Controlling Drug Partitioning in Individual Protein Condensates through Laser-Induced Microscale Phase Transitions

ABSTRACT Gelation of protein condensates formed by liquid-liquid phase separation (LLPS) occurs in a wide range of biological contexts, from the assembly of biomaterials to the formation of fibrillar aggregates and is therefore of interest for biomedical applications. Soluble-to-gel (sol-gel) transitions are controlled through macroscopic processes such as changes in temperature or buffer composition, resulting in bulk conversion of liquid droplets into microgels within minutes to hours. Using microscopy and mass spectrometry, we show that condensates of an engineered mini-spidroin (NT2repCT YF ) undergo a spontaneous sol-gel transition resulting in the loss of exchange of proteins between the soluble and the condensed phase. We find that liquid spidroin condensates absorb visible light, which enables us to control sol-gel transitions of individual droplets through laser pulses. Fluorescence microscopy reveals that laser-induced gelation significantly alters the interactions between droplet proteins and small molecules, which allows us to load single droplets with an anticancer drug. In summary, our findings demonstrate direct control of phase transitions in individual condensates opening new avenues for functional and structural characterization. SYNOPSIS TOC The liquid-to-solid transitions of phase-separated protein condensates are challenging to control. Leppert et al . show that condensates of engineered mini-spidroins gelate at slightly elevated temperatures. Using high-energy laser pulses at wavelengths that are absorbed by the droplets, the authors induce sol-gel transitions in single droplets. These gelated droplets are chemically stable and exhibit an increased ability to sequester drug molecules.