Freeze-Induced Protein\nAssembly of α‑Synuclein\ninto Stable Microspheres to Fabricate Light-Induced Cargo Release\nSystems

Stable hollow-type microspheres (MSs) have been fabricated\nusing\nα-synuclein (αS), an amyloidogenic protein, via freeze-induced\nprotein self-assembly. This assembly process involves three steps:\nrapid freezing to form spherical protein condensates from αS\noligomers, frozen annealing to form a crust on the condensate and\nfreeze-drying to create an interior lumen via the three-dimensional\n(3D) coffee-stain effect. The crust produced during the frozen-annealing\nstep is a β-sheet-mediated protein structure that is presumed\nto be created at the quasi-liquid layer of the protein–ice\ninterface and thus contributes to the stability of MSs in aqueous\nsolutions at room temperature without any additional surface stabilization.\nMSs transform into amyloid fibril condensates when heated to 70 °C,\nand the drug is loaded via centrifugal membrane filtration. Additionally,\nthe MSs were shielded with an iron-alginate layer embedded with gold\nnanoparticles (AuNPs) to prevent premature leakage and to control\ndrug release. This takes advantage of the photothermal effect of AuNPs,\nresulting in combined cytotoxicity between the drug and heat. Therefore,\ndrug-loaded MSs comprising αS and AuNPs can be suggested as\nlight-controllable drug delivery systems that exhibit chemical and\nphysical anticancer therapeutic effects.