样纤维化蛋白主要以β-淀粉样蛋白(Aβ)和Tau蛋白为主，还含有少量α-突触核蛋白(α-syn)，起始发病部位为脑内担任记忆学习与空间定位功能的海马体。而PD患者脑部的损伤起始于负责躯体运动功能的中脑黑质。起始发病部位的不同决定了两种疾病患者不同的症状。然而，研究表明，一半以上的AD患者在后期会出现运动功能障碍，而大多数PD患者在后期也会出现AD患者的症状，表明两种疾病发病机制和疾病进程存在内在的相关性。
脑内老年斑的形成是AD的基本病理特征之一。作为其主要组成物质的Aβ是由36-43个氨基酸组成的多肽，是纤维化蛋白前体蛋白(APP)的水解产物，其中Aβ(1-40)的含量约占Aβ总量的90％以上。当前研究已明确，虽然Aβ有正常的生理功能，可通过调控胆碱酯酶的催化活性调控神经突触间的乙酰胆碱能信号传递，但Aβ在脑内的过度聚集与纤维化可引起神经突触功能障碍，及后续的继发性炎症反应，导致神经元功能丧失和死亡。因此，研发能够抑制Aβ的聚集及纤维化，阻断其神经毒性的物质 是AD药物研发的重要思路之一。
PD的病理特征主要表现为黑质纹状体系统多巴胺(DA)能神经元进行性缺失，同时伴随路易小体的产生。路易小体主要由α-syn变性聚集而成的中空的放射状淀粉样纤维构成。α-syn处于神经元突触前膜末梢，在体内的自然状态是可溶的非折叠状态，在病理条件下会发生错误折叠，产生β-片层结构，进而聚集纤维化形成路易小体病变结构。研究表明，α-syn的淀粉样变性对该疾病的病理过程发挥关键的作用。因此，抑制α-syn的聚集与纤维化也成为PD预防与治疗药物研发中的思路之一。另一方面，1-甲基-4-苯基-1,2,3,6-四氢吡啶(MPTP)是一种神经毒素，其本身并无毒性，但当进入大脑后，其代谢产生的1-甲基-4-苯基吡啶阳离子(MPP+)能破坏黑质中的DA神经元，同时，MPP+还能干扰线粒体代谢中的呼吸链里一种重要物质NADH脱氢酶，进而导致细胞死亡和并引起自由基的积蓄。由此而引发的DA神经元大量死亡严重影响大脑皮质对运动的控制作用，导致PD的类似症状。因此MPTP及MPP+被广泛应用于PD相关动物模型及细胞模型的建立以及PD的药物研发中。
金纳米粒子是尺寸为纳米级(研究中所用的金纳米粒子的金核直径通常大于3nm)的金颗粒，因其具有独特的光学和电学性质，良好的生物相容性，并易于表面修饰，广泛应用于生物传感器、医学成像和肿瘤检测等生物及医学相关领域。由于其化学惰性和巨大的比表面，以及具有低浓度下穿透血脑屏障的能力，金纳米粒子也作为药物载体用于药物定向输运、药物可控释放等方面的研究。近年来，有研究将金纳米粒子与对纤维化蛋白的聚集有抑制作用的特定配体(如杂多酸、特定序列的多肽等)结合，在抑制蛋白纤维化变性的体外实验中取得一定效果(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 2013,13,6156；S.Hsieh,C.W.Chang,H.H.Chou,Colloids and Surfaces B:Biointerfaces,2013,112,525)，但细胞模型的结果表明，虽然金纳米粒子(金核尺寸5nm以上)与对纤维化蛋白损伤细胞具有保护作用的化合物共同使用时对细胞存活率提升存在一定协同作用(N.Gao,H.Sun,K.Dong,J.Ren,X.Qu,Chemistry-A European Journal 2015,21,829)，但其单独使用时作用并不明显。AD动物模型层面的实验则未见报道。并且在这些研究中，金纳米粒子主要作为药物载体使用，而不是起效成分。
金团簇是一种超微金纳米粒子，金核直径小于3nm。其中仅含有数个至数百个金原子，导致常规金纳米粒子中所具有的金原子的面心立方堆积结构坍塌，能级发生分裂，从而表现出与3nm以上的常规金纳米粒子完全不同的类分子的性质：一方面，由于能级分裂，金团簇不具备常规金纳米粒子所具有的表面等离子体效应及衍生的光学 性质，却表现出与半导体量子点相似的优异荧光发射性质；另一方面，金团簇的紫外可见吸收光谱中在520±20nm处的等离子体共振峰消失，而在560nm以上出现一个或多个新的吸收峰，而这类吸收峰在常规金纳米粒子中观察不到，因此紫外可见吸收光谱中等离子体共振吸收峰(520±20nm)的消失和560nm以上新吸收峰的出现是判断金团簇是否制备成功的重要标志(H.F.Qian,M.Z.Zhu,Z.K.Wu,R.C.Jin,Accounts of Chemical Research 2012,45,1470)。金团簇还具有与常规金纳米粒子明显不同的磁学、电学、催化性质和光热效应，因而在单分子光电、分子催化、光热转变等领域具有广阔的应用前景。
此外，金团簇由于优异的荧光发射性质在生物探针及医学成像领域也已获得应用。例如，Sandeep Verma课题组将嘌呤修饰的金团簇作为绿色荧光探针用于细胞核成像，(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)，该类文献利用的是金团簇的荧光特性，而未涉及其本身的药用活性。
发明内容
本发明的目的是针对现有技术中存在的技术缺陷，第一方面，提供一种具有药用活性的含金团簇的物质，包括金团簇及其外部包覆的配体Y。
所述金团簇的金核直径小于3nm，优选0.5-2.6nm。
所述配体Y包括但不局限于L(D)-半胱氨酸及其衍生物、含半胱氨酸的寡肽及其衍生物、其它含巯基的化合物中的一种或几种。
所述L(D)-半胱氨酸及其衍生物优选L(D)-半胱氨酸、N-异丁酰基-L(D)-半胱氨酸(L(D)-NIBC)或N-乙酰基-L(D)-半胱氨酸(L(D)-NAC)等。
所述含半胱氨酸的寡肽及其衍生物优选含半胱氨酸的二肽、含半胱氨酸的三肽或含半胱氨酸的四肽。
含半胱氨酸的二肽优选L-半胱氨酸-L-精氨酸二肽(CR)、L-精氨酸-L-半胱氨酸二肽(RC)、L-组氨酸-L-半胱氨酸二肽(HC)或L-半胱氨酸-L-组氨酸二肽(CH)等。
所述含半胱氨酸的三肽优选甘氨酸-L-半胱氨酸-L-精氨酸三肽(GCR)、L-脯氨酸-L-半胱氨酸-L-精氨酸三肽(PCR)、L-赖氨酸-L-半胱氨酸-L-脯氨酸三肽(KCP)或L-谷胱甘肽(GSH)等。
所述含半胱氨酸的四肽优选甘氨酸-L-丝氨酸-L-半胱氨酸-L-精氨酸四肽(GSCR)或甘氨酸-L-半胱氨酸-L-丝氨酸-L-精氨酸四肽(GCSR)等。
所述其它含巯基的化合物优选1-[(2S)-2-甲基-3-巯基-1-氧代丙基]-L-脯氨酸、巯基乙酸、巯基乙醇、苯硫酚、D-3-巯基缬氨酸、N-(2-巯基丙酰基)-甘氨酸或十二硫醇等。
所述物质为粉末或絮状物。
第二方面，本发明提供一种制备上述含金团簇的物质的方法，包括以下步骤：
(1)把HAuCl4溶于甲醇、水、乙醇、正丙醇、乙酸乙酯中的一种配成HAuCl4浓度为0.01～0.03M的溶液A；
(2)把配体Y溶于溶剂中配成浓度为0.01～0.18M的溶液B；
(3)将步骤(1)的溶液A和步骤(2)的溶液B混合，HAuCl4和配体Y的摩尔比为1:(0.01～100)(优选1：(0.1-10)，更优选1：(1-10))，在冰浴下搅拌0.1～48h(优选0.1-24h，更优选0.5-2h)，滴加0.025～0.8M的NaBH4溶液(优选NaBH4的水溶液、NaBH4的乙醇溶液、NaBH4的甲醇溶液)后，在冰水浴中继续搅拌0.1～12h(优选0.1-2h，更优选1-2h)，NaBH4与配体Y的摩尔比为1:(0.01～100)(优选1：(0.1-8)，更优选1：(1-8))；
(4)将步骤(3)的反应液以8000～17500r/min离心10～100min，即可得到不同平均粒径的金团簇沉淀；优选的，将步骤(3)的反应液用截留分子量为3K～30K的超滤管以8000～17500r/min梯度离心10～100min，即可得到不同平均粒径的金团簇；
(5)将步骤(4)得到的不同平均粒径的金团簇沉淀溶于水并装入透析袋中在室温下置于水中透析1～7天；
(6)将透析袋内的金团簇溶液冷冻干燥12～24h，得到含金团簇的物质。
步骤(2)中的所述溶剂为甲醇、乙酸乙酯、水、乙醇、正丙醇、戊烷、甲酸、乙酸、乙醚、丙酮、苯甲醚、1－丙醇、2－丙醇、1－丁醇、2－丁醇、戊醇、乙醇、乙酸丁酯、三丁甲基乙醚、乙酸异丙酯、二甲亚砜、乙酸乙酯、甲酸乙酯、乙酸异丁酯、乙酸甲酯、2－甲基－1－丙醇、乙酸丙酯中的一种或多种。
第三方面，本发明提供上述含金团簇的物质在制备催化剂或分子催化、手性识别、分子检测、生物医学检测与成像等领域中的近红外荧光探针中的应用。
第四方面，本发明提供上述含金团簇的物质在制备与Aβ的聚集及纤维化相关的疾病和或与α-syn的聚集及纤维化相关的疾病的药物中的应用。
第五方面，本发明提供上述含金团簇的物质在制备预防和治疗阿兹海默病药物中的应用。
第六方面，本发明提供上述含金团簇的物质在制备预防和治疗帕金森症药物中的应用。
第七方面，本发明提供金团簇在制备与Aβ的聚集及纤维化相关的疾病的药物中的应用。
所述与Aβ的聚集及纤维化相关的疾病为阿兹海默病。
所述金团簇为由L-谷胱甘肽(GSH)、N-乙酰基-L(D)-半胱氨酸(L(D)-NAC)、N-异丁酰基-L(D)-半胱氨酸(L(D)-NIBC)、L-半胱氨酸-L-精氨酸二肽(CR)、L-精氨酸-L-半胱氨酸二肽(RC)、1-[(2S)-2-甲基-3-巯基-1-氧代丙基]-L-脯氨酸(Cap)或L(D)-半胱氨酸(L(D)-Cys)等修饰的。
第八方面，本发明提供金团簇在制备与α-syn的聚集及纤维化相关的疾病的药物中的应用。
所述与Aβ的聚集及纤维化相关的疾病为帕金森症。
所述金团簇为由L-谷胱甘肽(GSH)、N-乙酰基-L(D)-半胱氨酸(L(D)-NAC)、N-异丁酰基-L(D)-半胱氨酸(L(D)-NIBC)、L-半胱氨酸-L-精氨酸二肽(CR)、L-精氨酸-L-半胱氨酸二肽(RC)、1-[(2S)-2-甲基-3-巯基-1-氧代丙基]-L-脯氨酸(Cap)或L(D)-半胱氨酸(L(D)-Cys)等修饰的。
本发明提供的含金团簇的物质在抑制Aβ和α-syn聚集的体外实验表现出优异的抑制Aβ和α-syn聚集的效果，在Aβ诱导的细胞AD模型和MPP+诱导的细胞PD模型实验中对改善细胞存活率表现出优异效果。在AD的转基因小鼠模型中，该含金团簇的物质可显著改善患病小鼠的认知行为能力，对小鼠海马区及脑皮质内Aβ(1-40)和Aβ(1-42)斑块的形成均有显著抑制作用。在MPTP诱导的PD小鼠模型中，该含金团簇的物质可显著改善并纠正MPTP损伤的模型小鼠的运动行为障碍，提升患病小鼠的运动行为能力，并大幅度抑制MPTP诱导的小鼠黑质与纹状体DA能神经元的特异性凋亡。并且在细胞层面和动物层面也具有良好的生物安全性。以上结果说明，本发明的含金团簇的物质除影响纤维化蛋白的聚集与纤维化外，还能在神经细胞的能量代谢及神经递质代谢相关的信号传导功能等更深的层面影响神经退行性疾病的进程，因此，本发明的含金团簇的物质对AD和/或PD等神经退行性疾病的新药研发有重要意义。
