Oxygen Biosensors Based on Block Copolymer Micelles Loaded with Phosphorescent Ir(III) Complexes: Design and Evaluation of <i>In Vivo</i> Performance

Molecular oxygen plays an important role in metabolism, and its deficiency (hypoxia) can be a marker of various diseases. Optical oxygen imaging techniques based on oxygen-sensitive phosphorescent probes provide a way for noninvasive visualization of intracellular and tissue oxygen distribution. Among potential oxygen probes, phosphorescent Ir(III) complexes are attracting increasing attention because they combine strong lifetime dependence on oxygen concentration with easy tuning of their spectral characteristics. Loading of iridium emitters into polymer micelles can provide their solubilization and protection, thus giving rise to phosphorescent oxygen nanosensors. In this work, we present oxygen nanoprobes based on a series of Ir(III) complexes embedded into poly(caprolactone-block-ethylene glycol) polymer micelles and evaluate their effectiveness for oxygen sensing in vitro and in vivo. We demonstrate that varying the structure of the ligands of the complexes leads to changes in the photophysical characteristics of the phosphors and the physicochemical properties of the nanosensor. The most promising system demonstrates high sedimentation and aggregation stability, unimodal and narrow size distributions, low zeta potentials, high sensitivity of phosphorescence lifetime to oxygen without cross-talk with other parameters, and linear Stern–Volmer plots with Stern–Volmer constants reaching up to 1 × 10–2 mm Hg-1. Evaluation of the best sensor candidate for its applicability for in vivo phosphorescence lifetime imaging revealed its ability to accumulate in tumors and internalize into cells without developing any toxic effects up to 0.3 mg/mL. The mean oxygen partial pressure inside CT26 tumor xenografts measured in vivo using the developed oxygen nanosensor was evaluated to be about 20 mm Hg.