# F127/Cisplatin Microemulsions: _In Vitro_, _In Vivo_ and Computational Studies
## 3 Results and Discussion
### Cytotoxicity Evaluation  
The cytotoxic activity of standard and encapsulated Cis were measured in malignant MCF7 and C152 cells compared to normal HUVECs. All three cell lines were treated with 0 to 40 ug/mL free Cis and F127/Cis microemulsions for 48 h. Figure 1 demonstrates the viability of the cells upon exposure to both agents as a function of concentration. Compared with untreated cells, free Cis significantly decreased the viability of MCF7 and C152 cells in a concentration-dependent manner (\(p<\) 0.05). The same pattern, but to a lesser extent, was observed when HUVECs were treated with increasing concentrations of free Cis. IC50 values for 48 h treatment of C152, HUVEC, and MCF7 cells with free Cis were 6.01, 22.42, and 6.81 ug/mL, respectively, while these values were 13.75 ug/mL (for C152 cells), 14.68 ug/mL (for MCF7 cells), and 24.27 ug/mL (for HUVECs) when exposed to F127/Cis microemulsions. Between the examined cell lines, C152 cells were the most susceptible to free- and encapsulated Cis. Based on MTT assay results, F127/Cis microemulsions induced less cytotoxic effects than free Cis on oral carcinoma cells and cancer cells derived from breast tissue (higher IC50s). Still, F127/Cis microemulsions induced less toxicity against normal HUVECs (higher IC50s), which might be a favorable outcome of using these microemulsions as an alternative tool for delivering Cis to target tissues. All cells used as controls (without treatment) maintained an integral cellular membrane and demonstrated a uniform distribution (Figures 2 and 3). Following 48 h of exposure of C152 cells to free Cis at 2.5 ug/mL, the number of viable cells was decreased. When treated with 10 ug/mL of free Cis, the C152 cells were morphologically rounded, and apoptotic bodies were formed. In higher concentrations of free Cis (20 and 40 ug/mL), cells were detached from the culture flask. Evidently, exposure of C152 cells to nano-Cis resulted in fewer morphological changes as cells were not detached from the culture flask, and cell shrinkage and roundness were only noticed when treated with the highest concentration (40 ug/mL) of F127/Cismicroemulsions (Figure 3). Similar morphological alterations were observed in MCF7 cells (Figure 4). Morphological alterations induced by free and nano-Cis on HUVECs were also examined (Data not shown). At concentrations below 10 ug/mL of encapsulated Cis, no evident change in HUVEC cell morphology was noticed. At concentrations between 20 and 40 ug/mL, a mild to moderate decrease in the number of living HUVECs were observed. In contrast, free Cis at lower concentrations (5 to 10 ug/mL) induced mild changes in the morphology of HUVECs. As compared with free Cis, we observed milder morphological changes and lesser cytotoxicity when normal human cells were treated with nano-Cis, which correlated with MTT assay results. Figure 1: Cytotoxicity assessment of free Cis and F127/Cis microemulsions on malignant (MCF7 and C152) cells compared with non-malignant HUVECs following 48 h exposure. (**_p_ < 0.05 compared with untreated cells). As a genotoxic agent, Cis is widely used alone or in combination with other anti-neoplastic agents/radiation as crucial first-line chemotherapy against a wide range of solid and hematological cancers [33]. However, the clinical use of free Cis is limited due to its severe organ toxicity (i.e., nephrotoxicity and peripheral neurotoxicity) [34]. Recently, researchers have attempted to improve the therapeutic efficacy of Cis while reducing its limitations and side effects [20; 33]. Insolubility and uncontrolled release of Cis are the two major obstacles limiting the anti-cancer potency of the drug [35]. [PERSON] et al. mixed two reverse microemulsions containing potassium chloride and a highly soluble precursor of Cis to formulate Cis lipid nanoparticles with controllable size. Their experiment showed promising invitro and in vivo anti-cancer activity of the synthesized nanoformulation [35].