另一方面，由于配体分子本身在体外抑制Aβ聚集的动力学实验并未表现出抑制作用，在Aβ损伤的AD细胞模型试验和MPP+损伤的PD细胞模型对细胞存活率也无提升作用，这表明对AD和PD的药效来自于金团簇本身，而不是配体。基于金团簇本身的药用活性，有望研发出具竞争力的新药。
附图说明
图1为不同粒径的配体L-NIBC修饰的金纳米粒子的紫外可见光光谱、透射电镜照片和粒径分布图；
图2为不同粒径的配体L-NIBC修饰的金团簇的紫外可见光光谱、透射电镜照片和粒径分布图；
图3为不同粒径的配体L-NIBC修饰的金团簇的红外光谱图；
图4为Aβ(1-40)与均为配体L-NIBC修饰的金纳米粒子或金团簇共同孵育48h后的AFM形貌图；
图5为不同粒径、不同浓度的均为配体L-NIBC修饰的金纳米粒子和金团簇的Aβ纤维化动力学曲线图；
图6为不同粒径、不同浓度的均为配体L-NIBC修饰的金纳米粒子或金团簇对Aβ诱导的AD细胞模型细胞存活率影响图；
图7为配体CR修饰的金团簇(CR-AuNCs)的紫外、红外、透射电镜和粒径分布图；
图8为配体RC修饰的金团簇(RC-AuNCs)的紫外、红外、透射电镜和粒径分布图；
图9为配体1-[(2S)-2-甲基-3-巯基-1-氧代丙基]-L-脯氨酸(即卡托普利(Cap))修饰的金团簇(Cap-AuNCs)的紫外、红外、透射电镜和粒径分布图；
图10为配体GSH修饰的金团簇(GSH-AuNCs)的紫外、红外、透射电镜和粒径分布图；
图11为配体D-NIBC修饰的金团簇(D-NIBC-AuNCs)的紫外、红外、透射电镜和粒径分布图；
图12为不同配体修饰金团簇对Aβ(1-40)聚集及纤维化的抑制效果图；
图13为实施例5中的水迷宫实验装置示意图；
图14为含金团簇的物质对APP/PS1双转基因C57BL/6小鼠模型认知行为学(给药第150天)影响图；
图15为含金团簇的物质对APP/PS1双转基因C57BL/6小鼠模型鼠海马区及脑皮质Aβ(1-40)表达(给药100天)的影响图；
图16为含金团簇的物质对APP/PS1双转基因C57BL/6小鼠模型鼠海马区及脑皮质Aβ(1-42)表达(给药100天)的影响图；
图17为含金团簇的物质对APP/PS1双转基因C57BL/6小鼠模型鼠海马区及脑皮质Aβ(1-40)表达(给药150天)的影响图；
图18为含金团簇的物质对APP/PS1双转基因C57BL/6小鼠模型鼠海马区及脑皮质Aβ(1-42)表达(给药150天)的影响图；
图19为含金团簇的物质对α-syn纤维化动力学的影响图；
图20为含金团簇的物质对MPP+损伤的PD细胞(SH-sy5y)模型细胞存活率影响图；
图21为含金团簇的物质对MPP+诱导的PD细胞(PC12)模型细胞凋亡的影响图；
图22为含金团簇的物质对MPTP损伤模型小鼠自发活动行为的影响图；
图23为含金团簇的物质对MPTP损伤模型小鼠游泳活动能力的影响图；
图24为含金团簇的物质对MPTP损伤模型小鼠滚轴行为学的影响图；
图25为含金团簇的物质对MPTP损伤模型小鼠的黑质及纹状体DA能神经元的影响图；
图26为不同粒径、不同浓度的含金团簇的物质对SH-sy5y神经母瘤细胞存活率的影响图。
具体实施方式
发明人在研究具有某些配体的金纳米粒子对Aβ聚集的作用时发现：当金纳米粒子金核直径从大变小时，表面相同配体修饰的金纳米粒子对Aβ的聚集从促进作用转变为抑制作用，当其粒径足够小转变为金团簇时，可实现Aβ的聚集的完全抑制。此外还发现，金团簇对α-syn也有完全的抑制效果。而这一效应中起抑制作用的并不是配体，而是金团簇本身。
通常，研究中所用的金纳米粒子金核直径在3nm以上，而当金核直径小于3nm时被称为金团簇，紫外可见吸收光谱中等离子体共振吸收峰(520±20nm)的消失和560nm以上新吸收峰的出现是判断金团簇是否制备成功的标志。金团簇不能脱离配体单独在溶液中稳定存在，其与含巯基的配体通过Au-S键结合，形成配体修饰的金团簇(或称金团簇)。
已有文献公开的配体修饰的金团簇有L-谷胱甘肽(GSH)、N-乙酰基-L(D)-半胱氨酸(L(D)-NAC)、N-异丁酰基-L(D)-半胱氨酸(L(D)-NIBC)等修饰的金团簇等，其制备过程见文献(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)；其应用集中于催化、手性识别、分子检测、生物传感、药物运输、生物成像等领域(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)。
本发明围绕金团簇对AD和/或PD的影响进行研究，至少包括：首先以具有不同配体(对Aβ的聚集没有抑制作用的配体)不同尺寸的金团簇为对象，通过抑制Aβ聚集以及抑制α-syn聚集的体外实验、Aβ诱导的AD细胞模型及MPP+诱导的PD细胞模型实验、AD转基因小鼠模型及MPTP诱导的PD小鼠模型实验三个层面的研究，并结合金团簇的细胞毒性、小鼠急性毒性实验、小鼠体内分布实验等，提供了配体修饰的金团簇， 发现其在制备治疗AD及PD的药物中的应用，并与金纳米粒子的实验结果对比，阐明直径大于3nm的金纳米粒子在这一用途中效果不佳，不能用于制备治疗AD或PD的药物，而配体修饰的金团簇可以用作制备治疗AD和/或PD的药物。
以下结合具体实施例，更具体地说明本发明的内容，并对本发明作进一步阐述，但这些实施例绝非对本发明进行限制。
下列实施例中所用原料的纯度只要达到化学纯以上即可，来源均可从市场购得。
实施例1：制备配体修饰的金团簇
本实施例介绍制备配体修饰的金团簇的方法，包括以下步骤：
(1)把HAuCl4溶于甲醇、水、乙醇、正丙醇、乙酸乙酯中的一种配成溶液A，其中HAuCl4的浓度为：0.01～0.03M；
(2)把配体Y溶于溶剂中配成溶液B，其中，配体Y的浓度为：0.01～0.18M；配体Y包括但不局限于L(D)-半胱氨酸及其他半胱氨酸衍生物，如N-异丁酰基-L-半胱氨酸(L-NIBC)、N-异丁酰基-D-半胱氨酸(D-NIBC)、N-乙酰基-L-半胱氨酸、N-乙酰基-D-半胱氨酸等；含半胱氨酸的寡肽及其衍生物,包括但不局限于含半胱氨酸的二肽、三肽、四肽及其它肽，如：L-半胱氨酸-L-精氨酸二肽(CR)、L-精氨酸-L-半胱氨酸二肽(RC)、L-半胱氨酸L-组氨酸(CH)、甘氨酸-L-半胱氨酸-L-精氨酸三肽(GCR)、L-脯氨酸-L-半胱氨酸-L-精氨酸三肽(PCR)、L-谷胱甘肽(GSH)、甘氨酸-L-丝氨酸-L-半胱氨酸-L-精氨酸四肽(GSCR)、甘氨酸-L-半胱氨酸-L-丝氨酸-L-精氨酸四肽(GCSR)等；及其他含巯基的化合物，如1-[(2S)-2-甲基-3-巯基-1-氧代丙基]-L-脯氨酸、巯基乙酸、巯基乙醇、苯硫酚、D-3-巯基缬氨酸、十二硫醇等中的一种或多种；溶剂为甲醇、乙酸乙酯、水、乙醇、正丙醇、戊烷、甲酸、乙酸、乙醚、丙酮、苯甲醚、1－丙醇、2－丙醇、1－丁醇、2－丁醇、戊醇、乙醇、乙酸丁酯、三丁甲基乙醚、乙酸异丙酯、二甲亚砜、乙酸乙酯、甲酸乙酯、乙酸异丁酯、乙酸甲酯、2－甲基－1－丙醇、乙酸丙酯中的一种或多种；
(3)将溶液A和溶液B混合，使得HAuCl4和配体Y的摩尔比为1:(0.01～100)，在冰浴下搅拌反应0.1～48h，滴加0.025～0.8M的NaBH4的水、乙醇或甲醇溶液，在冰水浴中继续搅拌反应0.1～12h，NaBH4与配体Y的摩尔比为1:(0.01～100)；
(4)反应结束后将反应液用截留分子量为3K～30K的超滤管以8000～17500r/min梯度离心10～100min，即可得到不同平均粒径的配体修饰的金团簇沉淀(具体的梯度离心如实施例2中(4)中所述，因不同截留分子量的超滤管的滤膜的孔径直接决定了能通过的金团簇的尺寸)，此步骤也可省略，即在步骤(3)结束后直接进入步 骤(5)，得到的是不同尺寸混合的金团簇；
(5)将步骤(4)得到的不同平均粒径的金团簇沉淀溶于水并装入透析袋中在室温下置于水中透析1～7天；
(6)透析后，冷冻干燥12～24h，得到的粉末状或絮状物质即为配体修饰的金团簇。
经检测(具体检测方法参见实施例2)，用以上方法得到的粉末或絮状物质，其粒径均小于3nm(一般分布在0.5-2.6nm)，其紫外-可见吸收光谱在560nm以上出现一个或者多个吸收峰，在520nm没有明显吸收峰，确定获得的粉末或絮状物为金团簇。
实施例2：不同配体修饰的金团簇的制备与确认
以配体L-NIBC为例，详述配体L-NIBC修饰的金团簇的制备与确认。
(1)称取1.00g的HAuCl4溶于100mL甲醇配成浓度为0.03M的溶液A；
(2)称取0.57g的L-NIBC，溶于100mL冰醋酸(乙酸)配成浓度为0.03M的溶液B；
(3)取1mL溶液A分别与0.5mL、1mL、2mL、3mL、4mL、5mL的溶液B混合(即HAuCl4与L-NIBC摩尔比分别为1:0.5、1:1、1:2、1:3、1:4、1:5)，冰浴搅拌下反应2h，溶液颜色由亮黄色变为无色，迅速加入新配制的0.03M(称取11.3mg NaBH4溶于10mL乙醇配制得到)的NaBH4水溶液1mL，溶液颜色变为深褐色后持续反应30min，加入10mL丙酮终止反应。
(4)反应结束后将反应液采用梯度离心法得到不同粒径的L-NIBC修饰的金团簇粉末，具体方法：反应结束后将反应液转移至截留分子量为30K的容积为50mL的超滤管中，用10000r/min的转速离心20min，取内管中的截留物溶于超纯水中即得到粒径为2.6nm左右的粉末，然后将外管中的混合溶液转移至截留分子量为10K的容积为50mL的超滤管中，用13000r/min的转速离心30min，取内管中的截留物溶于超纯水中即得到粒径为1.8nm左右的粉末，然后继续将外管中的混合溶液转移至移至截留分子量为3K的容积为50mL的超滤管中，用17500r/min的转速离心40min，取内管中的截留物溶于超纯水中即得到粒径为1.1nm左右的粉末。
(5)将通过梯度离心法得到的三个不同粒径的粉末沉淀，分别除去溶剂，并将粗品用N2吹干后溶于5mL超纯水，装入透析袋(截留分子量为3KDa)，置于2L超纯水中，隔天换水，透析7天，冷冻干燥后备用。
对以上制备得到的粉末(配体为L-NIBC的金团簇)进行表征实验，同时以配体同为L-NIBC的金纳米粒子作为对照。配体为L-NIBC的金纳米粒子的制备方法参照文献(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 Edition2014,53,4623)中的介绍。
1、透射电子显微镜观察形貌
把待测粉末(实施例2中制得的L-NIBC修饰的金团簇样品和配体同为L-NIBC的金纳米粒子样品)用超纯水稀释到2mg/L作为样品，然后采用悬滴法制样，具体方法为：取5μL样品滴到超薄碳膜网上，自然挥发直至水滴消失，然后在JEM-2100F STEM/EDS型场发射高分辨透射电子显微镜上观察金团簇的形貌。
