## 4 Results
### In Vivo Delivery of Single siRNA Integrins Loaded with CA Nanoparticles
#### 4.3.2 Tumor Regression Analysis  
Delivery of single integrin siRNAs using CA-NPs caused reduction in tumor volumes compared to both untreated and CA-NP treated groups. Compared to in vitro study where 1 nM of the siRNA was used to transfect the cells in each well of a 24-well plate, 50-fold more siRNA was administered intravenously per dose in the form of 100 ul of particle suspension. From Figure 10, we can see that, compared to untreated and CA controls, the tumor volume was lesser in the treatment groups. The significant decrease in tumor volume with \(p<0.05\) compared to CA-NP treated group was observed on Day 11, Day 13, Day 15, Day 17, Day 19, Day 21, and Day 23 in siRNA ITG\(\alpha\)v + CA and ITG\(\beta\)5 +CA treatments. For siRNA ITG\(\alpha\)6 +CA, the tumor regression was significant on Day 11, Day 13, Day 15, Day 17, Day 19, and Day 21 of measurements. For siRNA ITG\(\beta\)6 + CA treated group, while the trend for tumor regression was reduced, the significant difference with \(p<0.05\) was observed on Day 17, Day 19, Day 21, and Day 23. Table 5 shows the fold  
Figure 7: NP-loaded siRNA effect on cell viability against ITG\(\beta\)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. decrease in the tumor volume compared to CA-NP treated groups at different time points. Figure 11 shows reduced tumor volumes for ITG\(\otimes\)1, ITG\(\otimes\)3 and ITG\(\otimes\)4 siRNA treatments loaded with CA. As there was no significant difference between untreated and CA treated groups, the untreated group was not repeated for further experiments. Treatment of siRNA ITG\(\otimes\)1 + CA, siRNA ITG\(\otimes\)3 + CA, and siRNA ITG\(\otimes\)4 + CA showed significant reduction in tumor (\(p<0.05\)) at all the time points compared to CA. Table 6 shows the fold decrease in the tumor volume compared to CA groups on different days. Excised tumors at the end of the study of siRNA + CA treated and CA treated groups are shown in Figure 12, with tumor of treatment groups smaller compared to CA control. Figure 8: _Cont._  
Figure 8: Impact of CA loaded single integrin siRNA intracellular delivery in **(a)** MCF-7, **(b)** MDA-MB-231, and **(c)** 4T1 cells. Cells were incubated with CA loaded single integrin (ITG\(\alpha\)v, ITG\(\alpha\)6, ITG\(\beta\)1, ITG\(\beta\)3, ITG\(\beta\)4, ITG\(\beta\)5, and ITG\(\beta\)6) siRNAs for 48 h. Post incubation cells were lysed for Western blot assay. Then, 9 \(\upmu\)g of proteins were loaded uniformly on SDS-PAGE, and transferred onto nitrocellulose membrane for detection of p-AKT, total AKT, p-MAPK, total MAPK, and housekeeping gene GAPDH expression. Figure 9: Average body weight of mice on Day 8 and Day 23 of tumor regression for untreated, CA treated and **(a)** ITG\(\alpha\)v + CA **(b)** ITG\(\beta\)1+ CA treatment. Data are shown as average body weight of 5 mice + S.D. Figure 11: Tumor induced mice were treated intravenously with 100 \(\upmu\)L of CA-siRNA(s) formed in 4 \(\upmu\)L of 1 M CaCl\({}_{2}\) and individual siRNA against ITG\(\upbeta\)1, ITG\(\upbeta\)3, and ITG\(\upbeta\)4 genes. Two treatment doses were given, on Day 8 and on Day 11. A total of 5 mice per group were used and data were represented as mean \(\pm\) SD. Tumor outgrowth of mice treated with CA-siRNA against ITG\(\upalpha\)v, ITG\(\upbeta\)5, and ITG\(\upbeta\)6 genes values are significant for * representing \(p<0.05\) for ITG\(\upbeta\)1 + CA, * representing \(p<0.05\) for ITG\(\upbeta\)3 + CA, * representing \(p<0.05\) for ITG\(\upbeta\)4 + CA. Figure 10: CA bound siRNA treatments against integrin genes in 4T1 tumor induced mice. Subcutaneous injection of 4T1 cells was done in the mice mammary pad. Once tumors were palpable, mice were treated intravenously via tail vein with 100 \(\upmu\)L of CA-siRNA(s) generated in 4 \(\upmu\)L of 1 M CaCl\({}_{2}\) and individual siRNA against ITG\(\upalpha\)v, ITG\(\upalpha\)6, ITG\(\upbeta\)5, and ITG\(\upbeta\)6 genes.