Overcoming Non‐Specific Interactions for Efficient Encapsulation of Doxorubicin in Ferritin Nanocages for Targeted Drug Delivery

Abstract Due to its beneficial pharmacological properties, ferritin (Ftn) is considered as an interesting drug delivery vehicle to alleviate the cardiotoxicity of doxorubicin (DOX) in chemotherapy. However, the encapsulation of DOX in Ftn suffers from heavy precipitation and low protein recovery yield which limits its full potential. Here, a new DOX encapsulation strategy by cysteine‐maleimide conjugation is proposed. In order to demonstrate that this strategy is more efficient compared to the other approaches, DOX is encapsulated in Ftn variants carrying different surface charges. Furthermore, in contrast to the common belief, this data show that DOX molecules are also found to bind non‐specifically to the surface of Ftn. This can be circumvented by the use of Tris(2‐carboxyethyl)phosphine (TCEP) during encapsulation or by washing with acidic buffer. The biocompatibility studies of the resulting DOX Ftn variants in MCF‐7 and MHS cancer cells shows a complex relationship between the cytotoxicity, the DOX loading and the different surface charges of Ftn. Further investigation on the cell uptake mechanism provides reasonable explanations for the cytotoxicity results and reveals that surface charging of Ftn hinders its transferrin receptor 1 (TfR‐1) mediated cellular uptake in MCF‐7 cells.