## 4 Results
### Integrins Link with PI3-Kinase/AKT and MAPK Signalling Pathways in Breast Cancer  
To study whether delivery of single integrin siRNAs loaded with CA-NP in cell lines can regulate the expression of total and phosphorylated AKT and MAPK, we performed Western blot assay. In MCF-7 cells, from Figure 8a we can see the expression of phosphorylated AKT (p-AKT), and total MAPK was significantly reduced in \(\alpha\)6, \(\beta\)1, \(\beta\)3, \(\beta\)4, \(\beta\)5, and \(\beta\)6 integrin siRNA treatments delivered using CA complex compared to CA control. The expression of total AKT and phosphorylated MAPK (p-MAPK) was reduced in all the seven treatments. In MDA-MB-231 cells, the siRNA delivery with CA nanoparticles reduced the intensity of p-AKT and p-MAPK in ITG\(\alpha\)v, ITG\(\alpha\)6, ITG\(\beta\)1, ITG\(\beta\)3, ITG\(\beta\)4, and ITG\(\beta\)6 siRNAs-CA treatments compared to CA control. The intensity of total AKT was reduced with integrin ITG\(\alpha\)v, ITG\(\beta\)1, ITG\(\beta\)3, ITG\(\beta\)4, and ITG\(\beta\)6 siRNAs-CA treatment. On the other hand, the intensity of total MAPK was reduced with ITG\(\alpha\)v, ITG\(\beta\)1, ITG\(\beta\)3, and ITG\(\beta\)4 siRNA-CA treatments (Figure 8b). \begin{table}
\begin{tabular}{c c c c} \hline
**Treatment** & **Cell Viability (\%) \(\pm\) S.D.** & **(\%) Cell Death \(\pm\) S.D.** & **Actual Cytotoxicity (\%) \(\pm\) S.D.** \\ \hline ITG\(\alpha\)v +CA & \(61.5\pm 1.8\) & \(38.5\pm 1.8\) & \(21.04\pm 7.3\) \\ ITG\(\alpha\)6 +CA & \(87.2\pm 1.8\) & \(12.82\pm 1.8\) & \(3.6\pm 2.56\) \\ ITG\(\beta\)1 +CA & \(81.65\pm 3.36\) & \(18.3\pm 3.3\) & \(12.5\pm 3.0\) \\ ITG\(\beta\)3 +CA & \(94.38\pm 1.72\) & \(4.96\pm 0.63\) & \(-11.36\pm 0.50\) \\ ITG\(\beta\)4 +CA & \(87.11\pm 4.78\) & \(12.8\pm 4.7\) & \(0.805\pm 1.97\) \\ ITG\(\beta\)5 +CA & \(84.14\pm 4.36\) & \(15.86\pm 4.36\) & \(6.87\pm 3.44\) \\ ITG\(\beta\)6 +CA & \(60.79\pm 0.67\) & \(39.21\pm 0.67\) & \(33.01\pm 2.88\) \\ \hline \end{tabular}
\end{table} 
Table 4: Cytotoxicity enhancement of single integrin siRNA transfections in 4T1 cells. Figure 1: NP-loaded siRNA effect on cell viability against ITGav single gene after 48 h of MTT assay. CA-siRNA complexes were prepared at 1 mL final volume consisting of 1 nM siRNAs + 4 mM CaCl\({}_{2}\) into bicarbonate buffered DMEM (pH 7.4), followed by incubation for 30 min. The prepared complex was transfected for 48 h and absorbance was taken at 595 nm with 630 nm as reference wavelength. Data are shown as mean \(\pm\) S.D for (**a**) MCF-7 cells, (**b**) MDA-MB-231 cells, and (**c**) 4T1 cells. Figure 2: _Cont_. Figure 3: NP-loaded siRNA effect on cell viability against ITG\(\beta\)1 single gene after 48 h of MTT assay. Data are shown as mean \(\pm\) S.D for (**a**) MCF-7 cells, (**b**) MDA-MB-231 cells, and (**c**) 4T1 cells. Figure 2: NP-loaded siRNA effect on cell viability against ITG\(\alpha\)6 single gene after 48 h of MTT assay. Data are shown as mean \(\pm\) S.D for (**a**) MCF-7 cells, (**b**) MDA-MB-231 cells, and (**c**) 4T1 cells. Figure 4: NP-loaded siRNA effect on cell viability against ITG\(\beta\)3 single gene after 48 h of MTT assay. Data are shown as mean \(\pm\) S.D for (**a**) MCF-7 cells, (**b**) MDA-MB-231 cells, and (**c**) 4T1 cells. Figure 5: _Cont_. Figure 5: NP-loaded siRNA effect on cell viability against ITG\(\beta\)4 single gene after 48 h of MTT assay. Data are shown as mean \(\pm\) S.D for (**a**) MCF-7 cells, (**b**) MDA-MB-231 cells, and (**c**) 4T1 cells. Figure 6: NP-loaded siRNA effect on cell viability against ITG\(\beta\)5 single gene after 48 h of MTT assay. Data are shown as mean \(\pm\) S.D for (**a**) MCF-7 cells, (**b**) MDA-MB-231 cells, and (**c**) 4T1 cells. Western blot analysis showed reduced expression of p-AKT in 4T1 cells upon all the siRNA-CA treatments except siRNA ITG\(\beta\)6 + CA treatment, while as total AKT expression was reduced in all the groups excluding siRNA ITG\(\alpha\)v + CA treatment group. On the other hand, p-MAPK and total MAPK reduction was observed in all the treatment groups (Figure 8c).