How Do the Size, Charge and Shape of Nanoparticles Affect Amyloid β Aggregation on Brain Lipid Bilayer?

Here, we studied the effect of the size, shape, and surface charge of Au nanoparticles (AuNPs) on amyloid beta (Aβ) aggregation on a total brain lipid-based supported lipid bilayer (brain SLB), a fluid platform that facilitates Aβ-AuNP aggregation process. We found that larger AuNPs induce large and amorphous aggregates on the brain SLB, whereas smaller AuNPs induce protofibrillar Aβ structures. Positively charged AuNPs were more strongly attracted to Aβ than negatively charged AuNPs, and the stronger interactions between AuNPs and Aβ resulted in fewer β-sheets and more random coil structures. We also compared spherical AuNPs, gold nanorods (AuNRs), and gold nanocubes (AuNCs) to study the effect of nanoparticle shape on Aβ aggregation on the brain SLB. Aβ was preferentially bound to the long axis of AuNRs and fewer fibrils were formed whereas all the facets of AuNCs interacted with Aβ to produce the fibril networks. Finally, it was revealed that different nanostructures induce different cytotoxicity on neuroblastoma cells, and, overall, smaller Aβ aggregates induce higher cytotoxicity. The results offer insight into the roles of NPs and brain SLB in Aβ aggregation on the cell membrane and can facilitate the understanding of Aβ-nanostructure co-aggregation mechanism and tuning Aβ aggregate structures.


Supplementary
. The dark-field and TEM micrographs of citate-AuNPs (left) and amine-AuNPs (right). The particle diameter for amine-AuNPs and citrate-AuNPs were 39.06±2.06 nm and 37.67±1.52 nm, respectively. The sizes were measured from the TEM images. Scale bars in the dark-field images are 100 μm and those in TEM images are 100 nm.
Supplementary Figure S5. The dark-field and TEM images of AuNRs (left) and AuNCs (right) to verify the particle size and distribution. The scale bars in dark-field data are 100 μm, and the TEM images have 100 nm scale bars. Figure S6. TEM images obtained after 48-hr co-incubation of Aβ with three types of AuNRs. The scale bars are 100 nm.

Synthesis of Spherical Gold Nanoparticles (AuNPs).
We purchased citrate modified AuNPs whose sizes were 20-nm, 40-nm, 50-nm, and 80-nm AuNPs, and amine modified AuNPs was synthesized as following. under magnetic stirring at 80 rpm for 10 s. Before the solution color change from transparent to pink color, stirring bars in r.b.f. should be removed, followed by keeping undisturbed in 26℃ for more than 15min. The resulting purpled pink color solution is indicative of AuNCs having an edge length of 51.05nm. Then, the solutions were centrifuged twice at 5000 rpm for 10 min and were dispersed in deionized water.
Lyophilized FITC-Aβ was resolved in DMSO at first and the solution was stored at -80 °C as 30 μl of aliquots. This aliquot was diluted with 100 μl of 10mM PB (pH7.4) just before use, and then, the solution was centrifuged at 11000 rpm for 15 min along with acquiring supernatant only. In order to measure binding affinity of Aβ peptides to AuNPs, 10 μl of AuNPs was mixed with 100 μl of Aβ solution and incubated at room temperature for 30min.
Here, the concentration of AuNPs was same with dark-field and TEM experiments and concentration of Aβ peptides was varied from 10 nM to 50 μM. Thereafter, those samples were also centrifuged at 5000 rpm for 1 min except 20-nm AuNPs samples were centrifuged at 8000 rpm for 1 min, and supernatants of samples were collected and fluorescence signals were obtained through Synergy™ MX (BioTek Instruments, Inc, Winooski, VT, USA) at 532 nm.

Size and Zeta Potential of AuNPs with Aβ.
Aβ aliquot was diluted with 10mM PB (pH 7.4) for final concentration of 25 μM and centrifuged at 11000 rpm for 15 min. The size and surface charge of AuNPs were measured before and after 30 min co-incubation. In the case of 30 min incubation sample, it was usually centrifuged at 5500 rpm for 1min in order to precipitate only AuNPs enclosed by Aβ peptides except for 20-nm AuNPs which were centrifuged at 8000rpm for 1min. Supernatant was removed and then precipitated AuNPs were dispersed with 10 mM PB (pH 7.4). Then, the size and zeta potential were obtained through Zetasizer (Malvern, Worcestershire, UK).