Living image analysis software

All bright field, fluorescent, and merged (bright field and fluorescent) images were taken by a Curadel Lab-FLARE^® RP1 imaging system with 800 nm filter set (Curadel, LLC, Natick, MA, USA) or Spectrum IVIS^® camera with an ICG filter set (PerkinElmer, Inc., Waltham, MA, USA) and analyzed using Resvet Imaging software, v.4.0 (Curadel) or Living Image^® Analysis software (Perkin Elmer, Waltham, MA, USA). Fluorescent images were viewed with a Leica-DM4000B microscope (Leica Microsystems, Buffalo Grove, NY, USA) and analyzed with QCapturePro-7 software, v.5.0 (QImaging, Surrey, BC, Canada).

IR‐780 fluorescent dye was introduced to tag PTFL NPs (PTFL–IR780) for in vivo fluorescence imaging. IR‐780 was only used for in vivo fluorescence imaging experiments and was not used for in vivo therapy experiments. All the synthesis steps were the same as PTFL NPs synthesis except that 5 µL IR‐780 (10 mg mL⁻¹ in DMSO) was added in PFOB emulsion together with FeCl₃ · 6H₂O. The obtained PTFL–IR780 NPs were centrifuged to remove the unloaded IR‐780 and re‐dispersed in PBS. The successful labeling of IR780 and the stability of the labeling method were characterized by measuring the Vis–NIR extinction of the PTFL–IR780 NPs before and after dispersing in PBS for 7 days. After the tumor reached an approximate size of 100 mm³, 4T1 subcutaneous tumor‐bearing mice were i.v. injected with 0.2 mL PTFL–IR780 NPs (1 mg mL⁻¹), then imaged under an IVIS system with an excitation wavelength of 760 nm and an emission wavelength of 845 nm at different time intervals (0, 2, 4, 10, 24, 48 h). For the biodistribution study, the PTFL‐IR780 treated mice were sacrificed at 4, 10, 24, 48 h post‐administration. The major organs (lungs, kidneys, hearts, spleens, and livers) and tumor tissues were excised and imaged ex vivo with the same excitation (760 nm) and emission (845 nm). The PerkinElmer Living Image Analysis Software was used to further conduct the region of interest analysis.