Intracellular Temperature Sensing with RemarkablyHigh Relative Sensitivity Using Nile Red-Loaded Biocompatible Niosome

Accurate temperature sensing at the nanoscale within biological systems is crucial for understanding various cellular processes, such as gene expression, metabolism, and enzymatic reactions. Current temperature-sensing techniques either lack the temperature resolution and sensitivity necessary for intracellular applications or require invasive procedures that can disrupt cellular activities. In this study, we present Nile Red (NR)-loaded hybrid (span 60-L64) niosomes and Nile Red-loaded L64 niosomes as highly sensitive fluorescent nanothermometers. These niosomes are synthesized via the thin-layer evaporation method, forming thermoresponsive vesicles, and they demonstrate reversible phase transition behavior with temperature. When loaded with polarity-sensitive Nile Red, vesicles exhibit a strong temperature-dependent fluorescence response (change in intensity, emission maximum, and lifetime), suitable for noncontact temperature sensing in the biologically important temperature range of 25 to 50 °C. While NR-hybrid niosomes exhibit a high relative sensitivity of 19% °C^–1 at 42 °C, NR-L64 niosomes achieved extraordinary relative sensitivity of 36% °C^–1 at 40 °C. Using NR-L64 niosomes, the temperature resolution is found to be 0.0004 °C at 40 °C. The nanothermometers displayed excellent photostability, thermal reversibility, and resistance to variations in ion concentration and pH. Temperature-dependent confocal microscopy using FaDu cells confirmed the biocompatibility and effectiveness of the designed nanothermometers for precise intracellular temperature sensing. The results demonstrate the significant potential of Nile Red-loaded niosomes for temperature monitoring using live cell imaging in biological media.