## 3 Results
### Co-Delivery of siRNA against PRKCA and PTEN Plasmid Reduced Growth of Breast Cancer Cell Lines  
Treatment of breast cancer cells with CA NPs, PRKCA-specific siRNA+NPs, PTEN plasmid+NPs and PRKCA siRNA+PTEN plasmid+NPs for 48 h induced morphological changes in cell size and shape as observed under a light microscope. The images in Figure 2 demonstrated that untreated control cells are packed within 48 h of incubation, while in presence of 4 mM Ca\({}^{2+}\), the cells appear to be surrounded by the CA NPs. In addition, microscopic observation of the cells treated with siRNA and plasmid revealed decrease in cellular density in MCF-7 cell line, compared to the untreated control cells. This simple visualization of treated cells with less cell number indicated cell shrinkage and reduced cellular proliferation which was also reflected in the cell viability results.  
The cell viability data plotted in Figure 3a-c demonstrated that CA NPs were complexed with PRKCA-specific siRNA and PTEN plasmid for delivery into (a) MCF-7, (b) MDA-MB-231 and (c) 4T1 cell lines.  
In MCF-7 cells (Figure 3a), treatment with 1 nM PRKCA siRNA+NPs reduced cell viability to ~50% after 48 h of incubation. Again, 1 ug PTEN+NPs treated cells exhibited ~57% of cell viability. Interestingly, combined treatment with PRKCA siRNA+PTEN+NPs complex curtailed the cell viability to ~37%. In other words, compared to single siRNA and plasmid treated cells, co-delivery of PRKCA siRNA and _PTEN_ gene in this cell line  
Figure 1: (**a**) Size and (**b**) zeta potential measurements of NPs alone, PRKCA+NPs, PTEN+NPs and PKCA+PTEN+NPs formed with addition of 4 mM of exogeneous Ca\({}^{2+}\) in 1 mL DMEM medium and incubation at 37 \({}^{\circ}\)C for 30 min. Each of the measurements performed three times and mean and standard deviation were calculated.  
revealed statistically significant decrease (\(p<0.05\)) in cell growth, indicating additive effect of the treatment.  
In MDA-MB-231 cell line (Figure 3b), the PRKCA siRNA+NPs treatment declined cell viability to approximately 84%, and intracellular delivery of PTEN+NPs could lower the cell viability to ~85%. Moreover, PRKCA siRNA+PTEN+NPs complex reduced the cell viability to ~75%. Altogether, delivery of both PRKCA siRNA and _PTEN_ genes could induce additive effect compared to the individual siRNA or plasmid treatment in this cell line.  
Our observation in 4T1 cells (Figure 3c) showed that treatment with PRKCA siRNA+NPs and PTEN+NPs complexes for a consecutive period of two days led to cell viability of ~64% for both treatments. However, when co-delivered (PRKCA+PTEN+NPs), it reduced the cell viability to ~61% only, indicating no net decline in cell viability was observed compared to the individual siRNA or plasmid gene transfection in 4T1 cell line.  
In all of the three cell lines, NPs-only administration reduced cell viability, when compared to untreated cells. The cytotoxicity caused by NPs can be explained by the notion that particles associated with cell membrane could damage cell membrane, while the particles internalized into the cells release Ca2+ upon dissolution into the endosomes, enhancing cytosolic Ca2+ level which is associated with necrosis and apoptosis of the cells [35].