Versatile conjugated polymer nanoparticles for high-resolution O₂ imaging in cells and 3D tissue models

High brightness, chemical and photostability, tunable characteristics, and spectral and surface properties are important attributes for nanoparticle probes designed for live cell imaging. We describe a class of nanoparticles for high-resolution imaging of O₂ that consists of a substituted conjugated polymer (polyfluorene or poly(fluorene-alt-benzothiadiazole)) acting as a FRET antenna and a fluorescent reference with covalently bound phosphorescent metalloporphyrin (PtTFPP, PtTPTBPF). The nanoparticles prepared from such copolymers by precipitation method display stability, enhanced (>5-10 times) brightness under one- and two-photon excitation, compatibility with ratiometric and lifetime-based imaging modes, and low toxicity for cells. Their cell-staining properties can be modulated with positively and negatively charged groups grafted to the backbone. The "zwitter-ionic" nanoparticles show high cell-staining efficiency, while their cell entry mechanisms differ for the different 3D models. When injected in the bloodstream, the cationic and anionic nanoparticles show similar distribution in vivo. These features and tunable properties make the conjugated polymer based phosphorescent nanoparticles a versatile tool for quantitative O₂ imaging with a broad range of cell and 3D tissue models.