四个配体为L-NIBC的金纳米粒子样品的透射电子显微镜形貌照片见图1中的B幅、E幅、H幅、K幅；三个配体为L-NIBC的金团簇样品的透射电子显微镜形貌照片见图2中的B幅、E幅、H幅。
图2的照片表明L-NIBC修饰的金团簇样品粒径均匀，分散性好，L-NIBC修饰的金团簇的平均直径(指金核直径)分别为1.1nm、1.8nm和2.6nm，与图2中C幅、F幅、I幅结果相吻合。而相比较的配体为L-NIBC的金纳米粒子样品粒径较大，其平均直径(指金核直径)分别为3.6nm、6.0nm、10.1nm、18.2nm，与图1中C幅、F幅、I幅、L幅结果相吻合。
2、紫外-可见吸收光谱
把待测粉末用超纯水溶解到浓度为10mg·L-1，在室温下测定其紫外可见吸收光谱。扫描范围为190-1100nm，样品池为光程为1cm的标准石英比色皿，参比池盛放超纯水。
结果四个配体为L-NIBC的金纳米粒子样品的紫外-可见吸收光谱见图1中的A幅、D幅、G幅、J幅，粒径的统计分布对应见图1中的C幅、F幅、I幅、L幅；三个配体为L-NIBC的金团簇样品的紫外-可见吸收光谱见图2中的A幅、D幅、G幅，粒径的统计分布对应见图2中的C幅、F幅、I幅。
由图1可以看出，由于表面等离子体效应，配体为L-NIBC的金纳米粒子在520nm左右出现吸收峰，吸收峰的位置与粒径大小相关，其中3.6nm的紫外吸收峰在516nm处，6.0nm的紫外吸收峰在517nm处，10.1nm的紫外吸收峰在520nm处，而18.2nm的吸收峰则红移到523nm处，四个样品在560nm以上均无任何吸收峰。
图2中可以看到，实施例2三个不同粒径的配体为L-NIBC的金团簇样品的紫外吸收光谱中520nm附近的表面等离子体效应吸收峰消失，而在560nm以上出现两个明显的吸收峰，吸收峰的位置随金团簇的粒径不同而略有不同。这是因为金团簇由于面心立方结构的坍塌，表现出类分子的性质，导致金团簇的态密度不再连续，产生能级 分裂，等离子体共振效应消失，同时在长波方向出现新的吸收峰。由此可以判断实施例2得到的三个不同粒径的粉末样品均为配体修饰的金团簇。
3、傅里叶变换红外光谱
红外光谱在布鲁克公司生产的VERTEX80V型傅里叶变换红外光谱仪上采用固体粉末高真空全反射模式测定，扫描范围为4000-400cm-1，扫描64次。以实施例2中制得的L-NIBC修饰的金团簇样品为例，测试样品为L-NIBC修饰的三个不同粒径的金团簇的干燥粉末，对照样品为纯L-NIBC粉末。结果见图3。
图3为L-NIBC修饰的不同粒径的金团簇的红外光谱，对比纯的L-NIBC(最上面的曲线),L-NIBC修饰的不同粒径的金团簇在2500-2600cm-1之间的S-H伸缩振动均完全消失,而其他L-NIBC的特征峰仍可观察到，证明L-NIBC分子成功的通过金硫键锚定到金团簇表面。该图还表明配体修饰的金团簇的红外光谱与其尺寸无关。
用上述类似的方法制备其它配体Y修饰的金团簇，只是溶液B的溶剂、HAuCl4与配体Y的投料比、反应时间和NaBH4加入量稍作调整，如：L-半胱氨酸、D-半胱氨酸、N-异丁酰基-L-半胱氨酸(L-NIBC)、N-异丁酰基-D-半胱氨酸(D-NIBC)做配体Y时，选择乙酸作为溶剂；二肽CR、二肽RC、1-[(2S)-2-甲基-3-巯基-1-氧代丙基]-L-脯氨酸做配体Y时，选用水作为溶剂，等等；其余步骤类似，不再一一赘述。
本发明按上述方法制备得到一系列配体修饰的金团簇，所用的配体及制备过程的参数见表1。
表1本发明不同配体修饰金团簇的制备参数
Figure PCTCN2017093671-appb-000001
Figure PCTCN2017093671-appb-000002
采用上述相同的方法确认表1所列各实施例样品，图7-图11分别是配体CR、RC、1-[(2S)-2-甲基-3-巯基-1-氧代丙基]-L-脯氨酸(缩写：Cap)、GSH和D-NIBC修饰的金团簇相应的紫外光谱(图7-图11中的A幅)、红外谱图(图7-图11中的B幅)、透射电镜照片(图7-图11中的C幅)和粒径分布(图7-图11中的D幅)。
结果表明：表1得到的不同配体修饰的金团簇直径均在3nm以下，紫外光谱也表现为520±20nm处的峰消失，大于560nm以上范围出现吸收峰，只是该吸收峰的位 置随配体及粒径的不同而略有变化。同时，傅里叶变换红外光谱也显示配体的巯基红外吸收峰(位于图7-图11中B幅的虚线之间)消失，而其他红外特征峰均保留，说明各配体分子均成功锚定到金团簇表面，表明本发明成功获得了表1所列配体修饰的金团簇。
实施例3：体外Aβ聚集动力学实验
本实施例通过体外Aβ聚集动力学实验来验证配体修饰的金团簇的功能，并将其与配体修饰的金纳米粒子及单独使用配体分子时对Aβ聚集动力学的影响比较，证明其功能来自于金团簇，而不是来自于配体。实验采用ThT荧光标记法表征Aβ(1-40)纤维化聚集动力学。
硫磺素T(thioflavin T,简写：ThT)是一种专门染淀粉样纤维的染料。当其与多肽或蛋白单体共同孵育时，其荧光基本不发生变化，而当其碰到具有纤维结构的淀粉样多肽或蛋白时，会立即与淀粉样多肽或蛋白发生耦合，其荧光强度会呈指数级迅速增强。正是因为这个特性，ThT被广范用于监测多肽或者蛋白淀粉样变性的标记物。Aβ(1-40)的纤维化过程也是一种成核控制的聚合过程，因此，通过ThT荧光标记法测得的Aβ(1-40)纤维的生长曲线主要分为三个阶段：起始期、增长期和平台期。起始期主要是Aβ(1-40)发生构象转变形成错误折叠进而聚集成核的阶段；增长期是Aβ(1-40)单体沿纤维轴向方向累加到核或者寡聚体上形成纤维并快速增长的阶段；平台期是Aβ(1-40)分子全部形成了成熟的长纤维，即纤维不再生长的阶段。ThT荧光标记法可以方便的监测Aβ(1-40)分子的纤维化聚集的动力学过程。
1)Aβ(1-40)单体的前处理
将冻干的淀粉样多肽Aβ(1-40)粉末(Invitrogen Corp.)溶于六氟异丙醇(HFIP)中得到浓度为1g/L的Aβ(1-40)溶液，封口后在室温下孵育2-4小时，然后在通风橱中用高纯氮气(N2，99.9％)以适当的气流速度将六氟异丙醇吹干(大约耗时1小时左右)，最后将吹干后的Aβ(1-40)溶于200μL二甲亚砜(DMSO)中，密封后置于-20℃冰箱中保存备用，保存时间不得超过一周。使用前将淀粉样多肽的DMSO溶液用大量的磷酸盐缓冲液(PBS，10mM，pH＝7.4)稀释至Aβ(1-40)浓度为20μM，得到Aβ(1-40)的PBS缓冲溶液。所有实验用的Aβ(1-40)溶液均为新鲜配置，现配现用。
2)样品的制备和检测
将配体修饰的金团簇和金纳米粒子分别加入到20μM的Aβ(1-40)的PBS缓冲溶液中，形成不同浓度、不同粒径的不同配体修饰的金团簇样品和相应的不同配体修饰的金纳米粒子样品。采用ThT荧光标记法，在96孔板中37℃下连续孵育，用酶标仪每隔10分钟监测一次荧光强度。通过ThT的荧光强度变化来表征Aβ(1-40)聚集的动 力学过程。
实验组采用实施例2制备的粒径为2.6nm、1.8nm、1.1nm的三种L-NIBC修饰的金团簇，对照组采用粒径为18.2nm、10.1nm、6.0nm、3.6nm的四种L-NIBC修饰的金纳米粒子，以及未与金团簇或金纳米粒子结合的L-NIBC分子。对每种粒径的金团簇或金纳米粒子，所用的浓度均有6个，分别是：0ppm(不含金团簇、金纳米粒子或L-NIBC，用于对照)、0.1ppm、1.0ppm、5.0ppm、10.0ppm和20.0ppm，L-NIBC单独使用时，所用的浓度有2个，分别是：1.0ppm、10.0ppm。
结果见图4和图5。
图4分别显示Aβ(1-40)与各实验组和对照组共同孵育48h后的AFM形貌图，其中，A幅为仅有Aβ(1-40)单独孵育48h后的AFM形貌图，B幅为Aβ(1-40)与L-NIBC共同孵育48h后的AFM形貌图，C幅和D幅分别为Aβ(1-40)与平均粒径为6.0nm和3.6nm的金纳米粒子(用L-NIBC修饰)共同孵育48h后的AFM形貌图，E幅为Aβ(1-40)与平均粒径为1.8nm的金团簇(用L-NIBC修饰)共同孵育48h后的AFM形貌图。
图5中A幅为不同浓度L-NIBC存在时的Aβ(1-40)纤维化动力学曲线，B幅-E幅分别为不同浓度下粒径为18.2nm、10.1nm、6.0nm、3.6nm的金纳米粒子存在时的Aβ(1-40)纤维化动力学曲线，F幅-H幅分别为不同浓度下粒径为2.6nm、1.8nm和1.1nm的金团簇存在时的Aβ(1-40)纤维化动力学曲线。图5中A幅-H幅中□代表0ppm(即无金纳米粒子和金团簇)，○代表0.1ppm，△代表1ppm，▽代表5ppm，◇代表10ppm，☆代表20ppm的金纳米粒子或金团簇与Aβ(1-40)一起孵育时，Aβ的纤维化动力学曲线。
从图4可以看到作为对照的A幅里面布满了Aβ纤维；B幅里也布满了Aβ纤维；C幅中虽然纤维有所减少，但还是能看到较长的纤维，D幅中虽然看不到长纤维，但依然存在大量的Aβ短纤维。这说明，L-NIBC对Aβ(1-40)纤维的形成无明显影响，L-NIBC修饰的小尺寸金纳米粒子的加入虽然可以延缓Aβ(1-40)的纤维化进程，但无法实现完全抑制，因短纤维在更长时间后会继续生长成长纤维。图4的E幅可以看到其中既无长纤维也无短纤维，说明L-NIBC修饰的金团簇能够完全抑制Aβ(1-40)的纤维化进程。
图4为定性实验，图5为定量实验，图5的结果表明，L-NIBC的加入对Aβ(1-40)的纤维化动力学无明显影响(图5的A幅)；对金纳米粒子，当颗粒直径大于或等于10.1nm时，L-NIBC修饰的金纳米粒子的加入使得Aβ聚集动力学的增长期和平台期时间皆提前(当金纳米粒子的浓度为20ppm时，Aβ聚集动力学的增长期提前至12h，平台期时间提前至16h)，说明此时L-NIBC修饰的金纳米粒子能加速Aβ的聚集(图 5的B幅和C幅)；而当金纳米粒子颗粒尺寸小于或等于6.0nm(图5的D幅和E幅)时，则能延迟Aβ开始聚集的时间(当L-NIBC修饰金纳米粒子的浓度为20ppm时，Aβ聚集动力学的增长期延迟至54h)，说明此时金纳米粒子对Aβ的聚集有一定抑制作用。但从图5中看到，即使在很大浓度时(20.0ppm)L-NIBC修饰的金纳米粒子的加入也无法实现完全抑制(指不出现增长期，荧光曲线完全为平)的效果。另一方面，L-NIBC修饰的金纳米粒子加入后，由于ThT的荧光发射峰位于515nm处，而L-NIBC修饰的金纳米粒子的等离子体共振吸收峰位于520nm附近，因此此处观察到的ThT荧光强度的下降是金纳米粒子的等离子体共振效应对ThT荧光的部分淬灭，而不能归因于L-NIBC修饰金纳米粒子对Aβ(1-40)聚集的抑制作用。
