Plasmonic nanoparticle amyloid corona for screening Aβ oligomeric aggregate-degrading drugs

The generation of toxic amyloid β (Aβ) oligomers is a central feature of the onset and progression of Alzheimer’s disease (AD). Drug discoveries for Aβ oligomer degradation have been hampered by the difficulty of Aβ oligomer purification and a lack of screening tools. Here, we report a plasmonic nanoparticle amyloid corona (PNAC) for quantifying the efficacy of Aβ oligomeric aggregate-degrading drugs. Our strategy is to monitor the drug-induced degradation of oligomeric aggregates by analyzing the colorimetric responses of PNACs. To test our strategy, we use Aβ-degrading proteases (protease XIV and MMP-9) and subsequently various small-molecule substances that have shown benefits in the treatment of AD. We demonstrate that this strategy with PNAC can identify effective drugs for eliminating oligomeric aggregates. Thus, this approach presents an appealing opportunity to reduce attrition problems in drug discovery for AD treatment.

We performed gel electrophoresis as a suitable assay to ensure the degradation of the oligomeric aggregates on the PNAC surfaces ( Supplementary Fig. 2). Before electrophoresis, the bare AuNPs aggregated with each other because of their low stability in the salt conditions provided by the Trisacetate/ethylenediaminetetraacetic acid (TAE) 1× buffer. Thus, no band is observed. In contrast, the PNAC sample exhibit a distinct band. This is because the PNACs (AuNPs with Aβ oligomeric aggregates) are well dispersed, as the oligomeric aggregates provided good steric stability. With Aβdegrading agents, such as EPPS and protease XIV, the PNAC band is broadened, indicating that aggregation of the PNACs occurs with the removal of the oligomeric aggregates from the PNACs.
The aggregation of the PNACs causes slower movement in the agarose gel, resulting in band broadening. We performed gel electrophoresis to analyze the uniformity of the PNACs according to the size of the core AuNPs ( Supplementary Fig. 4). The narrow and clear band of the 20-nm PNAC indicates their high uniformity, demonstrating that 20-nm PNACs are suitable for our strategy of colorimetric Aβ-degrading drug screening. As the size of the PNACs is increased, their bands are broadened, indicating that the uniformity of larger PNACs is poor. In the case of single AuNPs, the hydrodynamic size of the AuNPs is increased by Aβ aggregation on the AuNP surface, and these larger AuNPs experience more Aβ aggregation, thereby increasing the local Aβ concentration around the AuNPs 1 . The increased local Aβ concentration at the surfaces of the AuNPs may enhance the probability of frequent contact between partially unfolded oligomeric aggregates, resulting in more rapid clustering of the AuNPs and Aβ 2 . This tendency occurs more easily with large-sized AuNPs.
The reaction between large-sized AuNPs and Aβ happens rapidly, which interferes with the fabrication of uniform and stable PNACs. For these reasons, the uniformity of PNACs with largesized core AuNPs is decreased, and their gel electrophoresis bands are broadened. We tested the salt resistance of differently sized PNACs in PBS ( Supplementary Fig. 5). The results showed that the absorbance peak of 50-nm PNACs is slightly decreased because the 50-nm PNACs become aggregated in PBS. In the case of 100-nm PNACs, their absorbance peak is dramatically decreased. The results indicated that large-sized PNACs became more unstable in salt conditions (PBS) as the PNACs became larger. However, as shown in Supplementary Fig. 8c, the aggregation of 20-nm PNACs is negligible, indicating that the 20-nm PNACs were stable in salt conditions and thus suitable for use in the colorimetric Aβ-degrading drug screening platform. We added the anti-aggregation agent (i.e., rutin hydrate) during the process of PNAC synthesis. As shown in Supplementary Fig. 7a, the formation of the amyloid corona on AuNPs is restricted compared to that on the PNACs without rutin hydrate (see Fig. 1c). For this reason, the steric stability of PNACs with rutin hydrate is significantly decreased, resulting in aggregation in PBS ( Supplementary Fig. 7b). We confirmed the aggregation of PNACs with rutin hydrate by a saltresistance test ( Supplementary Fig. 7c). The PNACs without rutin hydrate are stable in PBS because the amyloid corona on PNAC provides steric stability. In contrast, the PNACs with rutin hydrate are unstable, showing a plasmonic peak shift because rutin hydrate hinders the formation of the amyloid corona of PNAC. We also performed gel electrophoresis as a suitable assay to verify the antiaggregation effect of rutin hydrate ( Supplementary Fig. 7d). The PNACs without rutin hydrate exhibit a distinct band. This is attributed to the good dispersion of the PNACs arising from the good steric stability provided by the amyloid corona. With rutin hydrate during the PNAC fabrication process, the PNAC band is broadened, implying that the aggregation of PNACs occurs because of the low steric stability of PNACs synthesized in the presence of rutin hydrate.  Fig. 14a-d).
