# Green Approaches to Carbon Nanostructure-Based Biomaterials
## 2 Green Routes to Prepare Carbon Nanomaterials for Biomedical Applications
### Carbon Nanotubes (CNTs)  
CNTs can be considered as sheets of graphene rolled up in a tube, and comprise mainly single-walled CNTs or multi-walled CNTs, depending on the number of sheets that compose their walls. Similar to the other nanostructures, they have been noted as promising materials to innovate in the biochemical field. However, their morphological similarity to asbestos fibers has posed many barriers for their applications, despite the fact that functionalization can alleviate their toxicity, as recently reviewed [41]. In 2019, CNTs have been added to the ChemSec SIN (Substitute It Now) list, in light of the studies on their toxicity and resistance to biodegradation [166]. This prompted a strong response from the academic community active on CNT research that feared further obstacles to innovation, in which it was noted that toxicity was related to a specific type of CNT, and this class of nanomaterials is very diverse, as the biocompatibility profiles depend on a plethora of factors, which include functionalization and route of administration [167; 168; 169]. Indeed, CNTs have shown a unique ability to boost the activity of conductive cells [33], demonstrating an unmissable opportunity to regenerate the cardiac [170] and nerve tissues [34]. Furthermore, their high mechanical resilience and low mass density is promising for orthopedic applications [171]. CNT-coated surfaces effectively stimulated osteogenic differentiation of mesenchymal stem cells, which adhered and spread, with numerous visible focal adhesion and actin stress fibers, as shown in Figure 7[172]. Besides tissue engineering [173], CNTs have also been proposed to attain antimicrobial and anti-adhesive surfaces for medical applications [174]. Their bioconjugation can serve a variety of innovative applications that span from tissue engineering, to sensing and wearable electronics [175], and, in general, to prepare innovative biomedical electrodes [176]. Their biodegradation is possible, depending also on experimental conditions, route of degradation, and CNT functionalization [43]. Figure 6: Sonochemical treatment ensures dye adsorption onto CNH surface for theranostics, i.e., multi-modal imaging, photodynamic therapy (PDT), and photothermal therapy (PTT). Reproduced with permission from [161], copyright © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim, Germany. As discussed above for other nanostructures, also in the case of CNTs, oxidation is often the first step for their biological application to achieve good dispersibility in water, and this is typically obtained by acid treatment, as shown in a recent study where oxidized CNTs demonstrated antibacterial activity [177]. The process can be carried out also in a microwave oven [178]. However, this kind of treatment requires extensive washings, and greener alternatives include gas-phase methods that use radical oxygen species generated by UV irradiation in air [179]. Once carboxylic acid groups have been installed on CNTs, several subsequent functionalization routes can be undertaken, as needed. For instance, the cationic photosensitizer malachite green could be adsorbed by electrostatic and hydrophobic interactions by simple ultrasonication in water, for antibacterial photodynamic therapy [180]. With an analogous procedure, hyaluronic acid was added to CNTs and the resulting materials were sterilized by gamma irradiation before being used for bone healing [181]. Moreover, without ultrasounds, simple mixing with doxorubicin in phosphate buffered saline solution allowed for the drug adsorption onto CNTs [182]; doxorubicin sustained release by CNTs is indeed a hot topic of research in cancer therapy [183]. Alternatively, the carboxylic acid groups can be covalently coupled to biomolecules in water, as shown for ovalbumin to boost immune response to antigen presentation for vaccine development [178].