## 3 Results and Discussion
### MTT Assay and Morphological Changes  
The percentages of cells that survive in relation to various concentrations of GEF formulations were represented in Figure 8. The sigmoidal curve for the MTT assay shows the IC\({}_{50}\) values of GEF-containing formulations and the percentage viability (%) of treated A549 cells. Both the formulations (F1 and F3) illustrated remarkable reductions in the cell viability as indicated by the IC\({}_{50}\) values (2.239, 3.135 and 4.471 \(\upmu\)M for F3, F1 and GEF pure). The MTT assay has shown that the GEF pure, F1 and F3 had significantly reduced the population of A549 cells in dose-dependent way as represented by the sigmoid curve (Figure 8). However, no significant differences in the cell viability were noted between the pure-GEF and F1, while the F3-treated A549 cells showed significant (_p_ < 0.05) cell deaths. The changes in the treated A549 cells were more prominent when treated with F3, which was due to the complexation of GEF-TPGS SDs with _HP_\(\beta\)-CD (F3). The caging of the GEF SDs into _HP_\(\beta\)-_CD_ further enhanced the phase solubility of GEF, which in turn has shown improved cytotoxic potential regarding GEF [55].  
The encaging of the GEF into HP \(\beta\)-CD with TPGS further enhanced the solubility of the drug, which in turn has shown improved cytotoxic potential of GEF [55]. In addition to the MTT assay, the morphological changes in the cells (observed under microscopy) also support the cytotoxic potential of the developed formulations against the treated cells. The microscopic observations (Figure 9) endorsed the cytotoxic properties of GEF and F1 and F3. The morphological changes in the A549 cells were found after their treatment with GEF formulations. The IC\({}_{50}\) value of 3.135 \(\upmu\)M of GEF for A549 cells was chosen as the treatment dose for this experiment. The altered morphological topographies, such as blebbing of the membrane (blister-like), the shrinkage of the cells, cellular granularity, collapsing or condensation of nucleus and cellular volume, disintegration and break-up as well as morphological changes in cell surfaces, were considered as the standard apoptotic indicators [31; 32; 56].  
Figure 8: The MTT assay showed that GEF pure and GEF SDs (F1 and F3) significantly decreased the living population of A549 cells in dose dependent mode by assuming 100% of the cells were viable in the untreated (control) group. However, no significant differences in viability were noted between the pure-GEF and F1, while F3-treated A549 cells showed significant (\(p<0.05\)) cell deaths. The coefficient of correlation (\(R^{2}\)) values for GEF pure, F1 and F3 were 0.9823, 0.9825 and 0.9692, respectively. The data were reported as the mean and standard deviation of three measurements (mean \(\pm\) SD, \(n=3\)).  
Figure 9: The phase contrast images of morphological changes in A549 cells observed under phase contrast microscope after exposing them to different GEF formulations. Control or untreated cells (**a**); GEF-pure-treated cells (**b**); F1-treated cells (**c**) and F3-treated cells (**d**).  
The apoptosis and appearance of dead cell debris indicated a marked cytotoxic potential of GEF on A549 cells, where the treated cells have shown maximum morphological changes, such as blister-like blebbing of the membrane, shrinkage of the cells and condensation of the nucleus with F3 relative to F1 as compared to GEF pure at a particular drug concentration (F3 > F1 > GEF pure). The obvious changes in the morphology of the A549 cells treated with F3 followed by F1 might be associated with high solubility of the lipophilic drug (GEF) in the used media due to conversion of pure GEF to solid dispersions. The distinct morphological changes in the cells treated by F3 followed by F1 were due to the high phase solubility of GEF in solid dispersion states, which might increase the accumulation of GEF into the nucleus of the treated cells.