The results show a significant increase in the average height of the Aβ species as a function of the sucrose fraction density (Supplementary Fig. 14e); a drastic increase in the cross-sectional diameter (i.e., height) of Aβ species is also found in the 40% fraction. The average sizes of Aβ species in each sucrose fraction of 10%, 20%, 30%, and 40% are 1.64, 2.50, 2.23, and 7.28 nm, respectively.
The presence of Aβ oligomers in the 20% sucrose fraction is confirmed by the following: i) The 30% sucrose fraction ( Supplementary Fig. 14c) contains Aβ protofibrils as well as oligomers.
ii) The 2.50-nm average size of Aβ species in the 20% sucrose fraction is similar to that found in the amyloid corona on the PNACs. We performed a control experiment to reproduce the table in Fig. 2a using graphene-based sensors wherein each surface of the sensor was functionalized with the different antibodies of monoclonal 6E10, polyclonal A11, and polyclonal OC ( Supplementary Fig. 16). We treated the graphene sensors with each purified Aβ solution of monomers, oligomers, and fibrils ( Supplementary Fig. 16a-c) and monitored the relative resistance changes of each graphene sensor with the three different types of antibodies ( Supplementary Fig. 16d). The results indicate that each graphene sensor shows a specific interaction with each purified Aβ species. The results are expressed as a heatmap (Supplementary Fig.   16e) that is consistent with the table in supplementary Fig. 16d and with Fig. 2a    To validate corona formation in hCSF-PNACs, we cross-checked the amyloid corona formation of hCSF-PNACs ( Supplementary Fig. 21a) and PBS-PNACs by HRTEM (Fig. 1c). In both the images, each AuNP is covered with an amyloid corona of uniform thickness (~3 nm), which corresponds to the size of a single oligomeric aggregate.
To compare the conformational characteristics of hCSF-PNACs and PBS-PNACs, we used graphene sensors wherein each surface of the sensor was functionalized with 6E10, A11, and OC, as in Supplementary Fig. 16. Before the assay using hCSF-PNACs, we confirmed the affinities between the antibodies and bare AuNPs as a negative control. The relative resistance changes, representing the affinities between bare AuNPs and the antibodies, are negligible from all antibodies ( Supplementary   Fig. 21b). However, with hCSF-PNACs, the relative resistance values of the 6E10-and A11immobilized sensors are significantly changed by 2.77% and 3.25%, respectively, implying that 6E10 and A11 antibodies have strong affinities with the hCSF-PNACs. In contrast, the relative resistance value of the OC-immobilized sensor remains similar to that of AuNPs, meaning that OC antibodies do not capture hCSF-PNACs. These results indicate that the amyloid corona on hCSF-PNACs comprise not Aβ fibrils, but Aβ oligomeric aggregates. Moreover, we performed gel electrophoresis to analyze the uniformity of hCSF-PNACs compared to that of PBS-PNACs ( Supplementary Fig. 21c). The The values of EC 50 and maximal efficacy are estimated as 7.038 mM and 0.776 (a.u.), respectively, consistent with those from PBS-PNAC-based assays (see Fig. 4b).
Altogether, we confirmed that the hCSF-PNACs have the same characteristics as PBS-PNACs; therefore, Aβ-degrading drug screening can be achieved with hCSF-PNAC in a biological environment of hCSF solution.