Application Of Gold Nanoclusters In Parkinson's Disease Model

Parkinson's disease?PD?is a common neurodegenerative disease affecting more than 10 million people worldwide.It is characterized by motor symptoms such as tremor,rigidity,slowness of movement and problems with gait,which are often accompanied with fatigue,depression,pain and cognitive impairment.The presence of Lewy bodies composed mainly of abnormal?-Synuclein??-Syn?fibrillation in neurons and cell death of dopaminergic?DA?neurons in brain's basal ganglia?mainly in substantia nigra?SN?and striatum?are the main pathological characteristics of PD.Currently used therapeutic drugs,levodopa?L-DOPA?and dopamine agonists can temporarily relieve the motor symptoms of PD patients,but with the prolonged treatment time of drugs,the efficacy is gradually weakening.There is still no effective treatment or drug that could stop or retard the neurodegeneration process of PD up to now.Therefore,there is an urgent need to develop therapeutic drugs for PD.As a new type of nanomaterial in the 20^th century,gold nanoclusters?AuNCs?are usually composed of several to hundreds of yards Au atoms and are less than 3nm in size.Due to its strong quantum confinement effect,AuNCs exhibit HOMO-LUMO,electron-like energy states of molecules,and have good biocompatibility and physicochemical properties.They have been extensively studied in bioimaging,drug transportation,and photothermal therapy.However,the direct medicinal activities of AuNCs themselves have rarely been reported.To explore the potential of AuNCs as a drug in PD,we used biological-liked ligands to modify AuNCs to improve their stability and biocompatibility.AuNCs were used for PD therapy in the in vitro experiments,cell model experiments,and animal model experiments.The experimental content of this thesis is as follows:?1?Water-soluble N-isobutyl-L-cysteine-modified gold nanoclusters?L-NIBC AuNCs?were synthesized in organic phase,and the successful synthesis of L-NIBC AuNCs was proved by various characterization methods.The Thioflavine-T?ThT?binding assay,dynamic light scattering?DLS?were employed to monitor the fibrosis of?-Syn proteins incubated with different concentrations of AuNCs.Atomic force microscopy?AFM?,and transmission electron microscopy?TEM?were employed to study the morphology of?-Syn protein with AuNCs.The results showed that when the concentration of AuNCs reached 10 mg·L^-1,?-Syn protein fibrosis was completely inhibited?in 37oC,within 168 h?;AFM and TEM imges were detected respectively.It was found that the degree of?-Syn protein fibrillation was reduced with the concentration of AuNCs increasing.Namely,the long mature fibers were filled in the field of view without AuNCs,but when the concentration was high,there were almost random aggregates rather than fibrous shape;DLS monitored the particle size changes of?-Syn protein with/without 10 mg·L^-1 AuNCs in 168 h,showing that the size was maintained in a small range?32±15.3 nm?when co-incubated with AuNCs.All above results demonstrated that AuNCs with concentration over 10 mg·L^-1 could inhibit the fibrillation of?-Syn protein.?2?Cytotoxicityexperiments and PD cell model repair experiments were performed on the prepared L-NIBC AuNCs.The cytotoxicityof AuNCs was tested by three different cell lines?PC12,SH-Sy5y,INS-1 cells?.We found that AuNCs showed no cytotoxicityto the three cell lines(AuNCs concentrations below 40mg·L^-1),and even AuNCs had a certain nutritive effect on cell growth.The PD cell model was established by using 3 mmol·L^-1 MPP⁺on SH-Sy5y cells.AuNCs could improve the cell vablities of the lesion SH-Sy5y cells?from 59%to 96%?,and the apoptosis was detected by flow cytometry.When co-incubating with AuNCs,the percentage of apoptosis was 35.9±2.19%,significantly lower than that of the MPP⁺group.At the cellular level,AuNCs were shown to have low cytotoxicity and repair MPP⁺lesion cells.?3?The animal model of MPTP lesion in mice was established for studying the repair effect of AuNCs.The mouse model of PD was established by continuous injection of MPP⁺for 7 days in the peritoneal cavity of mice.After treatment with AuNCs,the behavioral disorder of PD was significantly improved by open field experiment,swimming experiment and rotating rod experiment;through immunohistochemical method and wertern-blotincreased dopamine content and protein expression levels of tyrosine hydrogenase in the SN and striatum,inhibited DA neuron damage;and AuNCs could cross the blood-brain barrier?BBB?using cryo-transmission electron microscopy to detect brain slices;however,AuNCs do not respond well to striatum inflammatory responses.From the animal level,it was shown that AuNCs could effectively improve the behavioral and pathological features of MPP⁺lesion mice.Based on the above experiment results,AuNCs have inhibitory effect on?-Syn fibrillation and neuroprotective effects on MPP⁺lesion cell models and MPTP lesion animal models at the cellular and animal levels.These results provided a proof of principle for the promising prospect of AuNCs in new drug discovery against PD,which also opened a new avenue for AuNCs in biological applications.