Light-Regulated Liquid-Liquid Phase Separation for Spatiotemporal Protein Recruitment and Cell Aggregation

We have previously shown that the upper critical solution temperature-type thermoresponsive ureido polymers such as polyallylurea and poly(2-ureidoethylmethacrylate) derivatives show liquid-liquid phase separation (LLPS), also known as simple coacervation, under physiological conditions below their phase-separation temperatures (T_p). The addition of the polymer-rich coacervate droplets that result from LLPS to a monolayer cell culture induced aggregation of cells into multicellular spheroids. In this study, we prepared a ureido copolymer, poly(vinylamine-co-vinylurea), with azobenzene substituents (Azo-PVU) and demonstrated light-guided assembly and disassembly of LLPS coacervates. Azo-PVUs with T_p values ranging from 10 to 52 °C were prepared by changing the azobenzene content. Ultraviolet light caused a decrease in the T_p of Azo-PVU because of trans-to-cis photoisomerization of the azobenzene and irradiation with visible light increased the T_p. Thus, LLPS of Azo-PVU was reversibly controlled. The coacervate droplets deposited on a dish surface were immediately dissolved by targeted UV irradiation (owing to a decrease in the T_p). Spatially controlled recruitment of proteins on the dish surface was achieved when protein solution was added to the light-patterned surface. Furthermore, the light-guided deposition of coacervates resulted in the spatiotemporal transformation of monolayer cells to aggregates. This light-controlled LLPS will allow the preparation of novel liquid-based materials for biomolecular and cellular engineering.