## 3 Results and Discussion
### Biocompatibility of the Nanocarrier
#### 3.4.2 Cytotoxicity Assay in Monolayer Culture of Cell Lines with Free and Loaded MTX  
The anti-proliferative effects of MTX, UiO-66-MTX, and UiO-66-NH\({}_{2}\)-MTX were assessed on all cells with a 10-fold serial dilution of six MTX concentrations (0.1 nM, 1 nM, 10 nM, 10 nM, 1 uM, and 5 uM) after 96 h of incubation with cancerous and non-cancerous cell lines. The efficacy of MTX-loaded MOFs was evaluated by comparing the IC\({}_{50}\) values obtained in all selected cell lines after treatment (Figure 9). The IC\({}_{50}\) value (Table 3) against MDA-MB-231 cells for MTX after 96 h of treatment was found to be 29 nM, consistent with prior studies [69,70]. UiO-66-MTX resulted in a decrease in the IC\({}_{50}\) value to 103 nM, while UiO-66-NH\({}_{2}\)-MTX exhibited a decrease to 1 uM after 96 h. Similarly, in MDA-MB-468 and MCF7 cells, the IC\({}_{50}\) values of MTX were the lowest, followed by UiO-66-MTX, and they were the highest for UiO-66-NH\({}_{2}\)-MTX. In MCF7 cells, MTX showed IC\({}_{50}\) values of 75 nM, which aligns with the results obtained in a previous study [71]. The incubation of MCF10A cells with MTX-loaded MOFs revealed that UiO-66-NH\({}_{2}\)-MTX exhibited a higher IC\({}_{50}\) value than free MTX, suggesting that the MOF formulations have a lower impact on normal cell proliferation in vitro. However, the IC\({}_{50}\) value for UiO-66-NH\({}_{2}\)-MTX was consistently higher than the IC\({}_{50}\) values for MTX and UiO-66-MTX in all cell lines. The sensitivity index varied with MOFs across different cell types. Dose-dependent cytotoxicity was observed across all cell lines for MTX and UiO-66-MTX. The increased cytotoxicity of MOFs with concentration was found to be correlated with the loading and release percentages of MTX in MOFs. UiO-66-MTX exhibited higher cytotoxicity than UiO-66-NH\({}_{2}\)-MTX, which had a lower loading profile. This result indicates the possibility of a more efficient internalisation of UiO-66-MTX inside the cell due to its  
Figure 8: Cytotoxicity data obtained in monolayer culture with pristine MOFs. Cytotoxicity profiles of UiO-66 and UiO-66-NH\({}_{2}\) on cell viability after 96 h of treatment. MDA-MB-231, MDA-MB-468, MCF7, and MCF10A cell lines were treated with 0.1 nM and 5 uM concentrations of (**A**) UiO-66 and (**B**) UiO-66-NH\({}_{2}\). The data points shown in each figure represent the average \(\pm\) SEM of three independent experiments normalised to the control value for untreated cells. smaller size as compared to UiO-66-NH\({}_{2}\)-MTX. This leads to a higher accumulation of the drug within the cell and consequently, a lower IC\({}_{50}\) value of UiO-66-MTX. However, the larger size of UiO-66-NH\({}_{2}\)-MTX as observed by DLS may have hindered the drug's entry into cells during culture [72]. The precise mechanism responsible for the internalisation of nanoparticles warrants further investigation. Overall, these results suggest that the loading percentage and size of MOFs play important roles in their cytotoxicity and can be optimised for therapeutic applications, with a preference for targeting neoplastic cells. The results show that the viability of all cell lines was negatively affected by increasing concentrations of the tested compounds. The slow-release nature of these nanocarriers could potentially reduce side effects and improve the preferential accumulation of the drug with efficacy due to enhanced permeation and retention effects for cancer treatment. The statistical analysis performed using two-way ANOVA indicated no significant differences between MTX and UiO-66-MTX at 1 uM and 5 uM concentrations, whereas UiO-66-NH\({}_{2}\)-MTX showed a significant decrease in cell viability inhibition from MTX and UiO-66-MTX at these concentrations (_p_ < 0.0001). This trend was consistent across all cell