# Green Approaches to Carbon Nanostructure-Based Biomaterials
## 2 Green Routes to Prepare Carbon Nanomaterials for Biomedical Applications
### Nanodiamonds (NDs)  
NDs differ from many of the other carbon nanostructures owing to the presence of a large number of sp\({}^{3}\)-hybridized carbon atoms, as the name suggests. However, they do also feature sp\({}^{2}\) carbon atoms [118] and various oxygen-containing functional groups on their surface (Figure 3) that can be exploited for functionalization [119], as recently reviewed [120]. Their production typically requires high temperatures and pressures to obtain sp\({}^{3}\) hybridization, such as those occurring in detonations. Other methods include chemical vapor deposition and milling of microsized diamonds [9]. Recently, a laser ablation route has been reported as a green alternative due to the possibility to carry it out at ambient temperatures [73]. Due to their low reactivity, functionalization is usually carried out in organic solvents under harsh conditions, or in the gas-phase, and can be promoted by microwaves, plasma, or UV irradiation [120]. Very few reports exist on their functionalization under green conditions. Often, the first step involves ND oxidation by strong-acid treatment [122], to provide functional groups for further derivatization. However, oxidation can also be attained through a green route, using atmospheric-pressure radio-frequency microplasma jet to deliver aqueous oxygen radicals to NDs suspended in water [74]. Then, further functionalization can occur under a variety of classical conditions, for instance to coat NDs with a suitable (bio)polymer for colloidal stability and improved biocompatibility [123]. In the majority of cases, non-environmentally friendly solvents are used, such as dimethylformamide; however, oxidized NDs can be further derivatized also in water [124], or other environmentally-friendly solvents, such as acetone and alcohols, as required for the other reagents, e.g., drugs [125]. Aqueous couplings were performed in this manner to attach a fluorescent protein and CRISPR-Cas9 components for gene editing [122], or to bind polyethylene glycol, and a lanthanide complex for enhanced bioimaging [126]. Alternatively, other useful functional groups for derivatization are alkynes to perform click-chemistry. In one example, NDs were first reacted with glycidol and glycidyl proparyl ether, which both served as reagents and solvents, to then allow the copper-catalyzed click reaction in an aqueous environment with a fluorophore for bioimaging [127]. In a similar approach, the click reaction was performed with a precursor of nitroxide radicals, which could be generated by sonochemical-promoted air oxidation, for redox sensing [128]. Mussel-inspired bioadhesives have also been effectively applied to coat and functionalize NDs in water, so that further molecules could be appended, such as polyethylene glycol polymer for colloidal stability, or DNA for particle tracking (Figure 4) [129]. Another biocompatible coating is mesoporous silica, which can be formed on the surface of NDs in aqueous conditions and allows to include a variety of functional groups, for instance for  
Figure 3: Blended atomistic model (**left**) and high-resolution TEM micrograph (**right**) of a nanodiamond (ND) to show its typical chemical structure. Reprinted from [121], copyright © 2015, with permission from Elsevier. the convenient grafting of a hydrogel polymer shell and metal nanoparticles for advanced sensing techniques [130]. Finally, NDs can be simply used as scaffolds to adsorb other molecular species, as shown for a hydrophobic magnetic-resonance contrast agent that required a mixture of DMSO and water to react with NDs, while the resulting product was water-soluble and could be tested for imaging [131]. They have been proposed as antibacterials [132; 133], for regenerative medicine [134], drug delivery [135; 136; 137], bioimaging [136; 138; 139], cancer therapy [137; 140; 141], theranostics [142; 143], and ultrasensitive diagnostics [144]. For many of these applications, they were envisaged to be used in composites [145; 146].