Phasor FLIM analysis of Thioflavin T fluorescence in protein amyloid aggregates: Mapping molecular interactions.

Thioflavin T (ThT) is a worldwide used dye to monitor protein aggregation as it stains with a certain specificity amyloid structures. The interactions between ThT and its hosts are largely studied suggesting that fluorescence properties of this dye critically depend both on the environment rigidity, electrostatic and hydrophobic properties as well as on molecular details binding site structure. Here FLIM and phasor approach analysis are used to exploit ThT amyloid interactions and, in turn, to address polymorphism and structural heterogeneity of amyloid species mapping aggregate-to-aggregate structural differences and revealing details of molecular architecture within the same aggregate.

Highly tunable protein microspheres for drug delivery

Traditionally protein aggregation has been related to several neurodegenerative diseases, however in the past few years there has been a grown interest to use them as biomaterials. Among the very broad range of different amyloid structures a special focus has been developed on protein particulates, spherical protein aggregates formed at a pH near the isoelectric point of the protein they are made of, whose radius ranges from hundreds of nm to few um. They are a generic feature for all globular proteins and besides, they have never been related to any disease. Among the years different methods for the functionalization of amyloid fibrils or microspheres have been unravelled, but normally the…

Molecular characterization of tunable microscale protein-based biomaterials

Highly tunable protein microspheres for drug delivery

It is well-known that protein amyloid aggregation has profound implications in several neurodegenerative diseases. In contrast, a natural role for amyloid structures as protection, adhesion and storage materials in living system is also reported, promoting protein aggregates as an interesting platform for the design of multifunctional biomaterials. Among the broad range of different amyloid structures protein particulates deserve special attention; they are spherical protein aggregates with radius ranging from hundreds of nm to few um which are readily formed in solution at pHs values near the isoelectric point of the protein they are made of. Interestingly, particulate appears to be a gene…

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

AbstractAmyloid 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 s…

Insights on amyloid spherulites structure at molecular level

Conformational Transitions upon Maturation Rule Surface and pH-Responsiveness of α-Lactalbumin Microparticulates

De novo designed protein supramolecular structures are nowadays attracting much interest as highly performing biomaterials. While a clear advantage is provided by the intrinsic biocompatibility and...

Direct formation of highly tunable and biocompatible protein microparticles

Probing ensemble polymorphism and single aggregate structural heterogeneity in insulin amyloid self-assembly.

Ensembles of protein aggregates are characterized by a nano- and micro-scale heterogeneity of the species. This diversity translates into a variety of effects that protein aggregates may have in biological systems, both in connection to neurodegenerative diseases and immunogenic risk of protein drug products. Moreover, this naturally occurring variety offers unique opportunities in the field of protein-based biomaterials. In the above-mentioned fields, the isolation and structural analysis of the different amyloid types within the same ensemble remain a priority, still representing a significant experimental challenge. Here we address such complexity in the case of insulin for its relevance…