Polysorbate 80 Controls Morphology, Structure and Stability of Human Insulin Amyloid-Like Spherulites

Abstract

Amyloid protein aggregates are not only associated with neurodegenerative diseases and may also occur as unwanted by-products in protein-based therapeutics. Surfactants are often employed to stabilize protein formulations and reduce the risk of aggregation. However, surfactants alter protein-protein interactions and may thus modulate the physicochemical characteristics of any aggregates formed. Human insulin aggregation was induced at low pH in the presence of varying concentrations of the surfactant polysorbate 80. Various spectroscopic and imaging methods were used to study the aggregation kinetics, as well as structure and morphology of the formed aggregates. Molecular dynamics simulations were employed to investigate the initial interaction between the surfactant and insulin. Addition of polysorbate 80 slowed down, but did not prevent, aggregation of insulin. Amyloid spherulites formed under all conditions, with a higher content of intermolecular beta-sheets in the presence of the surfactant above its critical micelle concentration. In addition, a denser packing was observed, leading to a more stable aggregate. Molecular dynamics simulations suggested a tendency for insulin to form dimers in the presence of the surfactant, indicating a change in protein-protein interactions. It is thus shown that surfactants not only alter aggregation kinetics, but also affect physicochemical properties of any aggregates formed.

Morphological integrity of insulin amyloid-like aggregates depends on preparation methods and post-production treatments

“…Aggregates consisting of misfolded proteins and amyloid-like structure may result in the break of immune tolerance in transgenic mice [39]. Amyloid-like spherulites produced in acetic acid contain a high amount of aggregated β-sheet, compared to native human insulin [11, 14], thus reducing the amount of aggregated β-sheet present in the spherulite could potentially reduce the immunogenicity risk of this aggregate type.…”

“…Moreover, several aggregates formed in vitro are generated in formulations, which are not suitable for certain characterization techniques or are not readily translatable to biological assays. Solvent exchange is thus required to allow for characterization or application in biological assays and it is often achieved via centrifugation-and dialysis-based methods [12][13][14]. Morphological integrity before and after solvent exchange is assumed intact, yet rarely investigated.…”

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

“…We previously reported that the addition of PS80 slows down the insulin amyloid aggregation process 19 . With the aim of determining how PS80 affects the energy barrier associated with the transition of soluble insulin to the aggregated form, we used the amyloid-specific dye Thioflavin T (ThT) 21 to detect the bulk aggregation kinetics at different temperatures in the range 37–60 °C.…”

“…In a previous study, we identified a surprising effect of the commonly used surfactant polysorbate 80 (PS80) on the stability and aggregation propensity of insulin in solution 19 . More specifically, it was shown that PS80 slows down the thermal aggregation of insulin, but induced the formation of highly stable spherulites with an increased content of β-sheet structure and increased protein packing density 19 .…”

“…In a previous study, we identified a surprising effect of the commonly used surfactant polysorbate 80 (PS80) on the stability and aggregation propensity of insulin in solution 19 . More specifically, it was shown that PS80 slows down the thermal aggregation of insulin, but induced the formation of highly stable spherulites with an increased content of β-sheet structure and increased protein packing density 19 . This leads to our general hypothesis that PS80 alters the mechanism of aggregate formation, the energetics related to the process, and the aggregate features, with these three aspects intrinsically related to each other.…”