## 5 Discussion  
Integrins maintain cellular architecture, survival, proliferation, migration, and overall tissue integrity by binding to ECM, cytoskeleton, and connecting via various cell adaptor proteins and ligands [16; 18]. Integrin \(\alpha\)v, \(\alpha\)6, \(\beta\)1, \(\beta\)3, \(\beta\)4, \(\beta\)5, and \(\beta\)6 subunits are the essential targets responsible for tumor proliferation, progression, and growth. High level of integrins or high activity of the signaling components associated with them synergize with oncogenes, growth factors, and other cell adhesion proteins, leading to the activation of signaling pathways and affecting both upstream and downstream molecules [6; 21; 28; 31; 44; 45; 46; 47; 48]. Therefore, they serve an essential targeting molecule to control tumor development. Dysregulation in the integrin expressions which participate in breast cancer development using siRNA-CA nano-formulation may lead to reduced association of the integrins with other cell adhesion molecules and proteins, thus hampering the cancer proliferation and survival. In our integrin siRNA transfection experiments MCF-7 cells showed no visible decrease in cell viability when they were transfected with CA loaded with ITG\(\alpha\)v siRNA (Figure 1a), while a significant reduction in viability of \(\sim\)57% was observed in MDA-MB-231 (Figure 1b), causing about 35% cytotoxicity (Table 3) and viability of \(\sim\)61% in 4T1 cells (Figure 1c) which caused about 21% cytotoxicity (Table 4). Research shows MDA-MB-231 cells contain high expression of ITG\(\alpha\)v causing different signaling [25]. Targeting this sub-unit in MDA-MB-231 cells might have reduced the viability by mainly targeting AKT and MAPK pathways, as from Western blot analysis we could see reduced band intensities after siRNA-CA transfection (Figure 8b). On the other hand, in MCF-7 cells phosphorylated AKT and total MAPK showed no decrease in band intensity while total AKT and phosphorylated MAPK showed significant reduction compared to CA (Figure 8a). Breast cancer is a heterogeneous disease, and each cell line represents a different model, with different characteristics and association with different protein molecules and signaling pathways. The expression of integrin \(\alpha\)v can vary among different metastatic and non-metastatic cells [25]. The upregulation of certain pathways in the cells upon delivery of \(\alpha\)v integrin might be due to cross talks with other pathways. One such pathway which could interfere is TGF-\(\beta\) pathway. TGF-\(\beta\) signaling mutually influence each other and its cross-talks with other pathways result in successful induction of cancer progression [48]. Upreguated in invasive and distant metastasis, studies show ITG\(\alpha\)v associates with various \(\beta\) sub-units and contributes towards breast cancer metastasis [25; 30]. When 4T1 cells were induced in the in vivo model of female Balb/c mice for tumor, after first dose of treatment of CA complexed with ITG\(\alpha\)v siRNA, reduced tumor volume trend was observed compared to CA treated and untreated groups (Figure 10) with highest fold decrease of 2.3 on Day 11 (Table 5). The tumor volume remained smaller compared to the control groups (Figure 12a). Statistical analysis performed between CA nanoparticle treated and siRNA-CA treated groups showed significant difference with \(p<0.05\) on selected time points (Figure 10). The CA loaded delivery of the integrin siRNA molecule increases its delivery efficiency to the target site. This is achieved by its ability to cause instability in the endosome, and release prior to its loss due to endosomal degradation, thus causing a sufficient decrease in the tumor volume [39]. The body weight remaining the same for both control and the treatment group indicates that there is no effect on the overall physiology of animals. One of the essential roles of ITG\(\alpha\)v is during angiogenesis and progression in breast cancer.