Cytokine Impregnated Biomatrix: A New Tool to Study Multi-Wall Carbon Nanotubes Effects on Invertebrate Immune Cells

The novel features of engineered nanoparticles, such as multi-wall carbon nanotubes (MWCNTs) are impressive and attractive for technology, however they dissolved in water and accumulate in soils through the application of sewage sludge, accidental spills, and deposition from the air, agrochemicals or soil remediation. Given that several studies have revealed that chronic exposure to these nanomaterials products through the ingestion of drinking water, inhalation and dermal contact may harbour potential risks to human health, risk assessment of this nanomaterials in the aquatic environment are becoming essential. Here we propose a freshwater invertebrate, the leech Hirudo medicinalis, as a model to assess the effects MWCNTs on the immune system by means of in vivo and in vitro experiments. For this study, we used a consolidated experimental approach based on injection in the body wall of the leech of the biomatrice Matrigel (MG), added with a specific macrophage chemoattractant, the cytokine Allograft inflammatory factor-1 (AIF-1) and/or with MWCNTs. MG sponges analysis show the presence of a larger number of cells positive for both CD68 and HmAIF-1, specific monocyte-macrophage markers. Ultrastructural analysis suggests that MWCNTs may be internalized by phagocytosis but they seem also to be able to pierce cell membranes during cells migration. Cells extracted from MG were also used for in vitro treatment with MWCNTs at different concentration (2.5, 5, 10, 25, 50 and 100 µg/ml) for 24 h to study cell morphology changes and production of amyloid fibrils in order to encapsulate the foreign bodies. Our results, not only confirm the ability of MWCNTs in inducing a potent inflammatory response, but highlight rapid colorimetric assays that can be successfully used as sensitive tools for aquatic pollution biomonitoring.