图5的F幅-H幅表明所有的L-NIBC修饰的金团簇均能大幅度抑制Aβ的聚集(推迟增长期开始的时间，当L-NIBC修饰的金团簇的浓度为5ppm时，20μM的Aβ聚集动力学的增长期开始的时间即可延迟至50h之后)，而且当L-NIBC修饰的金团簇的浓度达到10ppm及以上时，均能完全抑制Aβ的聚集(不出现增长期，荧光曲线完全为平)。至于完全抑制所需的L-NIBC修饰的金团簇最低浓度与配体的种类和金团簇直径有关，其中颗粒尺寸为1.1nm、1.8nm和2.6nm的L-NIBC修饰的金团簇所需的最低浓度分别为5.0ppm，5.0ppm和10.0ppm。此外，由于L-NIBC修饰的金团簇不存在等离子体共振效应，因而对ThT的荧光无淬灭作用，因此，此处观察到的荧光强度的降低完全是由于L-NIBC修饰的金团簇对Aβ(1-40)聚集的抑制作用。图5的定量结果与图4的定性结果完全吻合。
本实验表明：当L-NIBC修饰的金纳米粒子尺寸小于或等于6.0nm时，对Aβ的聚集及纤维化有一定的抑制作用，但作用有限；L-NIBC修饰的金团簇具有完全抑制Aβ聚集及纤维化的功能，由于L-NIBC分子本身不能影响Aβ的聚集及纤维化(结合图4的B幅与图5的A幅)，因此，这一功能来自于金团簇，而不是作为配体的L-NIBC。这为形成与Aβ的聚集及纤维化相关的疾病的药物打下了基础，可归为本发明定义的含有金团簇的物质。
本实施例还对表1所列其他不同配体修饰的金团簇的功能进行了验证，例如，图12的A幅-H幅分别是CR、N-乙酰基-L-半胱氨酸(L-NAC)、GSH、1-[(2S)-2-甲基-3-巯基-1-氧代丙基]-L-脯氨酸(Cap)、D-NIBC、RC、L-半胱氨酸和D-半胱氨酸修饰的金团簇(用量均为10ppm)对Aβ(1-40)的聚集和纤维化的抑制作用效果图。对不同配体修饰的金团簇也观察到类似现象，并能做出相同的结论：这些配体本身不能影响Aβ的聚集及纤维化，配体修饰的尺寸大于3nm的金纳米粒子对Aβ的聚集及纤维化抑制作用有限，更大的金纳米粒子甚至对Aβ的聚集及纤维化有促进作用；而配体修饰的金团簇对Aβ聚集及纤维化具有优异的抑制作用，当浓度达到5-10ppm以上时， 则可实现完全的抑制效果，完全抑制所需的最低浓度根据配体的不同和金团簇颗粒尺寸的不同而略有差别。这些配体修饰的金团簇同样归为本发明定义的含金团簇的物质。
实施例4：Aβ诱导的AD细胞模型实验
本实施例实验以细胞存活率为指标，通过CCK-8法检测的结果，反映配体修饰的金团簇或金纳米粒子样品对抗Aβ(1-40)的毒性作用的效果，以说明配体修饰的金团簇或金纳米粒子在淀粉样蛋白错误折叠致病机制中是否具有神经保护效果。实验所用的细胞为SH-SY5Y神经母瘤细胞株，Aβ诱导的AD细胞模型的构建根据文献(R.Liu,H.Barkhordarian,S.Emadi,C.B.Park,M.R.Sierks,Neurobiology of Disease2005,20,74)中的描述进行。具体方法为：
1)取对数生长期的SH-sy5y细胞(细胞传至第六代)，用完全培养基(MEM培养基+10％FBS+1％青霉素-链霉素)稀释成密度为5×104/mL的细胞悬液，每孔200μL接种于96孔板，置于37℃，5％CO2培养箱中培养。待细胞贴壁后，加入样品。
2)加入由维持培养基(MEM培养基+2％FBS+1％青霉素-链霉素)配制的不同粒径、浓度分别为0.04ppm、0.4ppm、4ppm、20ppm、40ppm和80ppm的配体修饰的金团簇样品或配体修饰的金纳米粒子样品100μL。在培养箱孵育2h后，加入浓度为80μM的Aβ(1-40)100μL，置培养箱中孵育24h。这样，配体修饰的金团簇或配体修饰的金纳米粒子的终浓度分别为0.01ppm、0.1ppm、1ppm、5ppm、10ppm和20ppm，而Aβ(1-40)的终浓度为20μM。同时设置不含SH-sy5y细胞的空白对照组、含有SH-sy5y细胞但不添加配体修饰的金团簇或配体修饰的金纳米粒子及Aβ(1-40)的阴性对照组、含有SH-sy5y细胞的只加入Aβ(1-40)(终浓度为20μM)的细胞模型对照组，以及含有SH-sy5y细胞、Aβ(1-40)(终浓度为20μM)和L-NIBC(终浓度为20ppm)的配体对照组。去除培养液，每孔加入100μL含10％CCK-8的维持培养基孵育4h，于450nm波长处测定各孔吸光度值，用来反映配体修饰的金团簇对Aβ(1-40)损伤的预保护及治疗作用。
以实施例2的L-NIBC修饰的金团簇为例，以L-NIBC修饰的金纳米粒子作为对比，结果见图6。
图6中A幅-C幅分别表示在不同浓度下粒径为1.1nm、1.8nm、2.6nm的L-NIBC修饰的金团簇对Aβ诱导的AD细胞模型中细胞存活率的影响；D幅-F幅分别表示在不同浓度下粒径为3.6nm、6.0nm、10.1nm的L-NIBC修饰的金纳米粒子对Aβ诱导的AD细胞模型中细胞存活率的影响。
由图6可知，单独L-NIBC的加入对细胞的存活率无任何改善作用。L-NIBC修饰的不同尺寸的金团簇(平均尺寸分别为1.1、1.8和2.6nm)在用量很低(如0.1-1ppm) 的情况下，即可使Aβ诱导的AD细胞模型的细胞存活率从接近60％提高至接近95％以上(P均小于0.05，图6中的A幅-C幅)。L-NIBC修饰的平均直径3.6nm的金纳米粒子随着所用浓度的增加对AD细胞模型的细胞存活率有所提高(图6中的D幅)，但均不明显(P>0.05)。而平均直径为6.0nm和10.1nm的L-NIBC修饰的金纳米粒子对细胞存活率均无作用(图6中的E幅、F幅)。以上结果说明，L-NIBC修饰的金团簇对Aβ诱导的AD细胞模型有显著的药效，而L-NIBC修饰的金纳米粒子无明显的药效。
本实施例还对表1所列其它配体修饰的不同尺寸的金团簇进行了实验，结果也均可使Aβ诱导的AD细胞模型的细胞存活率显著提高。说明至少在细胞模型层面，不同配体修饰的金团簇对阿兹海默病均具有优异的治疗效果，可归为本发明定义的含有金团簇的物质，并用于阿兹海默病治疗。
实施例5：AD转基因小鼠模型实验
实验一：
1)分别称取1.0g表1所列的配体修饰的金团簇，溶于100mL水中，作为母液放于4℃环境中冷藏备用，每次使用前取少量用水稀释后使用。
2)取180只B6/J-Tg(APPswe,PSEN1de9)85Dbo/MmNju品系的转基因小鼠(购自南京大学模式动物研究所)，随机分成三组，每组60只：对照组、低剂量给药组和高剂量给药组。从小鼠100日龄时，对照组每日正常喂养，低剂量给药组每日一次口服灌胃200μL浓度为0.5g/L的金团簇水溶液，高剂量给药组每日口服灌胃200μL浓度为2g/L的金团簇水溶液。
3)将对照组、低剂量给药组和高剂量给药组小鼠分别随机分为7批：分别在鼠龄为140天、160天、180天、200天、230天、260天和290天时，采用迷宫实验、开放场实验以及新物体识别实验等研究小鼠的学习和记忆行为的变化。其中，前4批实验每组6只，后3批实验每组6-8只(考虑到小鼠饲养过程中有一定死亡率，以下同)。
4)以上每批小鼠开展行为学研究后，检测血液中Aβ的含量：采用眼眶静脉丛采血，用血清Elisa方法检测Aβ及Aβ聚集体的含量。
5)以上每批小鼠检测血液中Aβ的含量后，检测海马区Aβ淀粉样沉积分布：眼球采血后麻醉，经心灌流固定，取全脑，蔗糖梯度沉降，冰冻切片，用免疫组化法检测海马区Aβ淀粉样沉积的分布。
结果表明本发明提供的配体修饰的金团簇能显著改善AD转基因小鼠的认知行为，抑制其脑内老年斑形成，抑制其病情的发展，可作为含有金团簇的物质用于对抗阿兹海默病。
实验二：
1.分别称取1.0g表1所列的配体修饰的金团簇，溶于100mL水中，作为母液放于4℃环境中冷藏备用，每次使用前取少量用水稀释后形成金团簇溶液使用，每两周配制一次母液。
2.取90只B6/J-Tg(APPswe,PSEN1de9)85Dbo/MmNju品系的转基因小鼠(购自南京大学模式动物研究所)，随机分成三组：模型对照组、低剂量给药组和高剂量给药组，每组30只(考虑到该品系转基因小鼠饲养过程中约有30％的死亡率，为保证后期实验时有足够的小鼠，因此最初小鼠的个数多于后期实验的小鼠个数)。从小鼠100日龄时，模型对照组每日正常喂养，低剂量给药组和高剂量给药组采用腹腔静脉注射方式根据小鼠体重按5mg/Kg体重和20mg/Kg体重的剂量分别给予金团簇溶液，每两日给药一次。
3.利用水迷宫实验测试小鼠的认知行为学。Morris水迷宫实验是一种强迫实验动物游泳，学习寻找隐藏在水中平台的实验，主要用于测试实验动物对空间位置和方向感知的学习记忆能力，被广泛应用于评价阿兹海默病药物开发及评价研究中，其中小鼠寻台潜伏期越短以及撤台后穿越平台次数越多，在目标象限线游泳路程及目标象限停留时间越长则表明小鼠对空间位置和方向感的记忆能力越好。模型小鼠给药150天后采用Morris水迷宫实验测试小鼠的行为学，实验方法参照文献(C.V.Vorhees,M.T.Williams,Nature Protocols 2006,1,848)。具体如下：
(1)定位航行实验：Morris水迷宫测试系统由圆形水池和自动录像及分析系统两部分组成，水池上方有摄像机与计算机连接(如图13)。水迷宫由直径为120cm，高60cm的圆形水池以及直径为9cm的站台构成，液面高出站台0.5cm，水位维持在22±0.5℃。使用白色色素将水染为乳白色。定位航行实验用于测量小鼠在水迷宫中的学习和记忆能力，历时4天。如图13所示，将水迷宫按东(E)西(W)南(S)北(N)四个方向十字交叉划分为4个象限。平台放置在SW象限中部，整个实验过程中平台位置固定不变。训练时，每天从不同象限1/2弧度处将小鼠头朝向池壁，靠近外壁轻轻放入水中。通过摄像跟踪系统记录小鼠爬到隐藏平台上的时间(寻台潜伏期)或到达60s时即停止实验。小鼠在平台上后让其在平台上停留30s，倘若小鼠60s内未找到平台(此时寻台潜伏期计为60s)，则实验者引导小鼠爬上平台，并让其停留30s。每只小鼠试验后移开并轻轻擦干。每只动物每天训练4次，训练之间间隔15-20min，连续训练4天。
(2)空间探索实验：第4天训练完毕后，第5天移走平台，将小鼠由NE弧的中点(平台最远端点)面朝池壁轻轻放入水中，用摄像机记录小鼠60s内的运动轨迹，软件分析小鼠的穿台次数,目标象限停留时间和目标象限游泳路程。
4.免疫组织化学实验检测鼠海马区和脑皮质Aβ(1-40)和Aβ(1-42)的淀粉样沉积分布。大脑皮质和海马出现Aβ在神经元外病理性沉积是AD的主要病理特征。其中，Aβ(1-40)和Aβ(1-42)是脑内老年斑的重要组成成分，具有神经毒性，可导致进行性认知功能障碍和记忆力减退。本实验采用免疫组织化学法检测了海马区及脑皮质内Aβ(1-40)和Aβ(1-42)斑块形成的变化。
