**3. Transferrin‐diphtheria toxin conjugates**

intracellular trafficking properties of the ligand. Moreover, although transferrin has been conjugated to liposomes [13] and nanoparticles [14] to enhance their targeting to cancer cells, they are not included in this review, since the intracellular trafficking properties of these drug delivery vehicles can vary from that of the ligand alone due to the large differences in size.

Micro-Nano Mechatronics — New Trends in Material, Measurement, Control, Manufacturing and Their Applications in

Human serum transferrin (Tf) has been investigated for several years as a targeting agent due to the overexpression ofits receptor on cancer cells. Tf has a molecular weight of approximately 80 kDa and is responsible for transporting free iron from the circulation to cells. Each Tf molecule has the capability to bind to two ferric (Fe3+) ions, one in the N‐terminal lobe (N‐lobe) and the otherin the C‐terminal lobe (C‐lobe). Each lobe binds to a ferric ion with an equilibrium dissociation constant (KD) of approximately 10‐<sup>22</sup> M [15, 16]. This iron‐bound Tf, or holo‐Tf, then binds to its cell‐surface receptor (TfR). After binding, The Tf/TfR complex is internalized, and holo‐Tf delivers its iron to the cell, promoting cellular growth and proliferation [17]. Since cancer cells require more iron to sustain their rapid proliferation, they have been found to overexpress TfR, and this high expression level of TfR has been exploited to achieve selective

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

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.

targeting of anticancer agents to cancer cells.

Tf/TfR trafficking pathway lasts only about 5 minutes [22].

**2. Intracellular trafficking**

Biomedical Engineering

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Diphtheria toxin (DT) is a protein toxin secreted by *Corynebacterium diphtheria*. DT acts by inhibiting protein synthesis through inactivation of elongation factor 2 (EF‐2) [27]. Tf conju‐ gated to a mutant of DT, known as CRM107, has been effective against malignant gliomas [28]. The mutation in CRM107 significantly inhibits the binding of the toxin to its native receptor, a heparin‐binding epidermal growth factor‐like growth factor precursor, thereby reducing the nonspecific toxicity of Tf‐CRM107 [29]. Youle and Oldfield's group performed *in vivo* studies using Tf‐CRM107 on solid human gliomas in the flanks of nude mice, and observed increased cytotoxicity exerted by the conjugate. The success of Tf‐CRM107 eventually led to phase III clinical trials, which were unfortunately canceled in late 2006 following the results of a conditional power analysis suggesting that its efficacy would not significantly improve upon the current standard‐of‐care treatments.

As mentioned above, Tf recycles very rapidly, and this short duration inside the cell can limit the ability of Tf to deliver its cytotoxic payload. Therefore, to identify new approaches to improving the efficacy of Tf‐CRM107, Kamei and coworkers used mass action kinetics to derive a mathematical model of the Tf/TfR trafficking pathway. Analysis of the model helped determine that, by reducing or inhibiting the iron release rate of Tf within the endosome, its drug delivery efficacy would be significantly improved [8]. In this scenario, iron would be retained by Tf upon recycling to the cell surface and the conjugate would be reinternalized to participate in another cycle of trafficking due to the preserved high affinity of holo‐Tf for TfR, increasing its cellular association. The drug delivery efficacy of Tf can therefore be improved, since a single Tf‐drug conjugate would undergo multiple trafficking cycles, thereby increasing the probability of delivering the drug. Kamei and coworkers engineered two Tf variants that satisfied the molecular level design criterion using site‐directed mutagenesis [9]. These Tf mutants were conjugated to DT and were shown to be more effective than wild‐type Tf in delivering DT *in vitro* to U87 and U251 human glioma cell lines [10]. Furthermore, Kamei and coworkers performed *in vivo* experiments, and demonstrated that both mutant Tf‐DT conju‐ gates were more effective than their wild‐type counterpart in shrinking glioma tumors on the flanks of mice [10]. Studies are currently being performed with these mutant Tf molecules conjugated to CRM107.
