# Preparation and Characterization of Gold Nanorods Coated with Gellan Gum and Lipoic Acid
## 3 Results and Discussion
### In Vitro Studies  
The cytocompatibility of the coated nanorods produced was tested on HCT116 human colon cancer cell line, via MTS assay. In particular, the metabolic activity of the cells was measured after 24 or 48 h incubation with the three types of coated AuNRs (Figure 7). All samples at the lowest concentrations tested appear cytocompatible during the whole experiment. The highest concentration of AuNRs/LA and AuNRs/LA,GG (Au: 5 ug\(\cdot\)mL\({}^{-1}\)) show medium/moderate toxicity, recording the minimum cell viability value after 24 h incubation with AuNRs/LA,GG (about 40%). On the other hand, no significant variations in cell viability are appreciated after AuNRs/GG-LA incubation, constantly recording high cytocompatibility values in all conditions tested. It could be hypothesized that the derivative that coats AuNRs/GG-LA is able to form a compact shell on the nanoparticles, which might lead to a reduction in the interactions with the cells and, as a consequence, to cell viability values almost overlapping the control samples. The comparison between AuNRs/LA and AuNRs/LA,GG, showed comparable values throughout the analysis. An interesting result was, however, highlighted after 48 h of incubation, as the incipient toxicity registered during the first 24 h of incubation seems to decrease in the following 24 h. In fact, cell viability data collected after 48 h appear a little increased in comparison to the first time point. The results obtained could suggest that the incubation with nanorods might disturb cell milieu, causing a decrease in metabolic activity. Figure 7: Cell viability of human colorectal cancer cell line (HCT116) line after 24 h (**a**) or 48 h (**b**) incubation with AuNRs/LA (red), AuNRs/LA,GG (blue), and AuNRs/GG-LA (fuchsia) at gold concentrations of 1, 2.5, and 5 ug\(\cdot\)mL\({}^{-1}\). Statistical significance (Student’s two-tailed \(t\) test): \({}^{*}p<0.05\). However, this settling phase is presumably transient, since during the second day, the cultured cells seem to enter in a recovery phase of the metabolic activity. Data collected are encouraging since they suggest a good overall cytocompatibility of the system tested. The in vitro hyperthermia study involved the incubation of HCT116 human colon cancer cells with the three types of systems at a concentration of 5 \(\upmu\)g-mL\({}^{-1}\). Cell viability after laser treatment (810 nm, 10 \(\times\) 10\({}^{-3}\) W/mm\({}^{3}\), and 100 s), was therefore evaluated at 20 s intervals using MTS colorimetric assay and expressed as a percentage with respect to the untreated cells used as control. As reported in Figure 8, the comparison between the three types of nanostructures revealed cytotoxicity in accordance with the hyperthermia studies. To confirm the ineffectiveness of the sole laser treatment, the viability data of cells irradiated without nanorods incubation have been reported. It is evident that the laser treatment is not harmful at all, detecting a high vitality during the whole experiment. Once again, AuNRs/LA and AuNRs/LA,GG demonstrate to induce a significant decrease in vitality. On the contrary, the data of metabolic activity after AuNRs/GG-LA treatment appear definitely high, for all the time points analyzed. Even this time, it is conceivable that the GG-LA coating is able to shield the energy administered, suggesting that AuNRs/GG-LA are not ideal for photothermal therapy. The AuNRs/LA and AuNRs/LA,GG nanorods have shown good efficacy in reducing cell viability. Cell death induced by hyperthermia is consistent, recording eradication percentages of 82% and 75%, respectively, for AuNRs/LA and AuNRs/LA,GG. Finally, the residual cell viability after laser irradiation was confirmed by LIVE/DEAD assay (Figure 9). After treatment lasting 100 s, the contents of the wells were incubated with the fluorescent probes calcein AM and ethidium homodimer-1, capable of discriminating respectively the live cells (green) from those that present irreparable damage to the cell membrane (red).