A safe by design approach for carbon nanomaterial biotechnological applications

As just described in the introductory chapter, the world of nanotechnology has been in great expansion in recent decades for many different reasons. First of all, it was the unique physical and chemical properties of these materials that enabled some applications, for example in medicine (CNTs are used for bone scaffolds, vascular stents, neuron growth and regeneration (Donaldson, et al., 2012); graphene showed good sensing properties for biological molecules, such as glucose, catecholamine neurotransmitter (Alwarappan et al. 2009), which were initially impossible. Moreover, as has just been pointed out, the global market for nanotechnology was expected to exceed a value of $125 billion by 2024, not including the case of graphene, whose market was estimated at 1.3 billion by 2023, with an annual growth rate of almost 47% compared to 2018 (De Marchi et al. 2018). The innovative power emanating from these new technologies and their predictable and incisive presence in many health and economic sectors, as well as the paucity of data on their toxicity, have led us to design an efficient approach to assess the toxicity of nanoparticles. Based on our own application project in biotechnology, we considered it fundamental to approach this application with a safe-by-design approach and evaluate the toxicity of the materials used in our application. The toxicity of different types of carbon nanomaterials was evaluated using two different cell models (MeT-5A and/or C6 cells). The selection was made considering the possible target of toxicity of the materials and the possible mode of exposure. The nanomaterials tested were: CNTs (different types with different dimensions - short and long; nanomaterial in fibrous form), N-doped graphene (2D carbon nanomaterial), carbon black (nonfibrous form). Cells were also exposed to a non-nanofibrous positive control (asbestos fibers). We used a multistage approach based on four levels of toxicity testing. The first tier was based on toxicity tests (Alamar blue, crystal violet, and LDH assay). The protocols had to be modified somewhat to adapt them to the use of nanoparticles. At this level, it is possible to define the range of action of nanomaterials and to study the effects of exposure on cell viability and metabolism. At the second level, it’s toxicogenomic analysis. This type of test allows a more comprehensive and complete view of what type of response the cells show to exposure to the materials. Moreover, toxicogenomics being a no-hypothesis driven approach, allows an unbiased investigation on the functional effecst of a given materials. As a third and fourth level, we looked at genotoxicity and ROS formation, which can be highly interconnected. Overall, this multi-level approach is the basis to build up an Integrated Approach to Testing and Assessment (IATA) of carbon nanomaterial.