Fluorescence Imaging of Nanoparticle Uptake into Liquid-Liquid Phase-Separated Droplets

Liquid-liquid phase separation (LLPS) in living cells has received considerable attention in the biomedical research field. This study is the first to report nanoparticle (NP) uptake into LLPS droplets. Fluorescent dye, Nile red loaded polystyrene NPs (NR-PSt NPs) uptake into model LLPS droplets consisting of adenosine triphosphate (ATP) and poly-L-lysine (PLL) was visualized using fluorescence imaging. Fluorescence imaging showed that the LLPS droplets had a quick NP uptake behavior. Furthermore, temperature changes (4-37 °C) significantly affected the NP uptake behavior of the LLPS droplets. Moreover, the NP-incorporated droplets displayed high stability under strong ionic strength conditions (1 M NaCl). ATP measurements displayed that ATP was released from the NP-incorporated droplets, indicating that the weakly negatively charged ATP molecules and strongly negatively charged NPs were exchanged, which resulted in the high stability of the LLPS droplets. These fundamental findings will contribute to the LLPS studies using various NPs.