Novel microbicidal agents derived from Naja atra cardiotoxin 1 (CTX-1)

During my PhD course, I focused my research on antimicrobial peptides (AMPs), in particular on the aspects of their computational design and development. This work led to the development of a new family of AMPs that I designed, starting from the amino acid sequence of a snake venom toxin, the cardiotoxin 1 (CTX-1) of Naja atra. Naja atra atra cardiotoxin 1, produced by Chinese cobra snakes belonging to Elapidae family, is included in the three-finger toxin family and exerts high cytotoxicity and antimicrobial activity too. This toxin family is characterized by specific folding of three beta-sheet loops (“fingers”) extending from the central core and by four conserved disulfide bridges. Using as template the first loop of this toxin, different sequences of 20 amino acids linear cationic peptides have been designed in order to avoid toxic effects but to maintain and strengthen the antimicrobial activity. As a result, the sequence NCP-0 (Naja Cardiotoxin Peptide-0) was designed as ancestor and subsequently other 4 variant sequences of NCP0 were developed. These variant sequences have shown microbicidal activity towards a panel of reference strains of Gram-positive and Gram-negative bacteria, fungi and an enveloped virus. In particular, the sequence designed as NCP-3 (Naja Cardiotoxin Peptide-3) and its variants NCP-3a and NCP-3b have shown the best antimicrobial activity together with low cytotoxicity against eukaryotic cells and low hemolytic activity. Bactericidal activity has been demonstrated by minimum bactericidal concentration (MBC) assay at values below 10 μg/ml for Pseudomonas aeruginosa ATCC 27853, Acinetobacter baumannii ( clinical isolates), Moraxella catharralis ATCC 25238, MRSA ATCC 43400, while towards Staphylococcus aureus ATCC 25923, Enterococcus hirae ATCC 10541 and Streptococcus agalactiae ATCC 13813 the bactericidal activity was demonstrated even below 1.6 μg/ml concentration. This potent antimicrobial activity was confirmed even for unicellular fungi Candida albicans, Candida glabrata and Malassezia pachydermatis (MBC 32.26-6.4 μg/ml), and also against the fast-growing mycobacteria Mycobacterium smegmatis DSMZ 43756 and Mycobacterium fortuitum DSMZ 46621 (MBC 100 μg/ml). Moreover, NCP-3 has shown a virucidal activity on the enveloped virus Bovine Herpesvirus 1 (BoHV1) belonging to herpesviridae family. The bactericidal activity is maintained in a high salt concentration (125 and 250 mM NaCl) medium and PB +20% Mueller Hinton Medium for E. coli, MRSA and Pseudomonas aeruginosa reference strains. Considering these in vitro obtained data, we propose NCP-3 and its variants NCP-3a and NCP-3b as promising antimicrobial candidates. For this reason, the whole novel AMPs family has been protected by a national patent (n°102015000015951).

Determinants of biological activity of nanomaterials in innate immunity cells: the role of aspect ratio, environmental contaminants and protein corona

As defined by the European Union, “ ’Nanomaterial’ (NM) means a natural, incidental or manufactured material containing particles, in an unbound state or as an aggregate or agglomerate, where, for 50 % or more of the particles in the number size distribution, one or more external dimensions is in the size range 1 nm-100 nm ” (2011/696/UE). Given their peculiar physico-chemical features, nanostructured materials are largely used in many industrial fields (e.g. cosmetics, electronics, agriculture, biomedical) and their applications have astonishingly increased in the last fifteen years. Nanostructured materials are endowed with very large specific surface area that, besides making them very useful in many industrial processes, renders them very reactive towards the biological systems and, hence, potentially endowed with significant hazard for human health. For these reasons, in recent years, many studies have been focused on the identification of toxic properties of nanostructured materials, investigating, in particular, the mechanisms behind their toxic effects as well as their determinants of toxicity. This thesis investigates two types of nanostructured TiO2 materials, TiO2 nanoparticles (NP), which are yearly produced in tonnage quantities, and TiO2 nanofibres (NF), a relatively novel nanomaterial. Moreover, several preparations of MultiWalled Carbon Nanotubes (MWCNT), another nanomaterial widely present in many products, are also investigated.- Although many in vitro and in vivo studies have characterized the toxic properties of these materials, the identification of their determinants of toxicity is still incomplete. The aim of this thesis is to identify the structural determinants of toxicity, using several in vitro models. Specific fields of investigation have been a) the role of shape and the aspect ratio in the determination of biological effects of TiO2 nanofibres of different length; b) the synergistic effect of LPS and TiO2 NP on the expression of inflammatory markers and the role played therein by TLR-4; c) the role of functionalization and agglomeration in the biological effects of MWCNT. As far as biological effects elicited by TiO2 NF are concerned, the first part of the thesis demonstrates that long TiO2 nanofibres caused frustrated phagocytosis, cytotoxicity, hemolysis, oxidative stress and epithelial barrier perturbation. All these effects were mitigated by fibre shortening through ball-milling. However, short TiO2 NF exhibited enhanced ability to activate acute pro-inflammatory effects in macrophages, an effect dependent on phagocytosis. Therefore, aspect ratio reduction mitigated toxic effects, while enhanced macrophage activation, likely rendering the NF more prone to phagocytosis. These results suggest that, under in vivo conditions, short NF will be associated with acute inflammatory reaction, but will undergo a relatively rapid clearance, while long NF, although associated with a relatively smaller acute activation of innate immunity cells, are not expected to be removed efficiently and, therefore, may be associated to chronic inflammatory responses. As far as the relationship between the effects of TiO2 NP and LPS, investigated in the second part of the thesis, are concerned, TiO2 NP markedly enhanced macrophage activation by LPS through a TLR-4-dependent intracellular pathway. The adsorption of LPS onto the surface of TiO2 NP led to the formation of a specific bio-corona, suggesting that, when bound to TiO2 NP, LPS exerts a much more powerful pro-inflammatory effect. These data suggest that the inflammatory changes observed upon exposure to TiO2 NP may be due, at least in part, to their capability to bind LPS and, possibly, other TLR agonists, thus enhancing their biological activities. Finally, the last part of the thesis demonstrates that surface functionalization of MWCNT with amino or carboxylic groups mitigates the toxic effects of MWCNT in terms of macrophage activation and capability to perturb epithelial barriers. Interestingly, surface chemistry (in particular surface charge) influenced the protein adsorption onto the MWCNT surface, allowing to the formation of different protein coronae and the tendency to form agglomerates of different size. In particular functionalization a) changed the amount and the type of proteins adsorbed to MWCNT and b) enhanced the tendency of MWCNT to form large agglomerates. These data suggest that the different biological behavior of functionalized and pristine MWCNT may be due, at least in part, to the different tendency to form large agglomerates, which is significantly influenced by their different capability to interact with proteins contained in biological fluids. All together, these data demonstrate that the interaction between physico-chemical properties of nanostructured materials and the environment (cells + biological fluids) in which these materials are present is of pivotal importance for the understanding of the biological effects of NM. In particular, bio-persistence and the capability to elicit an effective inflammatory response are attributable to the interaction between NM and macrophages. However, the interaction NM-cells is heavily influenced by the formation at the nano-bio interface of specific bio-coronae that confer a novel biological identity to the nanostructured materials, setting the basis for their specific biological activities.