Safety concerns and biodegradability of carbon and 2D nanomaterials
The behaviour of pristine CNTs have been reported to be very similar to asbestos fibers, opening the debate on the development and use of these materials that can potentially cause the onset of mesothelioma. However, we have demonstrated that an appropriate chemical functionalization of CNTs alleviates the pathogenic effects and the reactivity profile of long, pristine multi-walled CNTs, making them safer to use (1). We also discovered how the degree of functional groups determine the tissue distribution and elimination of CNTs. Indeed, as the number of functional groups covalently linked to CNTs increases, the excretion profile of functionalized CNTs increases, while the accumulation into the different organs is modulated (2). The different studies on the in vivo behaviour of CNTs have been complemented by in vitro cellular experiments aimed to better clarify the mechanisms of cellular uptake, intracellular trafficking and exocytosis of CNTs (3). Part of our activity was dedicated to study the impact of CNTs on immune cells. Functionalized CNTs were tested on human T and B lymphocytes, natural killer (NK) cells and monocytes, showing that the viability was not affected although a strong activation was evidenced on monocytes and NK cells, suggesting a possible use of these nanotubes as immune modulator systems (4).
Based on our thorough experience with CNTs, we have extended our studies to graphene-based materials (5). This is a part of the big EU project called Graphene Flagship in which our Team is involved to assess the impact of graphene and other 2D materials on immune cells. We have discovered an interesting behaviour of graphene oxide that we called mask effect (6). The work was then extended to human peripheral immune cells from healthy donors. Exposure to small sheets of graphene oxide was found to have a more significant impact on immune cells compared to large sheets (7). In parallel, we have reported the tissue distribution of functionalized graphene oxide labelled with radioactive indium after intravenous administration in mice (8). Intact sheets were detected in the urine of injected mice. These results offer a pharmacological understanding on how functionalized graphene oxide transport in the blood stream and interact with physiological barriers that will determine its body elimination and tissue accumulation. In addition, we studied if an extensive glomerular filtration of graphene oxide sheets can cause kidney damage (8). More recently, we have studied the impact of functionalized graphene oxide using single-cell mass cytometry, a powerful technique that allow to analyse several immune cell populations, and interrogate several markers contemporarily (9). We are now extending our studies to other 2D materials, like transition metal dichalcogenides (e.g., MoS2). We have compared the effect of few-layer graphene (FLG) and MoS2 on cellular activation and intracellular processes like autophagy. We looked at M1 and M2 human monocyte-derived macrophages and found minimal toxicity in term of cell viability and activation with secretion of inflammatory cytokines by MoS2, while FLG had minimal impact on autophagy secreting specific autophagy related genes (10). Studies in vitro have been complemented by injection of FLG in vivo (11). We investigated the tissue distribution and toxic effects of graphene in mice up to 30 days after intravenous injection. Histological analysis of the tissues revealed hepatic accumulation and excretion through the kidneys. The biochemical and hematological parameters remained within the reference range showing no hematotoxicity, and no signs of inflammation were detected in the cells isolated from the lymph nodes and spleen.
One fundamental aspect associated to the development of nanomaterials for therapy and diagnosis concerns their biodegradability profile and their biopersistence. CNTs have been considered biopersistent for many years, until the discovery that some peroxidases are able to degrade oxidized single-walled CNTs. This has given a new hope to develop CNTs as drug delivery carriers. We have discovered that also oxidized multi-walled CNTs can be degraded by peroxidases in test tubes (12). Functionalized multi-walled CNTs can be also degraded in vitro in macrophages. We have reported the in situ monitoring of reactive oxygen species-mediated CNT degradation by liquid-cell transmission electron microscopy. Two degradation mechanisms triggered by hydroxyl radicals were evidenced: a non-site-specific thinning process of the walls and a site-specific transversal drilling process on pre-existing defects of nanotubes (13). We have recently introduced a new concept, degradation-by-design, to render the nanotubes safer based on their surface modification with functional substrates that are able to enhance the enzymatic degradation activity of peroxidases (14). We are currently expanding the biogegradability studies to graphene and 2D materials (15). We have demonstrated that graphene oxide can be degraded by peroxidases, and that its degradation strongly depends on the dispersibility in water (16). We discovered that myeloperoxidase secreted by activated macrophages is able to degrade single- and few-layer graphene (17). Similar behaviour occurs with hexagonal boron nitride and MoS2 nanosheets (18). We have then extended the concept of degradation-by-design to graphene-based materials (19). We found that an appropriate covalent functionalization with ligands able to activate oxidative enzymes enhances the biodegradation of graphene oxide. Based on this approach we have recently modified graphene oxide with a peptide able to recognize receptors overexpressed in cancer cells and to trigger the activation of neutrophils (20). This platform with enhanced biodegradability was used to deliver doxorubicin to cancer cell and induce cell apoptosis. The design of multifunctional materials able to both selectively deliver a drug into cells in a targeted manner and display an enhanced propensity for biodegradation is a step forward in the development of biomedical applications of 2D materials.
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