(214d) 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.