Dottorato in BIOCHIMICA

Titolo della tesi: FERRITIN-BASED NANOVEHICLE FOR TARGETED DELIVERY OF BIOACTIVE CARGOES

In recent years, the development of protein nanoparticles has expanded into a broad range of biomedical applications. In this frame, ferritins occupy a very privileged place. Thanks to their hollow spherical cage and their unique 24-meric assembly, ferritins are used as drug delivery system. Compared to other ferritin-based nanocarriers, Archaeoglobus fulgidus ferritin is particularly interesting due to its unique ability to assemble-disassemble under very mild conditions. Recently, this ferritin was also engineered by grafting on it a surface loop of the human H ferritin containing the epitope for the recognition of human TfR1 receptor overexpressed in cancer cells. The resulting chimeric ferritin, named Humanized ferritin (HumFt), represents an ideal scaffold to host and selectively deliver different classes of therapeutic molecules. The present research work fits into this context. Specifically, as a first step, we have developed a new highly efficient, large-scale chromatography-free purification protocol mainly based on cross-flow ultrafiltration. This procedure allows to obtain high yields of purified protein avoiding expensive and time-consuming chromatographic steps. The purified HumFt was then tested as a nanocarrier for drug delivery. Specifically, the unique features of this chimeric ferritin have been exploited to encapsulate and deliver the therapeutic protein Cytochrome C (Cyt C) and a small nucleic acid (miRNA) to NB4 cells, an acute promyelocytic leukemia cell line typically hard to transfect and resistant to conventional liposome-based transfection methods. Cyt C is a basic protein capable of inducing apoptosis when released into the cytosol by activating the caspase cascade. This pro-apoptotic activity makes Cyt C an ideal candidate as a therapeutic protein in cancer therapy. However, its biomedical applications are mainly limited by the inability to cross cell membranes. In this frame, we have developed and characterized a ferritin-based self-assembling nanovehicle able to encapsulate and deliver Cyt C to cancer cells. The encapsulation of Cyt C is typically driven by electrostatic interactions between the negatively charged internal cavity of HumFt and the positively charged Cyt C. Once loaded, the protein nanoparticle is efficiently uptaken by NB4 cells and Cyt C is released triggering an apoptotic response. Along with the incorporation of this therapeutic protein, the ability of HumFt to incorporate small nucleic acids (microRNA) was also tested. HumFt is particularly suitable for incorporating positively charged molecules due to the anionic nature of the inner surface of the cage. So far, negatively charged molecules, such as nucleic acids, cannot be readily encapsulated within the protein cage. For this reason, we developed a self-assembling hybrid nanosystem in which the ferritin traps a positively charged polyamine dendrimer (Poly(amidoamine) or PAMAM) that acts as an anion sponge for negatively charged molecules, nucleic acids in particular. PAMAM positive charge typically enables the formation of a strong, yet reversible, complex with negatively charged phosphate backbones of nucleic acids leading to the formation of dendriplexes. The small nucleic acid can indeed reach the internal cavity of the hybrid nanoparticle through the 4 large triangular pores on the external surface of the protein cage. As a proof of concept, we tested a microRNA(pre-miR145-5p) whose cellular delivery to NB4 cells, and the induced phenotypic effects can be easily detected. Once the ternary system HumFt-PAMAM-miRNA is uptaken by leukemia cells, the pre-miRNA is released into the cytosol and processed by Dicer nuclease to mature miRNA, thus enhancing the expression of RARα, as well as inducing morphological changes typical of the granulocyte differentiation process. The self-assembling HumFt-based nanosystem here presented paves the way for the design of a new family of non-covalently modified nanoparticles that can be loaded with an extraordinarily high range of affinity molecules and that can specifically target a wide range of diseased cells.