具体方法如下：小鼠连续给药100天和150天后，每组取10-12只小鼠做海马及脑皮质免疫组织化学检测，其中给药第150天后的小鼠为做完水迷宫实验的小鼠。小鼠采用5％水合氯醛(10μL/g)腹腔注射麻醉后，在实验台上固定四肢，开胸，充分暴露心脏。注意开胸过程中不能剪到肝脏。经左心室先以0.1mol/L的PBS缓冲液50mL冲洗5min以除去血液，再用含4％多聚甲醛的0.1mol/L的PBS缓冲液灌注固定6min。灌注固定完毕取脑，置于4％多聚甲醛中4℃后固定过夜。将组织依次用10％、20％和30％的蔗糖溶液梯度脱水后-80℃保存备用。将组织进行石蜡包块参考小鼠脑图谱，中脑海马和脑皮质切片(厚度8μm)，用于免疫组化染色。其步骤如下：冰冻切片8μm，室温放置30min后，4℃丙酮固定20min，PBS洗3次(每次5min)，然后用3％过氧化氢孵育10min，消除内在过氧化物酶活性。PBS洗3次(每次5min)后用10％正常山羊血清室温封闭40min(用于做Aβ(1-42)免疫组化的切片在封闭前用10％蚁酸孵育10min修复抗原活性)。倾去血清，滴加抗Aβ(1-40)(ab20068，1:20稀释)或抗Aβ(1-42)工作液体(ab12267，1:200稀释)，室温孵育2h。PBS洗3次(每次5min)。滴加辣根酶标记链霉卵白素(PBS稀释)的二抗工作液，室温孵育1h。PBS洗3次(每次5min)后硫酸镍胺加强DAB蓝色反应法显色10min，当阳性产物呈深蓝色而背底清晰时用蒸馏水冲洗3次终止显色。后用苏木素复染1min，自来水冲洗干净后于通风处晾干，用中性树胶封片。共聚焦显微镜下观察并计数整个海马区域和脑皮质区域Aβ斑块的数量，每个样本分左右脑室，2张切片做平行实验，取平均值做统计分析。所有数据采用SPSS软件(SPSS 21)处理，采用t检验或单因素方差分析，P<0.05表示差异有统计学意义。
以实施例2中L-NIBC修饰的平均尺寸为1.8nm的金团簇为例，给药150天后的水迷宫实验结果如图14所示。结果表明，在定位航行试验训练的第1-2天，模型对照组小鼠与高、低剂量给药组小鼠的寻台潜伏期无统计学差异(P>0.05，n＝10-12/组)(图14的A幅)。随着训练时间延长，高剂量给药组小鼠在第3天和第4天寻台潜伏期明显低于模型组小鼠(P<0.01和P<0.05)，低剂量给药组小鼠寻台潜伏期虽低于模型组小鼠但无统计学差异(P>0.05，见图14的A幅)。小鼠定位航行实验结束后，撤掉平台进行空间搜索实验。结果发现，高剂量给药组的小鼠穿台次数和目标象限的游泳路程相对于模型对照组显著提高(P均小于0.05)，在目标象限的停留时间也显著增 加(P＝0.05)。而低剂量给药组小鼠的穿台次数、目标象限游泳路程和目标象限停留时间相对于模型对照组虽也有提升，但结果无显著性(P>0.05)(图14的B-D幅)。以上结果表明金团簇给药150天可显著提高了APP/PS1小鼠对空间位置和方向感的学习和记忆能力，并且该作用呈剂量依赖性。
免疫组织化学实验检测鼠海马区和脑皮质Aβ(1-40)和Aβ(1-42)的淀粉样沉积分布的实验结果如图15-图18所示。
图15的A幅、B幅和C幅分别是给药100天时高剂量给药组、低剂量给药组和模型对照组的Aβ(1-40)的海马区及脑皮质的典型免疫组织化学切片结果，图15的D幅是统计结果。实验结果表明，与模型对照组比较，给药100天时，高剂量给药能显著降模型小鼠海马区内Aβ(1-40)斑块形成(44.6±12.2％，P<0.05)，而对脑皮质内Aβ(1-40)斑块形成无显著影响(P>0.05)。低剂量给药对海马区及脑皮质内Aβ(1-40)斑块形成均无显著影响(P>0.05)。图16是则是相应的Aβ(1-42)的结果，结果表明，高剂量给药能显著降低脑皮质Aβ(1-42)斑块形成(减少了61.5±11.4％，P<0.05)，而未显著性降低海马区内Aβ(1-42)斑块形成(P>0.05)。低剂量给药对海马区及脑皮质Aβ(1-42)斑块形成均无显著影响(P>0.05)。这些结果说明，给药100天时，金团簇对Aβ(1-40)和Aβ(1-42)斑块的形成均已表现出显著抑制作用，且这一作用呈明显的剂量依赖关系。
随着给药时间延长，小鼠年龄增加，相比给药100天的模型对照组小鼠，给药150天时模型对照组小鼠海马区及大脑皮质内Aβ(1-40)和Aβ(1-42)斑块的形成显著增多，分别为Aβ(1-40)海马区增加57.2±7.2％(P<0.05)，脑皮质增加49.1±19.6％(P<0.05)，Aβ(1-42)海马区增加74.4±7.0％(P<0.05)，脑皮质增加65±11.1％(P<0.05)，表明模型小鼠随着年龄增加，记忆及认知功能受到的影响可能越大。图17的A幅、B幅和C幅分别是给药150天时高剂量给药组、低剂量给药组和模型对照组的Aβ(1-40)的海马区及脑皮质的典型免疫组织化学切片结果，图17的D幅是统计结果。结果表明，高剂量给药组小鼠海马区和脑皮质区内Aβ(1-40)均明显减少(海马区减少59.0±11.1％，P<0.05；脑皮质减少36.4±4.5％，P<0.05)，而低剂量给药对小鼠海马区Aβ(1-40)斑块形成无显著影响(P>0.05)，而显著性降低了脑皮质内Aβ(1-40)斑块量(降低26.9±2.1％，P<0.05)。这表明金团簇对150天时Aβ(1-40)斑块的形成有显著抑制作用，且这一作用也呈现剂量依赖关系。另外，通过SPSS软件分析Aβ(1-40)斑块数量与150天水迷宫实验中的小鼠穿台次数的相关性时发现，海马及皮质区域内Aβ(1-40)斑块的量均与小鼠平台穿越次数呈显著负相关(海马区：R＝-0.848，P<0.01；脑皮质：R＝-0.802,P<0.05)。该结果进一步支持金团簇给药所致海马和脑皮质区域内Aβ(1-40)斑块减少与金团簇给药提高小鼠记忆和学习能力存在 相关性。
图18则是相应的给药150天的Aβ(1-42)的结果。结果表明，金团簇高剂量给药明显抑制了小鼠海马区及脑皮质内Aβ(1-42)斑块的形成(海马区降低51.1±6.7％，P<0.05；脑皮质降低62.8±4.6％，P<0.05)。低剂量给药对小鼠海马区及脑皮质内Aβ(1-42)斑块形成无明显影响(P>0.05)。这表明金团簇对150天时Aβ(1-42)斑块的形成有显著抑制作用，且这一作用也呈现剂量依赖关系。SPSS相关性统计分析发现海马及皮质内Aβ(1-42)斑块数量与小鼠穿台次数也均呈显著负相关(海马区：R＝-0.794，P<0.05；脑皮质：R＝-0.802，P<0.05)。这也进一步支持了金团簇给药所致海马和脑皮质区域内Aβ(1-42)斑块减少与金团簇给药提高小鼠记忆和学习能力的相关性。
综上所述，金团簇对显著改善AD模型小鼠的认知行为能力，对小鼠海马区及脑皮质内Aβ(1-40)和Aβ(1-42)斑块的形成均有显著抑制作用，从而抑制患病小鼠病情的发展，可作为含有金团簇的物质用于AD的预防与治疗。
表1所列的其它不同配体修饰的金团簇也有相似作用，在此不一一赘述。
实施例6：体外α-syn聚集动力学实验
本实施例通过体外α-syn聚集动力学实验来验证配体修饰的金团簇的功能，并将其与单独使用配体分子时对α-syn聚集动力学的影响比较，证明其功能来自于金团簇，而不是来自于配体。
硫磺素T(thioflavin T,简写：ThT)是一种专门染淀粉样纤维的染料。当其与多肽或蛋白单体共同孵育时，其荧光基本不发生变化，而当其遇到具有纤维结构的淀粉样多肽或蛋白时，会立即与淀粉样多肽或蛋白发生耦合，其荧光强度会指数增强，因此广泛用于监测多肽或者蛋白淀粉样变性的标记物。本实施例采用ThT荧光标记法监测α-syn在金团簇存在下的纤维化聚集的动力学过程。具体实验方法如下：
α-syn单体的前处理：将冻干的α-syn粉末(Bachem Corp.)溶于六氟异丙醇(HFIP)中得到浓度为1g/L的α-syn溶液，封口后在室温下孵育2-4小时，于通风橱中用高纯氮气将六氟异丙醇吹干，将吹干的α-syn溶于200μL二甲亚砜(DMSO)中，密封后置于-20℃冰箱中保存备用，保存时间不得超过一周。使用前将α-syn的DMSO溶液用大量的磷酸盐缓冲液(PBS，10mM，pH＝7.4)稀释至α-syn浓度为20μM，得到α-syn的PBS缓冲溶液。所有实验中的α-syn的PBS缓冲溶液均现配现用。
样品的制备和检测：将不同浓度的表1所列的配体修饰的金团簇加入到35μM的α-syn的PBS缓冲溶液中，采用ThT荧光标记法，在96孔板中37℃下连续孵育，用酶标仪每10分钟监测一次荧光强度，通过ThT荧光强度变化表征α-syn聚集的动 力学过程。实验组以实施例2制备的粒径1.8nm的L-NIBC修饰的金团簇为例，配体对照组采用未与金团簇结合的L-NIBC分子。金团簇所用的浓度均有4个，分别是：0ppm(只含α-syn，不含金团簇或L-NIBC，作为模型对照组)、1.0ppm、5.0ppm和10.0ppm，L-NIBC单独使用时，所用的浓度有2个，分别是：1.0ppm、10.0ppm。
结果如图19所示。结果显示，35μM的α-syn在37℃孵育过程中，从第48h开始，ThT标记的荧光强度快速增加，说明α-syn发生聚集及纤维化，这与文献(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)报道的结果一致。而配体对照组的结果显示，单独使用的L-NIBC对α-syn的聚集动力学没有明显影响(图19A幅)。而对添加了金团簇的实验组，在较低浓度(如1.0ppm和5.0ppm)下，ThT标记的荧光强度相对于未添加金团簇的模型对照组和配体对照组均显著下降，且起始时间明显延后(图19B幅)，说明金团簇的加入能显著抑制α-syn的聚集和纤维化。当金团簇浓度达到10ppm时，在168小时的实验时间内，ThT标记的荧光强度一直保持在基线附近，未出现任何增长(图19B幅)，说明当金团簇浓度足够的情况下，可完全抑制α-syn的聚集和纤维化。
本实施例还对表1所列其他不同配体修饰的金团簇的进行了此实验，例如，图19的C幅-J幅分别是D-NIBC、CR、RC、1-[(2S)-2-甲基-3-巯基-1-氧代丙基]-L-脯氨酸(Cap)、GSH、N-乙酰基-L-半胱氨酸(L-NAC)、L-半胱氨酸(L-Cys)和D-半胱氨酸(D-Cys)修饰的金团簇(用量均为10ppm)对α-syn的聚集和纤维化的抑制作用效果图。对不同配体修饰的金团簇也观察到类似现象，并能做出相同的结论：这些配体本身不能影响α-syn的聚集及纤维化，而配体修饰的金团簇对α-syn聚集及纤维化具有优异的抑制作用，当浓度达到10ppm时，均可实现完全的抑制效果。实现完全抑制所需的最小浓度根据所用配体的不同而略有差别。这些配体修饰的金团簇同样归为本发明定义的含金团簇的物质。对表1所列的其它金团簇也有类似效果，只是完全抑制α-syn的聚集和纤维化所需的金团簇浓度有所不同，在此不一一赘述。
实施例7：MPP+诱导的PD细胞(SH-sy5y)模型实验
实验一：
本实验以细胞存活率为指标，通过CCK-8法检测的结果，反映配体修饰的金团簇或金纳米粒子样品对抗帕金森疾病常用神经毒素MPP+损伤SH-sy5y神经细胞模型的毒性作用大小，以说明其在帕金森神经衰退疾病中的神经保护作用。MPP+诱导的PD细胞模型的构建根据文献(Cassarino,D S；Fall,C P；Swerdlow,R H；Smith,T S；Halvorsen,E M；Miller,S W；Parks,J P；Parker,W D Jr；Bennett,J P 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)中的描述进行。具体方法为：
