Inhibition of Alzheimer's-Associated Aβ Aggregation by Gold Nanoparticles

Introduction: Alzheimer's disease (AD) is currently the most common type of dementia and the 6^th leading cause of death in the United States. One pathological hallmark of AD is amyloid plaques, which deposit around the neurons in the brain. These plaques are composed primarily of amyloid-beta protein (Aβ), which self-assembles to create aggregated structures in a process that is hypothesized to be closely tied to disease progression. During self-assembly, monomeric Aβ forms nuclei, which progress to form soluble aggregates and ultimately insoluble fibrils. Inhibition of Aβ aggregation is one therapeutic strategy for AD.

One potential therapeutic agent is gold nanoparticles (NP). Nanoparticles have emerged recently as effective therapeutics and diagnostic tools with applications in cancer, photo-thermal ablation, and enhanced medical imaging. In particular, gold NPs are highly attractive therapeutic candidates due to their stability, low toxicity, and ease of synthesis. In this study, we have examined gold nanospheres with varying electrostatic surface properties and particle diameters for their ability to attenuate Aβ aggregation.

Methods and Materials: 8nm, 18nm, or 40nm gold NPs were synthesized with citrate, cetrimonium bromide (CTAB), poly acrylic acid (PAA), and poly allylamine hydrochloride (PAH) surface properties. All NPs were tested for toxicity on SH-SY5Y cells using an XTT assay. NPs were further evaluated in a monomer aggregation assay. Briefly, 40µM Aβ monomer was agitated alone or in the presence of 200pM-20pM NPs. Reactions were monitored using thioflavin T (ThT), a fluorescent dye that specifically recognizes β-sheet structures of amyloid aggregates. Using this technique, the aggregation process, which includes a lag time, a period of growth, and an equilibrium plateau, can be observed. Reactions were periodically diluted into 10µM ThT and fluorescence was assessed. The effect of NP type on extension of the lag time and reduction of the equilibrium plateau was evaluated using a one-way analysis of variance with Dunnett's post test. p<0.5 was considered significant. After the completion of each reaction, samples were gridded for transmission electron microscopy (TEM) analysis.

Results and Discussion: SH-SY5Y cells treated with 100pM or 200pM CTAB NPs exhibited significant toxicity; however citrate, PAA and PAH NPs were non toxic. None of the NPs tested elicited any effect on monomer nucleation, which is evidenced by no extension of the lag time. 200pM citrate NPs were observed to have a small reduction of 19±8% on the equilibrium plateau, which is illustrative of their ability to attenuate the total quantity of amyloid material formed during monomer aggregation. PAH NPs had a stronger effect on abrogating aggregation with a 59±9% and 45±4% reduction of the equilibrium plateau at 200pM and 20pM, respectively. The greatest inhibition of monomer aggregation was observed in the presence of PAA NPs. At concentrations as low as 20pM, 96±2% inhibition was noted. This is equivalent to a substoichiometric ratio of NPs to Aβ_1-40 of 1:2,000,000. Inhibition was also monitored using TEM. Results from TEM images paralleled those of the ThT assay. In particular, no aggregates were observed in PAA reactions. To further study the positive inhibitory capabilities of PAA NPs, 8nm and 40nm particles were evaluated to determine whether NP size effects aggregation. 8nm PAA NPs proved to be equally effective at inhibiting monomer aggregation, with a 95±3% reduction in equilibrium plateau at 20pM. However, when particle diameter was increased to 40nm inhibitory capabilities decreased.

Conclusion: These results demonstrate that 8nm and 18nm PAA NPs are effective inhibitors of Aβ aggregation at concentrations as low as 20pM. It has also been evidenced that as particle diameter increases to 40nm, effectiveness decreases. Together, these results demonstrate that gold nanoparticles serve as effective inhibitors of Aβ self-assembly and modify aggregation at substoichiometric concentrations. Furthermore, inhibition of Aβ aggregation by gold nanoparticles is dependent upon the nanoparticle coating and particle diameter. Determining the mechanisms by which these various nanoparticles inhibit Aβ self-assembly could enable the development of an effective therapeutic for AD.

Figure  SEQ Figure \* ARABIC 1: Effect of 18nm NPs with varying surface chemistries on Aβ1-40 monomer aggregation. Aβ1-40 monomer diluted to 40μM was incubated alone (control,Â�), or in the presence of 200pM citrate NPs (¢), 200pM PAA NPs (p), or 200pM PAH NPs (z). Results represent three independent experiments.