**2. Intracellular trafficking**

The intracellular trafficking pathway of Tf and its receptor has been studied for several years and has been reviewed in many journals [18‐20]. After holo‐Tf binds to TfR on the surface of cells with nanomolar affinity (KD ~ 10‐<sup>9</sup> M), the Tf/TfR complex is internalized as part of an endocytic vesicle. The endosome then matures and acidifies to a pH between 5 and 6 [21], causing iron to be released from Tf. Once iron is released, it is reduced to the ferrous (Fe2+) form due to the presence of oxidoreductases in the endosome. A divalent metal transporter then shuttles Fe2+ into the cytosol. The iron‐free Tf/TfRcomplex recycles back to the cell surface, and since iron‐free Tf (apo‐Tf) has a low binding affinity for TfR at the near neutral pH of the cell surface, apo‐Tf quickly dissociates from the cell‐surface receptor. This entire cycle of the Tf/TfR trafficking pathway lasts only about 5 minutes [22].

While the rapid recycling of Tf contributes to the efficient transport of iron, it also limits the ability of the ligand to deliver its payload. Accordingly, drug delivery efficacy may be improved through a better understanding of the kinetics involved in the intracellular traffick‐ ing pathway of Tf [23]. Through *in vitro* experiments and mathematical modeling, Murphy and coworkers previously demonstrated that monoclonal anti‐transferrin receptor antibodies would be more effective drug delivery vehicles if they associated with cells for a greater period of time [24‐26]. In other words, this increased cellular association would lead to an increase in the exposure of cancer cells to the conjugated drug. Instead of investigating antibodies for TfR, the Kamei laboratory studied the Tf ligand itself. Specifically, by deriving and analyzing a mathematical model for Tf/TfR trafficking, Kamei and coworkers identified a novel design criterion for engineering Tf to enhance its drug delivery efficacy [8‐10], as discussed below.
