Freeze-Induced Protein Assembly of α-Synuclein into Stable Microspheres to Fabricate Light-Induced Cargo Release Systems

Stable hollow-type microspheres (MSs) have been fabricated using α-synuclein (αS), an amyloidogenic protein, via freeze-induced protein self-assembly. This assembly process involves three steps: rapid freezing to form spherical protein condensates from αS oligomers, frozen annealing to form a crust on the condensate and freeze-drying to create an interior lumen via the three-dimensional (3D) coffee-stain effect. The crust produced during the frozen-annealing step is a β-sheet-mediated protein structure that is presumed to be created at the quasi-liquid layer of the protein-ice interface and thus contributes to the stability of MSs in aqueous solutions at room temperature without any additional surface stabilization. MSs transform into amyloid fibril condensates when heated to 70 °C, and the drug is loaded via centrifugal membrane filtration. Additionally, the MSs were shielded with an ir…