Administration of therapeutic proteins holds significant promises in the treatment of threatening diseases, including cancer. The high intracellular activity and specificity of proteins endowed with catalytic activities has the potential to exert magnified effects at very low dosage if compared to more conventional, low molecular weight drugs. However, effective delivery of proteins and peptides to specific cell targets remains a challenging issue due to the presence of multiple diverse biological barriers, from circulating blood to tissues and cells.
In the present project, the chimeric archaeal-human ferritin (HumAfFt) will be used to deliver and release cytochrome c and induce apoptosis in different tumor cell lines. HumAfFt combines the versatility in 24-meric assembly and cargo incorporation capability of Archaeglobus fulgidus ferritin with specific binding to TfR1 receptor, the ¿heavy duty¿ carrier responsible for transferrin-iron uptake.
This project aims to set out the basis for the rational design of innovative protein-based carriers for therapeutic protein delivery and biotechnological applications. In this context, ferritin stood out as promising protein system due to its remarkable characteristics. Ferritins are versatile biocompatible protein scaffolds, displaying a cage-like structure that provides shielding of the cavity¿s content from harsh external conditions and are amenable to modifications in a relatively straightforward manner.
Up to now several works describe ferritin loading with small molecules such as doxorubicin, cisplatin, curcumin, etc. However, the possibility to encapsulate proteins could expand the technological importance of these systems considerably. Ferritin interior surface is rich in carboxylates, which naturally attract and bind iron ions and this feature can be exploited for the binding of basic proteins. However, the high stability of eukaryotic ferritin cages is a barrier to protein encapsulation as very acidic pH values (about pH 2) are needed for cage disassembly. These extreme experimental conditions can compromise the integrity of the reassembled 24mers as well as the therapeutic protein cargo. In contrast to mammalian ferritins, some microbial ferritins, such as AfFt from A. fulgidus naturally and reversibly disassemble into dimers at neutral pH values and low ionic strength. To allow the binding to TfR1 mammalian receptor, AfFt was genetically engineered grafting the BC loop sequence from human H ferritin into it, thus obtaining the humanized AfFt (HumAfFt). This novel humanized chimeric construct combines the self-assembly properties of AfFt with the typical human H-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 [Nanoscale]. Considering the unique properties of this ferritin, it could really represent a powerful nanodevice for therapeutic protein targeted delivery.
In this project we will set up the targeted delivery of a functional apoptosis-initiating protein, Cytochrome c, to cancer cells. This will be a keystone in therapeutic protein delivery because, to date, various nanovehicles have been explored to facilitate intracellular delivery of proteins (polymers, gold and silica nanoparticles, quantum dots and carbon nanotubes) but none of them has been proven to be ideal for all types of bioactive proteins. Moreover, they need extensive manipulations in order to confer both protein loading capabilities and selective cell targeting properties.
Compared to polymeric materials, proteins are highly biocompatible and biodegradable materials, displaying as well lower toxicity. Recombinant technologies allow to produce highly purified proteins at gram scale and several chemical or genetic manipulation can be employed to further modify their structure.