When scientists initially realized that CTAB would be an impediment to in-vivo applications, a tremendous amount of research was conducted on the development of surface functionalization methods aimed at the replacement of CTAB with bio-compatible surface ligands, such as thiol-terminated polyethylene glycol (HS-PEG), alkanethiols, glycols, and thiolated CTAB analogues (10, 11, 17, 18). Among these ligands, thiolated PEGs are the most commonly used molecules as they, theoretically, provide GNRs with a high degree of bio-compatibility (19). However, the main drawback of these PEGylation methods is that only the more weakly bound CTAB molecules at the tips of the rods are replaced with thiolated PEG, producing only partially functionalized GNRs (10,18).
Other methods have been developed to achieve higher PEGylation efficiencies, but these techniques leave a small quantity of CTAB on the GNRs (20, 21). Given the information on the toxic pathways for CTAB mentioned above, it has been stated that the amount of CTAB still present is enough to cause cytotoxicity at the elevated GNR concentrations required for their high cellular uptake, as in hyperthermal treatments. In fact, despite the above-mentioned advances, significant challenges for the surface modification of GNRs with bio-compatible molecules abound. First, many existing methods suffer from low PEGylation efficiencies that can limit the effectiveness of the PEG layer as surface coverage has been shown to be important for the effectiveness of PEG layers.