Scheme 1.

Schematic illustration of the possible mechanism of sub-5 nm uIONPs promoting active targeting that can be tested: At the early time points (1 and 3 hours) after co-injection, both ligand mediated active targeting and passive targeting uIONPs undergo fast extravasation into tumors from the leaky tumor vessels comparing to larger IONPs due to size advantage in permeation; At the late time point (24 hours), TfR targeting uIONPs, i.e., FITC-Tf-uIONPs, may exhibit longer tumor retention and intratumoral distribution within tumor environments due to the specific ligand-target interactions, while the majority of passive targeting uIONPs, i.e., TRITC-uIONPs, as well as other unbound or off-targeted FITC-Tf-uIONPs are cleared out of the tumor and intravasated back into blood vessels but not uptaken by macrophages that prefer larger NPs; However, both ligand mediated active targeting and passive targeting IONPs (30 nm core size) have similar tumoral accumulation due to the limited intravasation.