# Green Approaches to Carbon Nanostructure-Based Biomaterials
## 2 Green Routes to Prepare Carbon Nanomaterials for Biomedical Applications
### Carbon Nanohorns (CNHs)  
Carbon nanohorns (CNHs) or nanocones consist of short cones of sp\({}^{2}\) carbon atoms that aggregate into clusters of ca. 100 nm diameter and can be mainly of two types, i.e., \"dahlia-like\", when the cones protrude from the aggregate, or \"bud-like\" when they do not (Figure 5). They are produced by arc-discharge, laser ablation, or joule heating, from graphite [7], although greener alternatives at lower temperatures are continually sought [149]. Similar to the other carbon nanomaterials, they have been proposed for drug delivery [150], sensing [151], theranostics [152], and as components of nanocomposites for phosphoproteomics in cancer diagnosis [153], or for cancer treatment [154], or to yield patches for topical applications on skin [155].  
Figure 4: Dopamine can be polymerized in water to coat NDs and provide functional groups for the subsequent anchoring of polymers and biomolecules for imaging and detection. Adapted with permission from [129], copyright © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim, Germany.  
Functionalization is required to avoid further aggregation into larger clusters and ensure homogeneous dispersions in aqueous environments. It is typically carried out similarly to the other nanocarbons, with the most popular route being oxidation (for instance in air at high temperatures) and subsequent coupling to other (bio)molecules in water, as shown with a fluorophore-albumin conjugate to ensure colloidal stability and ease of tracking for bone-tissue regeneration studies [156]. Alternatively, treatment with hydrogen peroxide at 100 \({}^{\circ}\)C under UV-irradiation oxidizes CNFs, which then can be purified in water and stabilized with albumin [157], or attached with fluorophore-protein conjugates in aqueous environments, as shown in studies that tracked their cell entrance by endocytosis [75]. However, the most common route to oxidize CNFs is by treatment with nitric acid under mild heating [158], thanks to their high reactivity ensured by the highly curved, thus strained, cone tips. Once carboxylic acid groups have been installed, they can be coupled in aqueous environments to smart fluorophores, which allowed to confirm the endocytic entrance of CNFs into cells [159].  
Many other covalent functionalization approaches exist. Among these, 1,3-dipolar cycloaddition of an azomethine ylide, generated in situ from an amino acid and an aldehyde, can be performed in solvent-free conditions in a microwave reactor, for instance to attach oligothiophene to allow Surface-Enhanced Raman Spectroscopy (SERS) imaging [160].  
Non-covalent functionalization is also a popular approach. For instance, a simple sonochemical treatment of CNFs with a dye [161] or a natural photosensitizer [162] promoted adsorption onto the CNH surface, thanks to hydrophobic interactions, for combined multimodal imaging, photodynamic, and photothermal treatment of cancer cells, as shown in Figure 6. Similarly, when CNFs were added to albumin under sonication, a stable dispersion could be achieved at physiological conditions [163]. When compared against CNTs, interestingly, CNFs were shown to be more biocompatible due to their reduced ability to adsorb proteins on their surface [163], analogously to what observed for a self-assembling tripeptide [164]. Finally, colloidal stability of CNFs can be ensured also by surfactants, such as the biocompatible pluronic, by simple mixing in water, as envisaged to restore the mechanical integrity of tendon tissues after a sprain [165].  
Figure 5: TEM micrographs of (**a**) dahlia-like and (**b**) bud-like carbon nanohorms.