In vivo biodistribution and tumor cell uptake studies

NOD–SCID male mice (JAX, Ellsworth, Maine) were irradiated with 150 rad and injected subcutaneously with 5 million H929 cells. When tumors reached a volume of 150 mm³, mice were distributed randomly into groups and treated intravenously via retro-orbital injection with DiD-labeled or dox–lipid-labeled nanoparticles. Tumor volume was measured via calipers (volume = 0.5 × length × (width)²).

For biodistribution studies, mice were killed 24 h after nanoparticle injection, tumors and major organs were weighed, and ex vivo organ imaging was generated using a Kodak Multispectral FX (Kodak, Rochester, NY) with an excitation of 630 nm and an emission of 700 nm. Total fluorescence was calculated by ImageJ and normalized by tumor or organ mass. Excised tumors were then mechanically fragmented and then further treated with disaggregation solution (0.1% collagenase type IV (Life Technologies) and 0.003% DNase I (Sigma-Aldrich) in PBS) for 45 min at 37 °C with slow agitation. Samples were strained through a 40-µm mesh, washed three times, and analyzed via flow cytometry.