Long-Retention Fluorescent Probe for Plasma Membrane Imaging and Analysis of Membrane Remodeling

Abstract Reliable fluorescence analysis of plasma membrane (PM) remodeling is often compromised by probe internalization and intracellular redistribution, which can obscure genuine changes in membrane physicochemical properties. Herein, we report NIR-PMP-1, a near-infrared fluorescent molecular rotor designed for rapid, wash-free, and long-retention PM imaging and analysis. NIR-PMP-1 labels the PM within 30 s, remains predominantly membrane-confined for up to 24 h with minimal internalization, and retains membrane specificity in both live and fixed cells. Comparison with NIR-PMP-2, together with experimentally determined log P values of 0.65 ± 0.08 and 1.20 ± 0.10, respectively, indicates that balanced lipophilicity is critical for persistent membrane retention. Enabled by this stable localization, NIR-PMP-1 visualized progressive PM remodeling during erastin-induced ferroptosis and cholesterol-targeting drug treatments. HPLC–HRMS verified probe integrity, while immunoblotting and lipidomics revealed associated GPX4/Nrf2 alterations and distinct drug-specific lipid remodeling patterns. The probe further differentiated the tested cancer and normal cell lines in monoculture and coculture. Giant unilamellar vesicle experiments linked its fluorescence response to membrane lipid composition and order, providing mechanistic support for membrane-state discrimination. Collectively, NIR-PMP-1 provides a long-retention fluorescent platform for reliable analysis of dynamic PM remodeling and disease-associated membrane microenvironments.