Detection of CO Release by the Myoglobin (Mb) Assay

The Mb assay was used to determine the thermal-induced CO release from PPPPB–CO NPs based on the difference of deoxy-myoglobin (deoxy-Mb) and carbon monoxy-myoglobin (Mb-CO) in the UV–vis spectra.^40,41 First, a fresh solution of deoxy-Mb was prepared by mixing sodium dithionite (0.1% in PBS, 1 mL) and Mb (66 μM in PBS, 5 mL) under a nitrogen atmosphere and stirring at RT for 0.5 h. PPPPB–CO NPs bubbled with nitrogen for 0.5 h were added to the deoxy-Mb solution at the final concentration of 50 μg/mL PB. After incubation at 37 or 42 °C for different time, the UV–vis spectra were recorded.

Light-induced CO release from PPPPB–CO NPs was monitored similarly, wherein the mixture of deoxy-Mb and PPPB–CO NPs was irradiated (808 nm, 0.5 W/cm²) for different time before recording of the UV–vis spectra.