Engineered ferritin nanovectors for theranostic applications

Nanomedicine is the biomedical application of nanoscale materials for diagnosis and therapy of diseases. Recent achievements in cancer management include the development of highly precise and specific tools for early diagnosis and smart drug delivery systems for targeted delivery into tumor cells. For this purpose, protein-based nanocages have great potential as theranostic devices, used for both diagnostic and therapeutic applications. Among many nanovectors currently under investigation for biomedical purposes, ferritin has emerged as an excellent and promising nanocage due to its unique architecture, surface properties and high biocompatibility. It can be either genetically or chemically modified for encapsulating therapeutics or probes in its inner cavity. Ferritin H-homopolymers are naturally targeted toward TfR1 (or CD71) receptor highly expressed in iron avid, fast replicating, tumor cells. Only a few ferritin-based constructs have been devoted to design smart fluorescent probes for bioimaging, such as quantum dots gold nanoparticles. For this reason, I employed a new engineered ferritin nanoparticle to facilitate the incorporation of Eu3+ ions and create a nanotool with potential applications in bioimaging. Thus, the first part of this research project deals with the study of the interaction between the HFt-LBT and europium ions. HFt-LBT is an engineered H-ferritin nanoparticle carrying a lanthanide binding tag (LBT) at the C-terminal end of each subunit of the ferritin; it is a stretch of 17 aminoacids endowed with strong luminescence resonance energy transfer (LRET) sensitization properties since it has a tryptophan residue that can act as an antenna transferring the absorbed energy to the lanthanide ion. The tag has been designed to be located inside the inner cavity, so the lanthanide ions diffusing through the surface pores could bind to the LBT sequence. The construct would thus act both as carrier targeted to CD71 receptors and as a LRET sensitizer. Steady state emission measurements and time-resolved emission spectroscopy have been employed to investigate the interaction between the HFt-LBT and the Eu3+ ions. The results allowed me to identify the Eu3+ energy states involved in the process and to pave the way for the future use of HFt-LBT Eu3+ complex in diagnostics. In the second part, I studied another engineered ferritin-based nanotool. It is the complex HumAfFt-SPIONs, made up of the Humanized Archaeoglobus fulgidus Ferritin (HumAfFt) used as a coating material for 10 nm SPIONs. Thanks to their intrinsic physicochemical properties, SPIONs can be used simultaneously as therapeutic and diagnostic agents, and therefore they are considered as a nanotheranostic system. In particular, they attracted a great deal of attention for their potential application in biomedical fields such as cellular labeling, drug delivery, tumor treatment using magnetic hyperthermia and contrast enhancement for magnetic resonance imaging (MRI). The biocompatible coating of SPIONs is essential for most biomedical applications since this increases the stability of the iron oxide core, preventing aggregates formation and allowing functionalization of the surface of the nanoparticles with targeting ligand. For this reason, the HumAfFt was used as a coating material for SPIONs, in order to produce a new magnetic nanocarrier able to discriminate cancer cells from normal cells and maintain the potential theranostic properties of SPIONs. HumAfFt is an engineered ferritin characterized by the peculiar salt-triggered assembly-disassembly of the hyperthermophile Archaeoglobus fulgidus ferritin and which is endowed with the human H homopolymer recognition sequence by the CD71. Many biophysical, biological and biochemical techniques were employed to characterize the newly developed HumAfFt-SPIONs. The results indicate that they possess high stability and biocompatibility, making them an excellent theranostic nanotool.