Polysorbate 80 controls Morphology, structure and stability of human insulin Amyloid-Like spherulites

Zhou, Xin; Galparsoro, Dirk Fennema; Madsen, Anders Ø.; Vetri, Valeria; Weert, Marco van de; Nielsen, Hanne Mørck; Foderà, Vito

doi:10.1016/j.jcis.2021.09.132

Cited by 18 publications

(22 citation statements)

References 93 publications

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“…At high temperatures, the rate of dissociation may be comparable to the association rate, resulting in the absence of a temperature dependency for the overall rate of the process expressed by 1/𝑡 % . This effect is not detected in the presence of PS80, in agreement with the our previous observation that PS80 induces a higher stability of the aggregates 19 , likely minimizing the impact of the dissociation reaction.…”

Section: Table Of Contentssupporting

confidence: 91%

Section: Table Of Contentsmentioning

confidence: 99%

“…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.…”

Section: Table Of Contentsmentioning

confidence: 99%

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

Zhou

Sinkjaer

Zhang

et al. 2022

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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.

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Section: Table Of Contentssupporting

confidence: 91%

Section: Table Of Contentsmentioning

confidence: 99%

Section: Table Of Contentsmentioning

confidence: 99%

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

Zhou

Sinkjaer

Zhang

et al. 2022

Preprint

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“…Indeed the two species co-exist 17 and this is a further fact that justifies the need to have single-aggregate approaches to evaluate the heterogeneity of the system. Salt, pH, and protein concentration 28 , the addition of alcohols 22 , and surfactants 29 have been shown to contribute to the variability of the structure and morphology of spherulites. We choose the experimental conditions which guaranteed a time scale of the process allowing our real-time microscopy analysis.…”

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confidence: 99%

Direct observation of heterogeneous formation of amyloid spherulites in real-time by super-resolution microscopy

et al. 2022

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Protein misfolding in the form of fibrils or spherulites is involved in a spectrum of pathological abnormalities. Our current understanding of protein aggregation mechanisms has primarily relied on the use of spectrometric methods to determine the average growth rates and diffraction-limited microscopes with low temporal resolution to observe the large-scale morphologies of intermediates. We developed a REal-time kinetics via binding and Photobleaching LOcalization Microscopy (REPLOM) super-resolution method to directly observe and quantify the existence and abundance of diverse aggregate morphologies of human insulin, below the diffraction limit and extract their heterogeneous growth kinetics. Our results revealed that even the growth of microscopically identical aggregates, e.g., amyloid spherulites, may follow distinct pathways. Specifically, spherulites do not exclusively grow isotropically but, surprisingly, may also grow anisotropically, following similar pathways as reported for minerals and polymers. Combining our technique with machine learning approaches, we associated growth rates to specific morphological transitions and provided energy barriers and the energy landscape at the level of single aggregate morphology. Our unifying framework for the detection and analysis of spherulite growth can be extended to other self-assembled systems characterized by a high degree of heterogeneity, disentangling the broad spectrum of diverse morphologies at the single-molecule level.

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“…High temperature and acidic pH guarantee an aggregation process occurring in 1−3 days, thus avoiding experimental artifacts such as evaporation and major chemical degradation of the sample. 35 The CD signal in the near-UV region during a temperature ramp (from 250 to 320 nm at 1 °C/min) was used to determine the unfolding temperature. 36 Figure 1b−f summarizes the melting temperatures (T m ) for HEWL in the presence of the five chosen Hofmeister salts (see Supporting Information and Figure S1 for more information).…”

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confidence: 99%

Ion-Mediated Morphological Diversity in Protein Amyloid Systems

Chaaban

Vallooran

Weert

et al. 2022

J. Phys. Chem. Lett.

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Salt ions are considered among the major determinants ruling protein folding, stability, and self-assembly in the context of amyloid-related diseases, protein drug development, and functional biomaterials. Here, we report that Hofmeister ions not only determine the rate constants of the aggregation reaction for human insulin and hen egg white lysozyme but also control the generation of a plethora of amyloid-like morphologies ranging from the nanoscale to the microscale. We anticipate that the latter is a result of a balance between colloidal and conformational stability combined with an ion-specific effect and highlight the importance of salt ions in controlling the biological functions of protein aggregates.

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Polysorbate 80 controls Morphology, structure and stability of human insulin Amyloid-Like spherulites

Cited by 18 publications

References 93 publications

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

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

Direct observation of heterogeneous formation of amyloid spherulites in real-time by super-resolution microscopy

Ion-Mediated Morphological Diversity in Protein Amyloid Systems

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