## 4 Discussion
To the best of our knowledge, the correlation between PRKCA and PTEN proteins has been examined towards breast cancer gene therapy for the first time through our study. The novelty of our study reflected the additive effect upon co-delivery of siRNA against PRKCA and PTEN plasmid gene in reducing breast cancer cell proliferation, as reflected in cell viability assessment and Western blot experiments. However, for the in vitro study, we observed differences in cell viability results in three different breast cancer cell lines. Because breast cancer is a complex and heterogenous disease, gene expression profiling of the different representative cell lines also revealed heterogeneity at molecular level--explaining differential behavior of the treatments in cell lines [50]. A study by [PERSON] et al. established that different types of proteins interact with NPs, and the composition of protein corona could control uptake into specific cell types [51]. The in vivo testing of the potential nucleic acid-based nanothereeutics composed of PRKCA siRNA+NPs and PTEN plasmid+NPs in the mice bearing MCF-7-induced xenograft breast tumours revealed quite similar results, as observed in cell viability assessment and Western blot tests. Mouse groups treated with PRKCA siRNA+NPs and PTEN plasmid+NPs formulations exhibited significant reduction in tumour size compared to the control group of mice. As observed from the size and zeta potential results of the siRNA, plasmid and NSp complex, PTEN+NPs possessed slightly more negative zeta-potential compared to other complexes, and the mean tumour volume was smallest (162 mm\({}^{3}\)) in PTEN+NPs group of mice, implying that more negative zeta potential was advantageous in terms of longer retention of the formulation in blood stream [29]. However, the additive effect of the combined delivery could not be verified with the in vivo tumour regression results which rather followed the tumour reduction pattern similar as the mouse groups treated with individual elements of PRKCA siRNA+NPs and PTEN plasmid+NPs. Principally, the in vivo system is a relatively more complicated biological environment controlled by multiple factors and signaling pathways. A study by [PERSON] et al. demonstrated that upon systemic delivery, NPs come into interaction with serum proteins, forming a protein corona (PC). The composition of PC depends on size, charge and other physicochemical features of NPs, and in in vivo mouse studies, the PC can be remodeled with the change of proteome in different treatment formulations [28; 52; 53]. Another possibility of epigenetic changes in in vivo environment may affect gene expression and protein interaction in different treatment groups; hence, future study might focus on the in vivo mechanism of the combined therapy. Conception and Design: N.I., E.H.C. ; Acquisition of data: N.I., R.A.I. ; Analysis and interpretation of data: N.I. ; Manuscript drafting: N.I., R.A.I. ; Manuscript revising: all authors; Final approval of this version: all authors. All authors have read and agreed to the published version of the manuscript. This project was financially supported by a grant from Ministry of Higher Education, Malaysia (MoHE) (FRGS/1/2018/STG05/MUSM/02/3). The animal study was approved by the Monash University Animal Ethics Committee, with animal ethics approval No. MUM/2018/12. Informed Consent Statement: Not applicable. Data Availability Statement: Not applicable. We also thank [PERSON] and [PERSON] from Monash Animal Facility department for their technical assistance during the PhD thesis work. The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.