Synthesis, Characterization, and Application of Fluorescence Sensing Lipobeads for Intracellular pH Measurements

Abstract

This paper describes the synthesis and characterization of micrometric phospholipid-coated polystyrene particles, named lipobeads, with pH-sensing capability and their application for intracellular pH measurements in murine macrophages. The phospholipids used to coat the particles are labeled with fluorescein (a pH-sensitive dye) and tetramethylrhodamine (a pH-insensitive dye), which serves as a referencing fluorophore for increased accuracy of the pH measurements. The synthesis of the pH-sensing lipobeads is realized by the covalent attachment of the fluorescent phospholipids to the surface of carboxylated polystyrene particles. The pH dynamic range of the sensing particles is between 5.5 and 7.0 with a sensitivity of 0.1 pH unit. The excitation light intensity is reduced to minimize photobleaching of the fluorescein-phospholipid conjugates. The fluorescent lipobeads are used to measure the pH in single macrophages. The lipobeads are ingested by the macrophages and directed to lysosomes, which are the cellular organelles involved in the phagocytosis process. Despite the high lysosomal levels of digestive enzymes and acidity, the absorbed particles remain stable for over 6 h in the cells when they are stored in a phosphate-buffered saline solution at pH 7.4.

Design of fluorescent materials for chemical sensing

“…Rosenzweig and co-workers have used liposomes as fluorescent nanosensors 48 for intracellular measurements of pH, 164 Ca 2+ , 262 and O 2 . 263 The sensing reagents are encapsulated in the internal aqueous area of the liposomes and they retain their free solution properties.…”

Design of Fluorescent Materials for Chemical Sensing

“…These optical nanosensors have two main benefits: protection of the sensing component from interfering species within the intracellular environment and protection of the intracellular environment from toxic effects on the sensing component. Rosenzweig and co-workers have used liposomes as fluorescent nanosensors 48 for intracellular measurements of pH, 164 Ca 2+ , 262 and O 2 . 263 The sensing reagents are encapsulated in the internal aqueous area of the liposomes and they retain their free solution properties.…”

Non-invasive Characterization of Immune Responses to Biomedical Implants

“…Several fluorescence-labeled cells and particle-based sensors have also been developed for a wide range of applications in biological research and clinical diagnosis. 13,22,29,41,60,89,104,108 Of late, near Infrared (NIR) fluorescence has gained popularity for in vivo imaging because of reduced absorption and scattering of photons by tissues, and also reduced interference from auto-fluorescence signal. 31 NIR technique has been applied in several clinical studies in recent years.…”