Heterogeneous and Surface-Catalyzed Amyloid Aggregation Monitored by Spatially Resolved Fluorescence and Single Molecule Microscopy

Amyloid aggregation is associated with many diseases and may also occur in therapeutic protein formulations. Addition of co-solutes is a common strategy to modulate the stability and aggregation of proteins in pharmaceutical formulations. The purpose of this study is to establish a framework — combining a spatially resolved fluorescence approach and single molecule microscopy — to highlight the multiple effects of polysorbate 80 (PS80), a common surfactant in protein formulations, on the transition from native insulin to amyloid spherulites. The latter are amyloid superstructures, which can be formed both in vivo and in vitro . We show that PS80 addition increases the energy barrier for initiation of the amyloid aggregation process due to the shift from a surface-catalyzed reaction to a bulk aggregation. The shifted balance is mainly due to the ability of PS80 to prevent insulin adsorption at the liquid-solid and air-liquid interfaces. The kinetics of the spherulite formation was analyzed at single aggregate level and we identified two growth mechanisms for spherulites (isotropic and anisotropic), which are influenced by the amount of freely available PS80 molecules in bulk. Our framework provides a general tool for comprehensive analysis of the effect of co-solutes on self-assembly reactions and allows one to connect the changes in energy barriers with the mechanisms of action for the investigated molecules. This approach is particularly suitable for studying highly heterogeneous and surface-sensitive aggregation reactions. ![Figure][1] ### Competing Interest Statement The authors have declared no competing interest. [1]: pending:yes