1)取对数生长期的SH-sy5y细胞，用完全培养基稀释成密度为5×104/mL的细胞悬液，每孔200μL接种于96孔板，置于37℃，5％CO2培养箱中培养。待细胞贴壁后，加入样品。
2)第一组分别加入表1所列的由维持培养基配制不同粒径不同浓度的配体修饰的金团簇或金纳米粒子溶液100μL，使其终浓度分别为0.01ppm、0.1ppm、1ppm、5ppm、10ppm和20ppm，作为给药组；配体修饰的金团簇或金纳米粒子预处理2h后，在给药组和细胞模型对照组中分别加入MPP+(终浓度为1mM)，同时设置不含SH-sy5y细胞的空白对照组、含有SH-sy5y细胞但不添加金团簇或金纳米粒子及MPP+处理的阴性对照组、含有SH-sy5y细胞的只加入1mM的MPP+处理的细胞对照组，以及含有SH-sy5y细胞并加入1mM的MPP+处理的同时加入相应的配体分子(终浓度为20ppm)的配体对照组，37℃孵育24h，离心去除培养液，每孔加入100μL含10％CCK-8的维持培养基孵育4h，于450nm波长处测定各孔吸光度值，用来反映配体修饰的金团簇对MPP+损伤的预保护及治疗作用。
不同配体修饰的金团簇和金纳米粒子采用同样的步骤开展实验。结果表明本发明提供的配体修饰的金团簇对帕金森神经衰退疾病中的神经有保护作用，这一作用也是来源于金团簇自身，而不是配体，可作为含有金团簇的物质用于对抗帕金森疾病。
实验二：
本实验以细胞存活率为指标，通过CCK-8法检测的结果，反映配体修饰的金团簇或金纳米粒子样品对抗帕金森疾病常用神经毒素MPP+损伤SH-sy5y神经细胞模型的毒性作用大小，以说明其在帕金森神经衰退疾病中的神经保护作用。MPP+诱导的PD细胞模型的构建根据文献(D.S.Cassarino,C.P.Fall,R.H.Swerdlow,T.S.Smith,E.M.Halvorsen,S.W.Miller,J.P.Parks,W.D.Jr.Parker,J.P.Jr.Bennett,Biochimica et Biophysica Acta 1997,1362,77)中的描述进行。具体方法为：
1)取对数生长期的SH-sy5y细胞，用完全培养基稀释成密度为5×104/mL的细胞悬液，每孔200μL接种于96孔板，置于37℃，5％CO2培养箱中培养。待细胞贴壁后，加入样品。
2)第一组分别加入表1所列的由维持培养基配制不同粒径不同浓度的配体修饰的金团簇或金纳米粒子溶液100μL，使其终浓度分别为0.01ppm、0.1ppm、1ppm、5ppm、10ppm和20ppm，作为给药组；配体修饰的金团簇或金纳米粒子预处理2h后， 在给药组和细胞模型对照组中分别加入MPP+(终浓度为1mM)，同时设置不含SH-sy5y细胞的空白对照组、含有SH-sy5y细胞但不添加金团簇或金纳米粒子及MPP+处理的阴性对照组、含有SH-sy5y细胞的只加入1mM的MPP+处理的细胞对照组，以及含有SH-sy5y细胞并加入1mM的MPP+处理的同时加入相应的配体分子(终浓度为20ppm)的配体对照组，37℃孵育24h，离心去除培养液，每孔加入100μL含10％CCK-8的维持培养基孵育4h，于450nm波长处测定各孔吸光度值，用来反映配体修饰的金团簇对MPP+损伤的预保护及治疗作用。
以L-NIBC修饰的金团簇或金纳米粒子的实验结果为例，如图20所示。结果显示，经24小时培养后，添加了100mM金团簇但不用MPP+处理的样品对照组相对于空白对照组(设为100％)的细胞存活率上升至108.5±7.1％(P<0.01)，说明金团簇无毒。添加了1mM的MPP+但未添加金团簇的模型对照组的细胞存活率降为65.1±4.0％(对空白对照组P<0.01)，配体对照组细胞存活率为61.5±3.8％(对空白对照组P<0.01)，说明配体单独使用时对MPP+损伤的细胞模型存活率无提升作用。而添加了1ppm、5ppm、10ppm和40ppm金团簇的给药组细胞存活率分别上升至97.9±2.8％(对模型对照组P<0.01)，99.7±4.0％(对模型对照组P<0.001)，95.3±1.7％(对模型对照组P<0.01)和93.2±0.4％(对模型对照组P<0.01)，说明表明本发明提供的配体修饰的金团簇对帕金森神经衰退疾病中的神经细胞有保护作用，这一作用也是来源于金团簇自身，而不是配体。另一方面，相应配体的金纳米粒子在三个实验浓度对模型细胞的存活率均无提升无作用，说明金纳米粒子不能作为药物用于PD的预防与治疗。
表1所列的不同配体修饰的金团簇采用同样的步骤开展实验，有相似作用，在此不一一赘述。(注：在此处添加新实验结果)
实施例8：MPP+诱导的PD细胞(PC12)模型实验
本实验采用MPP+(100mM)诱导PC12细胞凋亡的模型，结合细胞流式技术,观察金团簇对MPP+致细胞损伤及凋亡的保护作用。具体实验方法为：实验设置不添加MPP+和金团簇的空白对照组、只添加MPP+的MPP+模型组、只添加金团簇的金团簇对照组和添加MPP+和金团簇的实验组。实验组中，PC12细胞PC12细胞悬浮液中预先半小时加平均粒径为1.8nm的L-NIBC修饰的金团簇溶液(终浓度为20ppm)，加入MPP+共同孵育24小时，用Annexin V-FITC/PI细胞凋亡检测试剂盒(购自Roch公司),FACSCalibur流式细胞仪检测细胞的生长活力和凋亡情况，CellQuest Pro获取数据并进行分析。
实验结果如图21所示。结果显示，MPP+作用24h后，细胞流式检测显示，未加MPP+的空白对照组细胞凋亡百分数为23.5±2.8％，20ppm的金团簇单独与PC12细胞 孵育时，细胞凋亡百分数为28.47±3.2％，与空白对照组比较，差异无显著性，提示金团簇无显著的细胞毒作用。而MPP+模型组细胞凋亡百分数为49.5±10.1％，模型组凋亡细胞显著增加(对空白对照组P<0.001)。PC12细胞预先与金团簇孵育半小时后再加入MPP+的实验组共同孵育24小时后，凋亡细胞百分比降为35.9±2.2％，与MPP+模型组比较，细胞凋亡显著降低(P<0.05)。
表1所列的不同配体修饰的金团簇采用同样的步骤开展实验，也有相似作用，在此不一一赘述。
实施例7和实施例8的结果共同显示，金团簇对MPP+诱导的PD细胞模型的细胞存活率有明显的提升作用，对细胞的凋亡有显著的抑制作用。
实施例9：MPTP诱导的PD小鼠模型实验
实验一：
实验动物：雄性C57bl/6小鼠80只，8周龄，体重25-30g；小鼠每笼3只，均待养于室温22-27℃的环境中，12h昼夜节律，自由进食和饮水，适应7天。
MPTP神经损伤小鼠模型:小鼠随机分成四组，每组20只，分为空白对照组，金团簇正常对照组，MPTP模型组，金团簇治疗组。MPTP模型组和金团簇治疗组每隔2h皮下注射20mg/kg(游离碱)MPTP，注射四次。生理盐水正常溶剂对照组每隔2h皮下注射20mg/kg生理盐水，注射四次。最后一次注射8h后，生理盐水正常溶剂对照组和MPTP模型组每日尾静脉注射10μL生理盐水，金团簇正常对照组和金团簇治疗组每日腹腔静脉注射10μL表1所列的配体修饰的金团簇的生理盐水溶液(金团簇的浓度10g/L)，连续注射7天，将动物置于有清洁垫料的饲养盒中，自由饮水，进食。
行为学检测：转轴实验，滚轴实验需要动物在滚轴上保持平衡并连续运动，是广泛用于检测运动协调性的实验，滚轴直径6cm，转速20rpm，适应五次后，每次检测间隔1min，记录其从滚筒上掉落的时间，连续测5次取平均值。
神经递质测定：行为学实验结束后，将动物处死，取小鼠纹状体组织，放置-80摄氏度冻存。测定时使用匀浆液(0.1M高氯酸，0.1mM EDTA-2Na)10μL/mg(纹状体)处理纹状体，冰浴下进行超声裂解，裂解30分钟后，置入低温离心机10000r/min，离心10min，提取上清液，用0.25μm的过滤器进行过滤后，注入HPLC的液相色谱柱，使用实验室建立的高效液相系统检测纹状体内多巴胺(DA)递质及其代谢产物3,4-二羟基苯乙酸(DOPAC)和高香草酸(HVA)的水平。每次检测前，必须用新鲜配置的流动相来维护色谱柱，持续2h。HPLC条件：流速：1mL/min；柱温30℃；荧光检测器激发光和吸收光波长分别为280和330nm。
酪氨酸羟化酶的测定：取出脑组织后固定于4wt％PFA+2wt％蔗糖中4-6h，然后浸 入30wt％蔗糖溶液中，待脑组织沉底后，用OCT包埋，冰冻切片机进行连续冠状贴片，采用ABC(Avidibiotin-peroxidase complex)法染色，取出黑质部位冰冻组织切片，进行TH染色，二联苯胺显色，显微镜观察拍照。
结果表明本发明提供的配体修饰的金团簇能显著改善MPTP诱导的PD模型小鼠的运动行为，提高多巴胺能神经元的数量，改善多巴胺神经递质的脑内水平，可作为含有金团簇的物质用于对抗帕金森疾病。
实验二：
实验动物：雄性C57bl/6小鼠80只，8周龄，体重25-30g；小鼠每笼3只，均待养于室温22-27℃的环境中，12h昼夜节律，自由进食和饮水，适应7天。
MPTP神经损伤小鼠模型：小鼠随机分成四组，每组20只，分为空白对照组，金团簇对照组(根据金团簇用量不同分为低剂量组和高剂量组)，MPTP模型组，金团簇实验组(根据金团簇用量不同分为低剂量组和高剂量组)。MPTP模型组和金团簇实验组每天腹腔静脉注射30mg/kg(游离碱)MPTP，连续七天。空白对照组每天皮下注射30mg/kg生理盐水，连续7天。金团簇对照组和金团簇实验组中的低剂量组每日腹腔静脉注射100μL浓度为1g/L的L-NIBC修饰的平均粒径为1.8nm的金团簇生理盐水溶液，而高剂量组则每日腹腔静脉注射100μL浓度为4g/L的L-NIBC修饰的平均粒径为1.8nm的金团簇生理盐水溶液，连续注射7天，将动物置于有清洁垫料的饲养盒中，自由饮水，进食。
1.行为学检测：
(1)自发活动计数实验：将动物从饲养笼中转移到自主活动检测仪,待动物适应新环境5min后,开始记录5min内其自发活动及变化,以5min内动物的活动里程及移动速度来衡量其活动能力的强弱。
(2)游泳实验：参照Donnan的测试方法(G.A.Donnan,G.L.Willis,S.J.Kaczmarezyk,P.Rowe,Journal of the Neurological Science 1987,77,185)，将受试小鼠放入Morris水箱中，水深60cm，水温为22℃。记录10min内其动物游泳的活动里程，游泳时间来衡量其活动能力的强弱。
(3)滚轴实验：需要动物在滚轴上保持平衡并连续运动，是广泛用于检测运动协调性的实验，滚轴直径6cm，转速20rpm，适应五次后，每次检测间隔1min，记录其从滚筒上掉落的时间，连续测5次取平均值。
