Intracellular oxygen monitoring and oxygen demand assessment using Ir(ppy)₃-encapsulated polymeric micelles

Intracellular oxygen concentration is a pivotal indicator of cellular metabolic and functional states that may be influenced by pathologic conditions, including hypoxia in solid tumors and hyperglycemia. Currently, methods for quantifying cellular oxygen levels-particularly within immune cells implicated in hypoxic dysfunction-are underdeveloped. Here, we introduce a novel biocompatible intracellular oxygen sensor developed by encapsulating the poorly water-soluble phosphorescent dye tris(2-phenylpyridinato)iridium(III) (Ir(ppy)₃) within micelles formed from 2-methacryloyloxyethyl phosphorylcholine (MPC) polymers. This micelle system enables efficient delivery and stable retention of the phosphorescent dye within the cytoplasm. Building on prior confocal microscopy studies characterizing the properties of MPC polymers, our findings reveal that cellular uptake of these polymers occurs via a cell-penetrating translocation mechanism, effectively bypassing significant endosomal sequestration and thus facilitating cytoplasmic oxygen sensing. Micelles prepared with a 30-μM Ir(ppy)₃ stock solution exhibited optimal phosphorescence and clear oxygen sensitivity. Using this sensor, we observed distinct oxygen consumption kinetics between KPL-4 and MDA-MB231 breast cancer cells. Moreover, analysis of the response of NK92-CD16 cells, an NK cell-derived immune cell line, to varying glucose levels revealed that high glucose conditions (300-400 mg/dL) significantly suppress the hypoxia-induced increase in phosphorescence, indicating reduced metabolic oxygen demand. Overall, this MPC micelle-encapsulated Ir(ppy)₃ platform serves as a robust tool for evaluating cellular metabolic states and in vitro responses to microenvironmental cues, such as hypoxia and hyperglycemia.