Dual-Ratiometric Fluorescent Nanoprobe for Visualizing the Dynamic Process of pH and Superoxide Anion Changes in Autophagy and Apoptosis

Abstract

Autophagy and apoptosis are closely associated with various pathological and physiological processes in cell cycles. Investigating the dynamic changes of intracellular active molecules in autophagy and apoptosis is of great significance for clarifying their inter-relationship and regulating mechanism in many diseases. In this study, we develop a dual-ratiometric fluorescent nanoprobe for quantitatively differentiating the dynamic process of superoxide anion (O₂^•-) and pH changes in autophagy and apoptosis in HeLa cells. A rhodamine B-loaded mesoporous silica core was used as the reference, and fluorescence probes for pH and O₂^•- measurement were doped in the outer layer shell of SiO₂. Then, chitosan and triphenylphosphonium were modified on the surface of SiO₂. The experimental results showed that the nanoprobe is able to simultaneously and precisely visualize the changes of mitochondrial O₂^•- and pH in HeLa cells. The kinetics data revealed that the changes of pH and O₂^•- during autophagy and apoptosis in HeLa cells were significantly different. The pH value was decreased at the early stage of apoptosis and autophagy, whereas the O₂^•- level was enhanced at the early stage of apoptosis and almost unchanged at the initial stage of autophagy. At the late stage of apoptosis and autophagy, the concentration of O₂^•- was increased, whereas the pH was decreased at the late stage of autophagy and almost unchanged at the late stage of apoptosis. We hope that the present results provide useful information for studying the effects of O₂^•- and pH in autophagy and apoptosis in various pathological conditions and diseases.