**3. Results**

## *3.1. Optimal Dosage Determination*

In our previous study [16], we assessed bi-HNTs cytotoxicity and observed that cytotoxicity increased with bi-HNT concentration. In order to confirm the optimal dosage range for bi-HNTs, we used an MTS assay and XTT assay to assess cell proliferation and viability with cells co-cultured with pure HNTs and bi-HNTs at different concentrations. Both tests presented similar results: cell viability changed with the concentration of HNTs nanoparticles. In the MTS assay (Figure 3A,B), cell proliferation was improved when the concentration of nanoparticles was below 100 mg/mL. However, when the concentration was increased to 150 mg/mL, the proliferation ability decreased by 20%. XTT assay showed similar results (Figure 3C,D), cell viability decreased with the increasing concentration of HNTs. However, the XTT assay showed that CT26WT cells have some tolerance to higher concentrations of HNTs, with significant cytotoxicity exhibited above 200 mg/mL. The cumulative results suggest that the highest dosage of bi-HNTs is 150 mg/mL.

**Figure 3.** XTT Cell Proliferation Assay. CT26WT cells were cultured with pure HNTs (**A**) and bi-HNTs (**B**) at different concentrations. Cell proliferation at each different concentration was calculated with comparison to the control group. CT26WT cells cultured with pure HNTs (**C**) and bi-HNTs (**D**) at different concentrations. Cell viability at each different concentration point was calculated by comparison to the control group. (error bar with standard deviation, *n* = 6, \* represents *p* < 0.05).
