Characterization of IR780-Loaded PEG-b-PPS Micelles

The concentration of IR780 loaded into the micelles was confirmed by UV–vis spectroscopy (Spectamax i3). Lyophilized IR780-loaded micelles were dissolved in DMSO to destabilize them and release the loaded IR780, then the released contents were determined using a free IR780 calibration curve. The micelle size distribution was recorded by dynamic light scattering (DLS, Malvern Zetasizer Z). The morphology of the IR780-loaded PEG-b-PPS micelles was characterized by transmission electron microscopy (TEM, BIO TEM JEM-1011). A sample was prepared by drying a drop of the IR780-loaded micelle solution (0.5 mg/mL) on a copper grid coated with amorphous carbon. For negative staining, 10 uL of uranyl acetate was placed on the grid and dried again. The sample grid was dried in a desiccator for one day before TEM observation. A serum stability test of IR780-loaded PEG-b-PPS micelles under conditions mimicking blood was conducted^71,72 and confirmed that the micelles maintained stable size for up to 72 h under 10% serum conditions. The PA sensitivity according to the concentration of micelle-loaded IR780 after subcutaneously injection was conducted. After subcutaneously injecting 50 μL of micelle-loaded IR780 at various concentrations into the right flank of nude mice, the PA imaging was performed at 780 nm. Figure S5 was represented using the same color scale, and the representative signals of the materials were analyzed by designating polygonal ROIs on the PA MAP images. The single-pixel sensitivity was derived by multiplying the volume of one pixel with the sensitivity threshold concentration, using a 380 μm isotropic resolution.