## 4 Discussion  
As a promising cancer therapeutic strategy, siRNA has numerous advantages over chemotherapeutic drugs including high degree of safety and specificity. siRNA inhibits the post-transcriptional stage of gene expression by complementary base pairing with a target mRNA through RNA interference system, without adverse side effects [37]. Another crucial advantage of siRNA is its high efficacy, restraining the expression of a target gene markedly in a single cancer cell with just several copies [38]. However, 'free' or 'naked' siRNA can hardly penetrate the cell or tissue because of its negatively charged phosphate backbone that could be repelled by anionic extracellular matrix molecules. In addition, its half-life is very short due to the risk of degradation by serum nucleases, demanding for a robust delivery vector to protect it from cleavage and deliver to the target site with high efficacy [39]. In a recent study, [PERSON] et al. investigated the application of siRNA nanotherapeutics in breast cancer targeting several oncogenes such as EGFR, ERBB2 and BCL-2 using CA NPs of size ranging 100-300 nm. This siRNA mediated simultaneous silencing of cell-surface growth factor receptors and anti-apoptotic genes synergistically killed breast cancer cells through inhibition of MAPK and PI-3 kinase pathways [40]. In addition, [PERSON] et al. reported that CA NP-mediated intracellular delivery of c-ROS1 siRNA sensitized MCF-7 breast cancer cells to chemotherapy drugs cisplatin and paclitaxel [41]. In order to further investigate other oncogene signaling pathways, in this study, CA NPs were employed to deliver the PRKCA siRNA, which showed great implication in minimizing breast cancer cell growth both in vitro and in vivo. Evidence has emerged from several studies describing importance of PRKCA knockdown using siRNA targeted to PRKCA gene in different types of cancer. Specific and efficient silencing of PRKCA gene was achieved using chemically synthesized siRNAs, resulting in prolonged obstruction of neuroblastoma cell division [42]. [PERSON] et al. demonstrated that PKC-alpha siRNA transfection nearly abrogated PKC-alpha expression and significantly reduced melanoma cell migration compared to siRNA controls [43]. PTEN, another potential target in our study, is considered as a candidate tumour suppressor gene based on the finding that mutation or loss of this gene has been linked to a variety of common human cancers, including breast, prostate, glioblastoma, melanoma and lymphoid cancer [44]. A group of researchers affirmed that using Lipofectamine 2000 transfection method, over-expression of PTEN induced cell growth arrest and apoptosis in PTEN-null human breast cancer cells [45]. Similarly, a study by [PERSON] et al. concluded that exogenous PTEN suppressed the growth of human breast cancer cell and prompted apoptosis by phosphatase activity. They also used Lipofectamine 2000 for delivery of PTEN gene in breast cancer cells [46]. Unlike Lipofectamine 2000, we utilized inorganic pH sensitive CA NPs which are small in size, convenient, cost-effective, biodegradable and biocompatible nano-vectors for transfection of the PTEN plasmid in breast cancer cell line with very noticeable outcomes. Our cell viability and protein expression results showed that over-expression of PTEN in MCF-7 cells caused growth suppression by downregulating P-AKT expression, in agreement with the study by [PERSON] et al. as mentioned above [46]. Delivering double elements to maximize the treatment outcome is an attractive concept in clinical medicine. The additive response of the combined formulation in our study can be explained by their opposite regulatory roles in PI3K-AKT-mTOR pathway. Upon activation, PKC\(\alpha\) catalytically phosphorylates a variety of protein substrates (e.g., AKT to P-AKT) [47; 48], thus activating the protein of interest towards cellular proliferation, whereas PTEN catalyses the opposite reaction, dephosphorylation (e.g., P-AKT to AKT), and implies deactivation of the proliferative signal [49].