2.黑质及纹状体免疫组织化学检测：行为学检测后，每组取5只小鼠行黑质及纹状体免疫组织化学检测。0.5％戊巴比妥钠1mL腹腔麻醉后，开胸经主动脉先以0.9％生理盐水15mL冲洗血液，再用含4％多聚甲醛的0.1mol/L的磷酸缓冲液(PBS，pH 7.2)100mL先快后慢灌注固定1h。灌注固定完毕取脑，置于4％多聚甲醛中，将组织进行石蜡包块参考小鼠脑图谱，中脑黑质和纹状体冠状切片，脑片的厚度为3μm/张，切好的脑片用于免疫荧光、超敏二步法免疫组化等实验。免疫组织化学染色步骤如下：0.3％过氧化氢甲醇溶液(30％过氧化氢1mL+甲醇80mL+PBS 19mL)30min，0.3％Triton X-100的PBS 30min，浸入小鼠抗酪氨酸羟化酶(TH)单克隆抗体(1：200)或IBa1(稀释比例1:250)孵育48h(4℃)，浸入生物素化兔抗小鼠二抗(1：500)孵育2h(室温)，蒸馏水快速冲洗后硫酸镍胺加强DAB蓝色反应法显色20～30min，当阳性产物呈深蓝色而背底清晰时蒸馏水冲洗3次终止显色。以上步骤每步后均需0.01mol/L PBS清洗3次，每次10min。其中一抗用含1％牛血清和0.3％的Triton X-100的PBS稀释，二抗和ABC复合物用PBS稀释。贴片，脱水，透明，中性树胶封片。
3.纹状体蛋白免疫印迹(WB)检测：酪氨酸羟化酶(TH)是多巴胺(DA)生物合成途径的关键酶，用TH免疫组织化学法可以显示黑质及纹状体中DA能神经元的变化(D.Luo,J.Zhao,Y.Cheng,S.M.Lee,J.Rong,Molecular Neurobiology 2017,DOI:10.1007/s12035-017-0486-6)。行为学检测后每组取5只做纹状体WB检测，于冰上取出所需脑部位，RIPA裂解液裂解，匀浆4℃12000g条件下离心30min，提取蛋白，制备样品，SDS-聚丙烯酰胺凝胶电泳，电压55V-60V，电泳时间为4.5h，半干法恒流转膜，电流设为60mA，时间约1.5h。5％脱脂奶粉室温封闭1h；加入TBST稀释的兔抗TH(稀释比例1:300)，4℃过夜；回收抗体，TBST洗3次，10min/次；加入TBST稀释的IRDye R 680RD Goat anti-Rabbit(稀释比例1:3000)；TBST洗3次，10min/次；双色红外激光成像系统扫描蛋白信号。
小鼠行为学自发活动计数实验结果如图22所示。小鼠在给与MPTP后3～5min，出现震颤、运动减少、弓背、后肢张开、步态不稳、竖尾、竖毛等改变，个别出现癫痫样发作，约30～60min后上述症状逐渐减轻，24h后基本恢复正常，但随着给药次数的增加，急性反应表现反倒减轻，但24h后其运动减少、步态不稳、反应迟缓的表现越来越明显。连续MPTP注射七天，小鼠自发活动路程和移动速度较空白对照组显著降低(P<0.01)，表现出运动迟缓的症状。金团簇单独给药对正常小鼠的自发活动路程和移动速度无显著影响(图22的A幅和C幅)。而MPTP模型小鼠合并给予金团簇(高剂量给药)可显著增加小鼠自发活动路程和移动速度(图22的B幅和D幅)，表明金团簇对MPTP模型小鼠自发活动的改善作用显著，与MPTP模型组比较，差异具有显著性(自发活动路程：P<0.05；移动速度：P<0.01)。
小鼠行为学游泳实验结果如图23所示。小鼠在连续MPTP注射七天后,将小鼠置于在水箱中,进行游泳能力测试。小鼠游泳的时间越多，游泳距离越远，说明小鼠肢体运动协调情况越好。空白对照组和金团簇单独给药组对小鼠游泳时间和游泳距离无显 著影响(图23的A幅和C幅)。与空白对照组比较，MPTP模型组10min内游泳距离显著缩短(P<0.05)，同时在水箱中运动时间显著减少(P<0.05)，表明MPTP可显著降低小鼠的游泳运动能力。与MPTP模型组相比，金团簇(高剂量给药)与MPTP伴随给药组，游泳路程增加(P<0.05)，游泳时间也显著增加(P<0.05)(图23的B幅和D幅)，表明金团簇对MPTP诱发的小鼠游泳行为障碍具有显著的改善作用。
小鼠行为学滚轴实验结果如图24所示。小鼠在连续MPTP注射7天后进行滚轴行为学测试，生理盐水正常对照组小鼠落棒潜伏期和落棒百分率分别为12.1±4.6min和33.3±1.5％(图24的A幅和C幅)；与空白对照组相比，MPTP模型组小鼠落棒潜伏期显著缩短为5.5±3.7min，落棒百分率显著增加至83.3±3.4％。表明MPTP给药导致小鼠运动协调能力下降，抓棒不稳，易于落棒(图24的B和D幅)。金团簇单独给药对小鼠落棒潜伏期无显著性影响(图24的A幅)，但在长时间滚轴运动条件下，小鼠的落棒百分率显著增加(对空白对照组P<0.001),表明金团簇自身给药对小鼠的滚轴行为具有一定的影响(图24的C幅)。但与MPTP模型组比较，MPTP与金团簇伴随的给药组，落棒潜伏期显著延长(低剂量给药：P<0.01；高剂量给药：P<0.05)，落棒百分率显著下降(高剂量给药和低剂量给药均P<0.001)，结果见图24的B和D幅。这表明金团簇具有改善MPTP诱发的运动协调功能障碍的作用。
黑质及纹状体免疫组织化学检测及纹状体WB检测结果如图25所示。MPTP模型组与空白对照组相比，黑质TH免疫阳性经元(即DA能神经元)数目明显减少，残留神经元皱缩，突起减少或消失，纹状体TH免疫阳性细胞及神经纤维密度减低，WB分析结果表明纹状体DA能神经元减少至55.8±5.6％(以空白对照组为100％)(对空白对照组P<0.01，见图25的C幅)。金团簇单独用药对黑质及纹状体TH免疫阳性细胞及神经纤维密无显著影响(图25的A幅和B幅)。金团簇与MPTP合并给药，可显著抑制MPTP下调黑质及纹状体细胞及神经纤维TH免疫阳性表达作用，WB分析结果表明采用低剂量金团簇时纹状体DA能神经元比例为空白对照组的65.6±6.3％(对MPTP模型组P<0.01，见图25的C幅)，采用高剂量金团簇时，纹状体DA能神经元达到空白对照组的84.7±4.5％(对MPTP模型组P<0.001)，结果表明金团簇具有显著的抗MPTP细胞毒作用，对黑质及纹状体内DA神经元的特异性丢失具有显著地保护作用。
表1所列的不同配体修饰的金团簇采用同样方法开展实验，也有相似作用，在此不一一赘述。
以上结果表明，本发明提供的配体修饰的金团簇能显著改善MPTP诱导的PD模型小鼠的自发活动能力、运动能力和身体协调能力，对黑质及纹状体内DA能神经元的特异性丢失具有显著的保护作用，说明含有金团簇的物质可用于对抗PD。
实施例10：生物安全性评价
1、采用SH-sy5y细胞株评价细胞层面含金团簇的物质的生物安全性。
具体方法如下：收集细胞繁殖处于对数期的SH-sy5y细胞(细胞传至第六代)，调整细胞悬液浓度，每孔加入100μL，铺板使待测细胞调密度至1000-10000孔，将细胞培育板(96孔平底板边缘孔用细胞培养液填充)置于细胞培育箱中，在5％CO2，37℃环境中孵育24h让细胞贴壁。将96孔板取出，酒精消毒后置于生物安全柜内，吸出原细胞培养液,分别加入用细胞培养液稀释至1ppm、10ppm、50ppm，100ppm、200ppm、500ppm的表1所列的配体修饰的金团簇溶液，对照组(无金团簇)加入等量的新鲜细胞培养液，然后放入细胞培育箱中继续孵育48h,实验组和对照组每组均设6个复孔。培养48h后，离心除去培养液，用PBS冲洗2-3遍后，在每孔加入100μL新鲜培养液和20μL噻唑蓝(MTT)溶液(5mg/ml，即0.5％MTT)，继续培养4h后终止培养，取出96孔板，离心(1000r/min)10min，吸出上清液，每孔加入200μL DMSO，置于摇床上低速振荡10min至孔内颜色均匀使结晶物充分溶解；用酶标仪测量在490nm处各孔的吸光值。以上操作都必须在无菌环境中执行，所有步骤除检测外均在生物安全柜内完成，实验用品在使用前必须用高温蒸汽灭菌锅消毒处理。
以实施例2的L-NIBC修饰的金团簇为例，结果见图26，其中A幅-C幅分别是粒径为2.6nm、1.8nm、1.1nm，终浓度分别为1ppm、10ppm、50ppm，100ppm、200ppm、500ppm的金团簇对SH-sy5y细胞存活率的影响。可以看到，在相当高的浓度(如100ppm)下，L-NIBC修饰的金团簇的加入对细胞存活率几乎无影响，在更高浓度(如200和500ppm)下，L-NIBC修饰的金团簇的加入会造成较小程度的细胞损伤(细胞死亡率小于20％)。由于100ppm已远大于药物的起效浓度(0.1～1ppm或更低)，因此，可以认为L-NIBC修饰的金团簇在细胞层面具有很高的安全性。
表1所列的其它配体修饰的不同尺寸的金团簇也有相似的效果，在此不一一赘述。
2、采用小鼠急性毒性实验评价含金团簇的物质的急性毒性。
具体方法如下：对于表1所列的不同配体修饰的的金团簇(以实施例2中L-NIBC修饰的平均直径为1.8nm的金团簇为例)，取60只成年小鼠，分成四组，每组15只，分别为对照组及三个实验组。其中对照组正常喂养，而三个实验组在正常饮食的情况下，按每天0.1g/Kg体重、0.3g/Kg体重和1g/Kg体重的量采用口服灌胃的方法喂食金团簇，持续一星期。停止喂食金团簇后再继续正常饲养30天。观察小鼠的异常反应。
在小鼠实验中，三种不同浓度的不同尺寸的金团簇摄入对小鼠的存活及活动性均无影响。即使是1g/Kg体重的高剂量摄入，小鼠依然保持健康。
表1所列的其它不同配体修饰的金团簇也有类似结果，在此不一一赘述。从以上结果可以得到结论，金团簇是非常安全的。
实施例11：含金团簇的物质在小鼠体内组织分布及代谢分布
实验一：
操作步骤：80只小鼠随机分成四组，每组20只，采用口服灌胃的方式喂食表1所列的配体修饰的金团簇，每组金团簇喂食的量分别是100mg/kg、20mg/kg、5mg/Kg和1mg/kg。喂食金团簇后再将每组的20小鼠随机分成4组，每组5只，分别按喂食后2h、6h、24h和48h的时间点处死小鼠，分离心、肝、脾、肺、肾和脑组织。将各组织称重，然后加入2mL水进行组织匀浆，匀浆后加入2mL王水涡旋混匀，再放在振荡器上振荡72h后，加入2wt％的稀硝酸溶液定容到10mL，15000rpm离心15min。吸取上清液4mL，用原子吸收光谱法测定组织液中金元素的含量。
结果表明金团簇可通过血脑屏障到达大脑，并随着时间的延长能排出体外因而在体内无明显蓄积，因此，本发明提供的含有金团簇的物质在制备治疗AD或PD的应用中有良好前景。
实验二：
操作步骤：80只小鼠随机分成四组，每组20只，采用腹腔静脉注射的方式对小鼠给予表1所列的配体修饰的金团簇，每组金团簇(以L-NIBC修饰的平均直径为1.8nm的金团簇为例)的用量相对于小鼠的体重分别是100ppm、20mg ppm、5ppm和1ppm。注射金团簇后再将每组的20小鼠随机分成4组，每组5只，分别按喂食后2h、6h、24h和48h的时间点处死小鼠，分离心、肝、脾、肺、肾和脑组织。将各组织称重，然后加入2mL水进行组织匀浆，匀浆后加入2mL王水涡旋混匀，再放在振荡器上振荡24h后，加入2wt％的稀硝酸溶液定容到5mL，15000rpm离心15min。吸取上清液1mL，用原子吸收光谱法的石墨炉法测定组织液中金元素的含量。
针对表1所列的其它不同配体的金团簇均采用上述实验步骤开展实验。
