Magnetic nanoparticles functionalized by amino acids as anti-amyloid agents

Alzheimer´s disease, Parkinson´s disease, type II Diabetes Mellitus and more than 50 other disorders known as amyloidoses are incurable protein misfolding diseases [1]. Magnetic nanoparticles (MNPs) gained massive attention in the last decades. Amino acid functionalization of their surface provides a more extensive surface area to interact with macromolecules allowing them to be used as anti-amyloid agents [2].

The aim of the presented work was to study the anti-amyloid effect of MNPs functionalized by proline (Pro-MNPs), cysteine (Cys-MNPs), and poly-L-lysine (PLL-MNPs) on the amyloid aggregation of α-lactalbumin (α-LA) in vitro. α-LA, a small globular, acidic and Ca²⁺-binding protein present in the mammalian milk, can be used as a model protein for amyloid aggregation in vitro [3]. The interaction of amino acid-functionalized magnetic nanoparticles (aa-MNPs) with amyloid fibrils can be influenced by their physico-chemical properties and type of functionalized aa [2]; therefore, the aa-MNPs were analyzed by dynamic light scattering (DLS) technique to obtain their hydrodynamic diameter (D_H) and by laser Doppler velocimetry to characterize their zeta potential (ζ) and isoelectric point (pI). The results showed that the biggest Cys-MNPs have the lowest ζ and pI; the smallest Pro-MNPs have lower ζ and pI; and smaller PLL-MNPs have the lowest ζ and pI.

Thioflavin T (ThT) fluorescence assay was used to investigate destroying and inhibitory capability of aa-MNPs on α-LA amyloid aggregation. Our results revealed that aa-MNPs destroy α-LA fibrils (α-LAF) and inhibit the formation of α-LAF in a concentration-dependent manner. The efficiency of destroying and inhibitory ability of aa-MNPs were quantified by DC₅₀ and IC₅₀ values (concentration of aa-MNPs causing a 50% decline in fluorescence intensities corresponding to 50% destruction or inhibition of fibrils). Results showed no correlation between destroying ability of aa-MNPs and their physico-chemical properties. However, the inhibitory effect of aa-MNPs negatively correlates with their D_H (the smallest Pro-MNPs had the highest inhibitory activity (the lowest IC₅₀), the lower inhibition was observed for bigger PLL-MNPs followed by Cys-MNPs). Atomic force microscopy was used to visualize the effects of aa-MNPs on the morphology of α-LA. There were no morphology changes of fibrils observed after their interaction with aa-MNPs. On the other hand, shorter fibrils and aggregates were formed due to the inhibition of α-LAF fibrillization by aa-MNPs. Moreover, aa-MNPs´ cytotoxicity was tested on human kidney (HEK293) cells by lactate dehydrogenase assay and optical microscopy. We have found that PLL-MNPs were the most cytotoxic.

To conclude, we studied aa-MNPs and their anti-amyloid potential and we found out that Cys-MNPs with a low DC₅₀ value and relatively low cytotoxicity effect are a suitable candidate for amyloid fibrils destruction. Instead, Pro-MNPs may serve as a potential inhibitor of amyloid fibrils formation due to their low IC₅₀ and relatively low cytotoxicity.