# Use of Ferritin-Based Metal-Encapsulated Nanocarriers as Anticancer Agents
## 4 Discussion  
The ability of HFt to pass through the BBB and to enter into cells via transferrin receptor, together with its ability to bind hundreds of metal molecules into very stable complexes, make HFt-based nanocarriers the ideal candidates for the delivery of toxic molecules to brain tumors. In the present work, we have studied: (i) the ability of HFt to bind toxic metals into HFt-metal complexes and the efficiency of HFt-metal complexes production; (ii) the ability of HFt to enter readily into neuroblastoma SH-SY5Y cells; (iii) the differential effects of iron-, silver, gold- and cisplatin-containing HFt nanocarriers on cell viability, cell cycle, mitochondrial transmembrane potential and redox homeostasis. We have limited the present study to metal-containing compounds in cultures of SH-SY5Y cells, and have tested the uptake of metal-containing HFt from neuroblastoma cells and the differential effect of the different metals, among which include the commonly used cisPt, on this cancer cell line. The natural ability of HFt to bind iron was used to test the efficiency of binding of other metals and metal-containing compounds. Here, we demonstrate that HFt is able to bind efficiently high amounts (tens or even hundreds) of molecules of metal ions, and to protect them from the fast release. These materials are fully soluble in water and buffer solutions. This, together with the high yields and the overall low-cost production, makes metal-containing HFt-NCs excellent nanocarriers. The second point tested is the ability of neuroblastoma human cells to internalize HFt-based nanocarriers from the culture medium. The level of expression of TfR1 (CD71) on the surface of cells reflects their metabolic requirements for iron [32]. Upregulation of CD71 in cancer cells is associated with malignant transformation. CD71 expression is particularly increased in metastatic and drug resistant tumors [33; 34; 35; 36; 37; 38]. This makes CD71 both a tumor marker and a possible system for the delivery of small-molecule drugs to these cells by CD71-mediated endocytosis [39; 40; 41; 42; 43; 44]. Recently, CD71 was identified as the receptor for HFt [22], with distinct binding sites for HFt and transferrin [45]. After binding of HFt to CD71 on the cell surface, membrane invagination takes place, and vesicles are formed. HFt is localized sequentially into endosomes and lysosomes [22]. Neuroblastoma cells, including SH-SY5Y cells, express high levels of CD71 [46; 47], and are therefore able to incorporate high amounts of HFt. Molecules as sodium ascorbate, which are able to decrease the expression of CD71, cause cell death within 24 h [47]. Here, we show that SH-SY5Y neuroblastoma cells are able to incorporate HFt NCs within hours, taking advantage of their high levels of expression of transferrin receptors. This result, together with the presence of a transferrin-mediated vesicle-based transcytosis system through the BBB [19; 20; 48; 49] makes HFt-based NCs potentially good carriers for toxic molecules to the brain tumors. Many metals (e.g., sodium, potassium, magnesium, calcium, iron, zinc, copper, manganese, chromium, molybdenum and selenium) are required for normal biological functions in humans. We show that HFt containing small amounts of iron is not toxic, and has a slightly proliferative effect. However, most metals have toxic effects on the cells, and many metal-containing molecules are used in medicine [50], cisPt being the most commonly used in antitumor therapy. Following administration of cisPt, one of the chloride ligands is slowly displaced by water, in a process termed aquation, resulting in [PtCl(H\({}_{2}\)O)(NH\({}_{3}\))\({}_{2}\)]\({}^{+}\). Water is itself easily displaced, allowing the platinum atom to bind to DNA bases, in particular to guanine. DNA crosslinking often occurs via displacement of the other chloride ligand, typically by another guanine. CisPt-dependent DNA crosslinking interferes with cell division and elicits DNA repair mechanisms, which, in turn, activate apoptosis when repair proves impossible.