## 3 Results
### Effect of Co-Delivery of PRKCA siRNA and PTEN Gene on AKT Signaling Pathway: Western Blot Analysis  
In order to further verify our cell viability results in MCF-7 cells and to interpret the effect of the co-treatment on cellular proliferation pathway, after treatment of the cancer cells with NPs (control), PRKCA siRNA+NPs, PTEN+NPs and PRKCA siRNA+PTEN+NPs, cell lysates were prepared using a cell lysis buffer. The protein content of the lysates was measured by Bradford protein assay, and 10 \(\upmu\)g of proteins for each treatment was loaded onto a precast gel for their separation. Afterwards, the proteins were transferred onto a PVDF membrane using the Trans-Blot Turbo Transfer System (Bio-Rad) and the PVDF membrane was incubated overnight with monoclonal antibodies directed against phospho-AKT (P-AKT), total AKT and GAPDH as housekeeping loading control. After incubating the PVDF membrane with P-AKT antibody, the same membrane was stripped with a stripping buffer and incubated with another antibody for detection of total AKT in the lysates. As evident from the Western blot image and densitometry analysis (Figure 4a,b), the expression levels of P-AKT protein in the cells treated with PRKCA siRNA+NPs and PTEN+NPs were lower than the NPs control. Notably, the band was almost faded or invisible in the lysate of the cells treated with PRKCA siRNA+PTEN+NPs formulation. Figure 2: Light microscopic images of MCF-7 cell line following treatment with CA NPs, PRKCA siRNA+NPs, PTEN plasmid+NPs and PRKCA siRNA+PTEN plasmid+NPs. The scale bars set at 500 \(\upmu\)m.  
Figure 3: Cell viability assessment of PRKCA siRNA and PTEN plasmid delivery in **(a)** MCF-7 (**b**) MDA-MB-231 and **(c)** dT1 cells treated with PRKCA+NPs, PTEN+NPs and PRKCA+PTEN+NPs formulations for 48 h. The experiments were performed three times in each of the cell lines and values were presented as mean \(\pm\) SD of triplicates in MTT assay. * \(p\) < 0.05 for PKCA+PTEN+NP compared to PRKCA+NPs and or PTEN+NPs treated cells. The significantly reduced amount of P-AKT protein was a direct indication of the successful co-delivery effect of the siRNA and the plasmid. Reduced AKT activation refers to subsided survival signal via AKT/mTOR pathway and thus retarded cellular growth [36]. Influence of NPs-Facilitated PRKCA siRNA and PTEN Tumor Suppressor Gene Delivery on Tumor Regression  
Based on cell viability and Western blot results, PRKCA has been revealed as a potential target for siRNA-mediated knockdown in reducing proliferation and/or survival of breast cancer cells. In addition, gene augmentation therapy by delivering PTEN tumour suppressor plasmid with the aid of NPs also showed promising cell viability profile. Moreover, co-treatment of the siRNA and plasmid remarkably exhibited significant decrease in breast cancer cell viability as well as diminished level of proliferation signal by downregulating P-AKT. Considering all the results, we further examined the feasibility of the nano-formulations in an in vivo setting. Because our in vitro cell viability results  
Figure 4: (**a**). Western blot images following intracellular delivery of PRKCA siRNA+NP, PTEN+NP and PRKCA siRNA+PTEN+NP in MCF-7 cell line. Cells were incubated with the treatment formulations for 48 h prior to cell lysis for protein extraction and Western blotting. Detection of Phospho-AKT (P-AKT), Total-AKT (T-AKT) and GAPDH was performed using respective polyclonal antibodies. (**b**). Densitometry analysis showing ratio of P-AKT/Total AKT expression. P-AKT expression was significantly lower in PRKCA siRNA+PTEN+NPs treatment group compared to PRKCA siRNA+NPs and PTEN+NPs treatment groups. Statistical analysis was very significant, *** when \(p<0.01\). in MCF-7 cell line were very prominent compared to other two cell lines, we established MCF-7 xenograft mouse model to test our nucleic acid-based numberapeeutics, PRKCA siRNA+NPs, PTEN+NPs and PRKCA siRNA+PTEN+NPs in comparison with the control (NPs) on the tumour-bearing mice randomly assigned to different groups.