Ferritin nanocages for theranostic applications

Ferritin is a ubiquitous protein involved in iron storage composed of 24 subunits assembled in a hollow spherical nano-cage architecture. Channels formed between the intersection of peptide subunits are lined with polar aminoacids and allow for the entry and exit of cations. Ferritin can be successfully used as an highly biocompatible nanocarrier, due to the ability of being recognized and uptaken by TfR-1 overexpressing tumour cells. Furthermore, both inner or outer surface can be easily functionalized conferring multiple functionalities onto a single molecule. For these reasons, ferritins are emerging as novel biotech platforms for biomedical applications (both diagnostical and therapeutic) due to their ability to encapsulate cargo molecules, broad functionalization possibilities and selective targeting properties. In this framework, the present work has been focused on the development and characterization of engineered recombinant mammalian and archaeal ferritin constructs to expand the scope of their nanotechnological applications. With the aim of investigating the biological and biophysical properties of prokaryotic homopolymers and characterizing the permeability of the prokaryotic protein shell toward diffusants, two ferritins from Archaea have been chosen as model. A set of engineered mutants of Pyrococcus furiosus ferritin (Pf-Ft) and Archaeoglobus fulgidus ferritin (Af-Ft) have been obtained by placing a reactive cysteine residue per subunit in the same topological positions either inside or outside the internal cavity. These mutants differ from each other by the aminoacid composition of ferritin channels and the related “open” versus “closed” ferritin architecture. The molecular diffusion through the ferritin cavity has been characterized by studying within these mutants the cysteine reactivity toward the bulky and negatively charged DTNB molecule (5,5'-dithiobis-2-nitrobenzoic acid). Moreover, Archaeoglobus fulgidus ferritin has been genetically engineered by changing the surface exposed loop connecting helices B and C to mimic the sequence of the analogous human H-chain ferritin loop. This novel “humanized” chimeric construct (named HumAf-Ft) thus combines the unique open structure and self-assembly properties of Af-Ft with the typical humanH-ferritin ability to bind the Transferrin Receptor TfR-1, which is overexpressed in several types of tumor cells. HumAfFt has been structurally and biophysically characterized and the improved cellular uptake has been demonstrated on HeLa cell line. Lastly, to exploit lanthanide fluorescence properties and develop an intrinsically fluorescent nanoparticle, a novel construct has been developed by genetically fusing at the C-terminal end of mouse H-ferritin a lanthanide binding tag (LBT). LBTs are short peptides that selectively bind lanthanide ions at low-nanomolar affinities and, due to the presence of a tryptophan residue, provide strong FRET sensitization. This novel construct (named HFt-LBT) has been designed by locating the tag inside the inner cavity, so that the lanthanide ions diffusing through the surface pores can eventually bind to the LBT sequence. HFt-LBT would thus act both as carrier targeted to TfR-1 receptor and as a FRET sensitizer. Fluorescence improvement and lanthanide binding properties have been investigated by spectrophotometric measurements using Tb+3 as lanthanide probe. The structural characterization has been carried out and cellular uptake by HeLa cell line has been assessed as well.