## 3 Results and Discussion
### Cytotoxicity of HB-AuNPs  
Investigation of the cytotoxicity of nanomaterials is pivotal to assess how cells will respond when exposed to the nanomaterials either intentionally or accidentally. Various meth  
Figure 5: In vitro stability testing of HB-AuNPs. The AuNPs were incubated for 0 to 420 h in water (**A**), DMEM (**B**), RPMI-1640 (**C**), and PBS (**D**); and their stability was measured through UV-Vis spectroscopy.  
ods have been used to evaluate cytotoxicity [17; 51]. Among others, the tetrazolium-based assays have dominated since the introduction of the 3-(4,5-dimethyl-2-thiazol-lyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay in 1983. These assays do not actually measure the number of viable cells but rather provide information about the enzymatic activities that are related to cellular metabolism in live cells [52]. In the current study, the cytotoxicity of HBE and HB-AuNPs was investigated on a panel of human cancer cells (Caco-2, MCF-7 and PC-3) and non-cancer cells (KMST-6 and HEK-293) using the WST-1 assay. The WST-1 assay is based on the cleavage of tetrazolium salt to a yellow formazan product by cellular mitochondrial dehydrogenases, which is directly related to a number of metabolically active or live cells [32; 52]. The WST-1 assay offers various advantages over the other assays when used in the investigation of the cytotoxicity of AuNPs [32]. Unlike the MTT assay, the WST-1 assay excludes the addition of DMSO as it converts tetrazolium salt to water-soluble formazan products. It is also considered to be less toxic compared with other water-soluble variants such as 2,3-bis-(2-methoxy-4-nit-ro-5-sulfophenyl)-2H-tetrazolium-5-carboxaniline (XTT) assay. Most importantly, the formazan product produced in the MTT assay absorbs at the same wavelength range (570 nm) [52] as AuNPs and might interfere with the assay [31].  
The HBE was non-toxic to KMST-6 cells up to 4 mg/mL and showed a significant toxicity only at 8 mg/mL (Figure 6). On the contrary, the viability of Caco-2 cells was reduced in a dose-dependent manner after HBE treatment. Similarly, 2 mg/mL_HB-AuNPs were not toxic to KMST-6 cells but were effective in inhibiting the cell viability of Caco-2 cells. The 1 and 4 mg/mL HB-AuNPs were least effective in inhibiting the cell viability of both KMST-6 and Caco-2 cells, and showed low cytotoxicity towards cancer cells when compared with the 2 mg/mL_HB-AuNPs.  
HBE was non-toxic to HEK-293 and MCF-7 cells and exhibited selective cytotoxicity towards PC-3 cells at \(\geq\)0.5 mg/mL (Figure 7A). The 2 mg/mL_HB-AuNPs also displayed a selective and significant cytotoxicity towards the PC-3 and MCF-7 cells as shown in Figure 7B; at 100 \(\mu\)g/mL the PC-3 cell death (63%) was 2.5-fold higher than the MCF-7 cells (25%). These selective cytotoxic effects were also reported for HB-AuNPs_Ci against U87 (brain glioblastoma) and PC-3 cells. Interestingly, HB-AuNPs_Ci were also non-toxic to non-cancer breast (MCF-12A) cells [4]. This confirmed that the HB-AuNPs might be biocompatible when used in vivo and will selectively hamper or disrupt the activities of diseased cells. Biogenic AuNPs have been reported to have negligible cytotoxicity towards normal cells and will have therapeutic potential at concentrations that are non-toxic to healthy cells.  
Figure 6: Cytotoxicity of HBE and HB-AuNPs on KMST-6 and Caco-2 cells. \(p\leq 0.05\) was considered as statistical significant for untreated vs. treated samples, * \(p\leq 0.05\), *** \(p\leq 0.001\).