结果表明2h后，大脑中的金元素含量达到初始给药浓度的1％-10％，6h后，脑内含量能维持在相似的水平，24h后其脑内含量显著下降，48h时除100ppm给药量的样本外，均降低至检测限附近或以下。以上结果说明，含金团簇的物质在动物层面也具有良好的生物安全性，能穿透血脑屏障，并在体内无明显蓄积。
综上所述，以上实验结果说明了以下几点(以下提及的“金纳米粒子”和“金团簇”均指有配体修饰)：
(1)在抑制Aβ聚集的体外实验(实施例3)中，发现金纳米粒子对Aβ聚集动力学的影响与尺寸相关。当颗粒直径大于或等于10.1nm时，金纳米粒子的加入能加速Aβ的聚集，而当粒子尺寸小于或等于6.0nm时，则对Aβ的聚集有抑制作用，但无法实现Aβ聚集的完全抑制。但当所用的为金团簇(平均直径小于3nm)时，所有的金团簇均能在体外大幅度抑制Aβ的聚集，而且这一作用与金团簇的浓度有关。当金团簇的浓度达到5-10ppm时，就能完全抑制Aβ的聚集，完全抑制所需的最低浓度与配体的种类和金团簇直径有关。当金团簇的浓度达到5-10ppm时，就能完全抑制Aβ的聚集。在抑制α-syn聚集的体外实验(实施例6)中，也发现金团簇对α-syn的聚集与纤维化有同样的完全抑制的效果。
(2)在Aβ诱导的细胞AD模型及MPP+诱导的细胞PD模型实验中(实施例4、实施例7和实施例8)，发现当采用小尺寸的金纳米粒子(如平均直径3.6nm或6.0nm的金纳米粒子)时，对Aβ诱导的细胞AD模型及MPP+诱导的细胞PD模型细胞存活率的提升均无显著作用，说明金纳米粒子在细胞层面对AD和PD均未表现出明显药效，因而不能直接作为有效成分用于制备治疗AD或PD的药物。然而，对本发明所用的各种不同配体修饰的不同尺寸的金团簇(平均直径均小于3nm)，发现在金团簇用量很低(如0.1-1ppm)的情况下，均可使两种模型细胞存活率从50％-65％提高至95％以上。说明细胞层面，金团簇的药效显著。由于所用的配体本身均对Aβ的聚集及两种细胞模型均无作用(实施例4，实施例7和实施例8)，因此，可以推出金团簇的药效来自于其自身的结论，这为金团簇的应用提出了新的思路。
(3)进一步地，本发明采用了AD的转基因小鼠模型和MPTP诱导的PD小鼠模型(实施例5、实施例9)进一步验证金团簇的药效，说明所用的金团簇对改善小鼠认知行为能力、运动行为能力、抑制脑内老年斑的形成和抑制MPTP诱导的黑质与纹状体DA能神经元的特异性凋亡等均有显著作用，能作为相关疾病的预防或治疗药物。
(4)进一步评价生物安全性的实验中(实施例10)，金团簇在100ppm重量百分比浓度下与神经细胞共同培养时对细胞的存活率无明显影响，超过100ppm(远大于药物起效的浓度)时，细胞存活率略有下降。由于金团簇的起效浓度(0.1-1ppm)远低于100ppm，因此，可以认为金团簇在细胞层面具有优异的生物安全性。而在小鼠急性毒性实验中，采用每天一次1g/Kg体重(相当于1000ppm)给药量连续服用七天，小鼠未表现出不良反应。在小鼠体内分布及药代动力学实验(实施例11)中，2h后，大脑中的金元素含量达到初始给药浓度的1％-10％，6h后，脑内含量能维持在相似的水平，24h后其脑内含量有显著下降，48h时除100ppm给药量的样本外，均降低至检测限以下。以上结果说明，含金团簇的物质在动物层面也具有良好的生物安全性，能穿透血脑屏障，并在体内无明显蓄积。以上结果说明，含金团簇的物质在动物层面 也具有良好的生物安全性，能穿透血脑屏障，并在体内无明显蓄积，因此在制备治疗AD或PD的药物的应用中有良好前景。
(5)相对于现有技术，本发明所用的配体并非针对Aβ和α-syn的聚集行为特殊设计，且对比实验表明所用的配体对Aβ和α-syn的聚集均无明显作用(实施例3)，但由于金团簇的尺寸小于蛋白本身的尺寸，因此可通过尺度效应与弱分子间相互作用的结合大幅度抑制Aβ和α-syn的聚集。Aβ诱导的AD细胞模型与转基因动物模型中的优异效果更验证了含金团簇的物质应用于制备治疗AD的药物中的可行性。此外，含金团簇的物质在MPP+诱导的PD细胞模型及MPTP诱导的PD动物模型中的优异效果则说明含金团簇的物质在制备治疗其他神经退行性疾病的药物中也有广阔应用前景，而且由于MPP+诱导的PD细胞模型及MPTP诱导的PD动物模型并不涉及蛋白纤维化，而是作用于神经细胞的能量代谢及神经递质代谢相关的信号传导功能等更深层的机制，因此，可以推测，含金团簇的物质除影响蛋白纤维化外，还能在更深的层面影响神经退行性疾病的进程，这将对神经退行性疾病的新药研发有重要意义。
工业应用性
本发明提供的含金团簇的物质能够在AD的转基因小鼠模型和MPTP诱导的PD小鼠模型中改善小鼠认知行为、运动行为能力、抑制脑内老年斑的形成等，并在动物层面也具有良好的生物安全性，适于工业应用。

Claims (22)

  1. 一种含金团簇的物质，其特征在于，包括金团簇及其外部包覆的配体Y。
  2. 根据权利要求1所述物质，其特征在于，所述金团簇的金核直径小于3nm，优选0.5-2.6nm。
  3. 根据权利要求1或2所述含金团簇的物质，其特征在于，所述配体Y包括但不局限于L(D)-半胱氨酸及其衍生物、含半胱氨酸的寡肽及其衍生物、其它含巯基的化合物中的一种或几种。
  4. 根据权利要求3所述含金团簇的物质，其特征在于，所述L(D)-半胱氨酸及其衍生物优选L(D)-半胱氨酸、N-异丁酰基-L(D)-半胱氨酸(L(D)-NIBC)或N-乙酰基-L(D)-半胱氨酸(L(D)-NAC)等。
  5. 根据权利要求3所述含金团簇的物质，其特征在于，所述含半胱氨酸的寡肽及其衍生物优选含半胱氨酸的二肽、含半胱氨酸的三肽或含半胱氨酸的四肽。
  6. 根据权利要求5所述含金团簇的物质，其特征在于，含半胱氨酸的二肽优选L-半胱氨酸-L-精氨酸二肽(CR)、L-精氨酸-L-半胱氨酸二肽(RC)、L-组氨酸-L-半胱氨酸二肽(HC)或L-半胱氨酸-L-组氨酸二肽(CH)等。
  7. 根据权利要求5所述含金团簇的物质，其特征在于，所述含半胱氨酸的三肽优选甘氨酸-L-半胱氨酸-L-精氨酸三肽(GCR)、L-脯氨酸-L-半胱氨酸-L-精氨酸三肽(PCR)、L-赖氨酸-L-半胱氨酸-L-脯氨酸三肽(KCP)或L-谷胱甘肽(GSH)等。
  8. 根据权利要求5所述含金团簇的物质，其特征在于，所述含半胱氨酸的四肽优选甘氨酸-L-丝氨酸-L-半胱氨酸-L-精氨酸四肽(GSCR)或甘氨酸-L-半胱氨酸-L-丝氨酸-L-精氨酸四肽(GCSR)等。
  9. 根据权利要求3所述含金团簇的物质，其特征在于，所述其它含巯基的化合物优选1-[(2S)-2-甲基-3-巯基-1-氧代丙基]-L-脯氨酸、巯基乙酸、巯基乙醇、苯硫酚、D-3-巯基缬氨酸、N-(2-巯基丙酰基)-甘氨酸或十二硫醇等。
  10. 根据权利要求1-9任一所述含金团簇的物质，其特征在于，所述物质为粉末或絮状物。
  11. 一种制备权利要求1-10任一所述含金团簇的物质的方法，其特征在于，包括以下步骤：
    (1)把HAuCl4溶于甲醇、水、乙醇、正丙醇、乙酸乙酯中的一种配成HAuCl4浓度为0.01～0.03M的溶液A；
    (2)把配体Y溶于溶剂中配成浓度为0.01～0.18M的溶液B；
    (3)将步骤(1)的溶液A和步骤(2)的溶液B混合，HAuCl4和配体Y的摩尔比 为1:(0.01～100)(优选1：(0.1-10)，更优选1：(1-10))，在冰浴下搅拌0.1～48h(优选0.1-24h，更优选0.5-2h)，滴加0.025～0.8M的NaBH4溶液(优选NaBH4的水溶液、NaBH4的乙醇溶液、NaBH4的甲醇溶液)后，在冰水浴中继续搅拌0.1～12h(优选0.1-2h，更优选1-2h)，NaBH4与配体Y的摩尔比为1:(0.01～100)(优选1：(0.1-8)，更优选1：(1-8))；
    (4)将步骤(3)的反应液以8000～17500r/min离心10～100min，即可得到不同平均粒径的金团簇沉淀；优选的，将步骤(3)的反应液用截留分子量为3K～30K的超滤管以8000～17500r/min梯度离心10～100min，即可得到不同平均粒径的金团簇；
    (5)将步骤(4)得到的不同平均粒径的金团簇沉淀溶于水并装入透析袋中在室温下置于水中透析1～7天；
    (6)将透析袋内的金团簇溶液冷冻干燥12～24h，得到含金团簇的物质。
  12. 根据权利要求11所述方法，其特征在于，步骤(2)中的所述溶剂为甲醇、乙酸乙酯、水、乙醇、正丙醇、戊烷、甲酸、乙酸、乙醚、丙酮、苯甲醚、1－丙醇、2－丙醇、1－丁醇、2－丁醇、戊醇、乙醇、乙酸丁酯、三丁甲基乙醚、乙酸异丙酯、二甲亚砜、乙酸乙酯、甲酸乙酯、乙酸异丁酯、乙酸甲酯、2－甲基－1－丙醇、乙酸丙酯中的一种或多种。
  13. 权利要求1-10任一所述含金团簇的物质在制备催化剂或分子催化、手性识别、分子检测、生物医学检测与成像等领域中的近红外荧光探针中的应用。
  14. 权利要求1-10任一所述含金团簇的物质在制备与Aβ的聚集及纤维化相关的疾病和/或与α-syn的聚集及纤维化相关的疾病的药物中的应用。
  15. 权利要求1-10任一所述含金团簇的物质在制备预防和治疗阿兹海默病药物中的应用。
  16. 权利要求1-10任一所述含金团簇的物质在制备预防和治疗帕金森症药物中的应用。
  17. 金团簇在制备与Aβ的聚集及纤维化相关的疾病的药物中的应用。
  18. 根据权利要求17所述应用，其特征在于，所述与Aβ的聚集及纤维化相关的疾病为阿兹海默病。
  19. 根据权利要求17或18所述应用，其特征在于，所述金团簇为由L-谷胱甘肽(GSH)、N-乙酰基-L(D)-半胱氨酸(L(D)-NAC)、N-异丁酰基-L(D)-半胱氨酸(L(D)-NIBC)、L-半胱氨酸-L-精氨酸二肽(CR)、L-精氨酸-L-半胱氨酸二肽(RC)、1-[(2S)-2-甲基-3-巯基-1-氧代丙基]-L-脯氨酸(Cap)或L(D)-半胱氨酸(L(D)-Cys)等修饰的。
  20. 金团簇在制备与α-syn的聚集及纤维化相关的疾病的药物中的应用。
  21. 根据权利要求20所述应用，其特征在于，所述与Aβ的聚集及纤维化相关的疾病为帕金森症。
  22. 根据权利要求20或21所述应用，其特征在于，所述金团簇为由L-谷胱甘肽(GSH)、N-乙酰基-L(D)-半胱氨酸(L(D)-NAC)、N-异丁酰基-L(D)-半胱氨酸(L(D)-NIBC)、L-半胱氨酸-L-精氨酸二肽(CR)、L-精氨酸-L-半胱氨酸二肽(RC)、1-[(2S)-2-甲基-3-巯基-1-氧代丙基]-L-脯氨酸(Cap)或L(D)-半胱氨酸(L(D)-Cys)等修饰的。
PCT/CN2017/093671 2016-08-05 2017-07-20 一种含金团簇的物质及其制备方法与应用 WO2018024111A1 (zh)

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