Washing-free fluorescent probes for visualizing the process of lipophagy and identifying non-alcoholic fatty liver disease

Lipid droplets (LDs) are organelles involved in various physiological processes, and real-time monitoring of their dynamic behavior is essential for diagnosing and treating related diseases. Herein, we designed and synthesized five morpholine-based fluorescent molecules with a strong electronic push-pull effect, referred to as WZX1-WZX5, and explored their LDs-targeting ability. By modifying the substituents of the molecules, we identified that WZX2, WZX4, and WZX5 exhibited remarkable efficacy in specifically targeting cellular LDs. Among these, WZX5 was chosen for further investigation due to its significantly longer emission wavelength (677 nm in DMSO and 664 nm in PBS) compared to the other probes. Notably, probe WZX5 demonstrated favorable biocompatibility and rapid accumulation in LDs within 20 s. Additionally, probe WZX5 exhibited a high signal-to-noise ratio and produced consistent results without the need for washing with PBS. Importantly, probe WZX5 successfully visualized the process of lipophagy, facilitating the study of LDs-lysosome interactions. This capability was further confirmed by identifying nonalcoholic fatty liver disease (NAFLD) at the tissue and organ levels through fluorescence imaging. The properties of probe WZX5 render it a valuable tool for diagnosing LDs-associated diseases and conducting in-depth investigations into lipophagy processes.