Synthesis and application of submicrometer fluorescence sensing particles for lysosomal pH measurements in murine macrophages

Phagocytosis of bioparticles such as bacteria and viruses by macrophages is a critical component of the immune response against infections. In this paper we describe the synthesis of submicrometer fluorescent particles with pH sensing capability. The particles are used to measure the pH and to monitor the effect of chloroquine, an antimalarial drug, on the pH in the lysosome, the cellular organelle involved in the phagocytosis process. The synthesis of the pH sensing particles is realized by the covalent attachment of amine reactive forms of Oregon Green (pH sensitive dye) and Texas Red (pH insensitive dye) to the surface of amino-modified submicrometer polystyrene particles. The particles are absorbed by J774 Murine Macrophages through phagocytosis and directed to lysosomes. Despite the high lysosomal levels of digestive Enzymes and acidity, the absorbed particles remain stable for 12 h in the cells when they are stored in a PBS buffer solution at pH 7.4. The pH dynamic range of the sensing particles is between pH 4.5 and 7 with a sensitivity of 0.1 pH units. Exposure of the cells to chloroquine increases the lysosomal pH from 4.8 to 6.5. The effect is concentration-dependent.