Unlocking the Treasure Box: The Role of HEPES Buffer in Disassembling an Uncommon Ferritin Nanoparticle

Ferritins are ideal nanoparticles as drug delivery systems due to their hollow-sphere structure and the ability to target specific receptors on the cell surface. Here, we develop and characterize a new ferritin derived from the chimeric humanized A. fulgidus one, already designed to recognize the TfR1 receptor. Starting from the synthetic gene of this chimeric protein, we replaced two positively charged amino acids with two alanine residues to close the large triangular pores on its surface. These mutations make the protein nanoparticle suitable to incorporate even small therapeutics without leakage. Size-exclusion chromatography shows that the assembling/disassembling of this new protein cage can be easily fine-tuned by varying the HEPES buffer and MgCl2 concentration. The protein cage can be opened using 150 mM HEPES buffer without magnesium ions. Adding this divalent cation to the solution promotes the quick assembly of the ferritin as a 24-mer. The development of this new protein cage paves the way for encapsulation and delivery studies of small molecules for therapeutic and diagnostic purposes.