## 1 Introduction
In fact, just a 20% reduction in the protein level was sufficient to induce tumourigenesis in a hypermorphic mouse model with the highest penetrance for breast cancer [20; 21]. A mutant protein without its lower level of expression due to the loss of an allele was proved to be more tumourigenic. In a heterozygous mutant case, the catalytically inactive mutant protein dimerizes with the wild-type protein, and predictably, the resultant dimer is catalytically less active. Interestingly, the mutant protein slowly replaces the normal protein to make a homodimer, ultimately removing the brake on proto-oncogenic PI3K/AKT pathway [22; 23]. Loss of PTEN is associated with tumourigenesis. Germline loss was reported in a group of diseases collectively described as PTEN hamartoma tumour syndromes (PHTS), of which Cowden syndrome is the most studied one. Of Cowden syndrome patients, 80% possess PTEN germ-line mutations, and females develop a 25-50% lifetime risk of having breast cancer. In sporadic breast carcinomas, the occurrence of PTEN loss is 30-40% [24]. PTEN loss is associated with poor outcome in hormone-positive, HER2-positive or -negative breast cancer [17]. In breast cancer, PTEN loss contributes to drug resistance and helps evade immune regulation by inducing overexpression of programmed cell death ligand 1 (PD-L1) [17; 25]. In view of the importance of PTEN function as a tumour suppressor, the wild type PTEN gene containing a functional version of the lost gene was planned here to be delivered into the breast cancer cells, using carbonate apatite nanoparticles (CA NPs). Moreover, PTEN acts as a major antagonist of PI3K activity. Therefore, we tried to combine the treatment of siRNA against PRKCA gene with tumour suppressor PTEN plasmid in order to enhance PTEN protein expression, with a view of obtaining a synergistic or additive effect of the formulation in decreasing breast cancer cell growth both in vitro and in vivo. In order to protect the siRNA/DNA from nuclease-mediated degradation in intracellular and extracellular environment, smart pH-sensitive inorganic CA NPs have been employed for effective nucleic acid transfer onto the cancer cells with positive therapeutic impact. [PERSON] et al. had established development of proficient, biodegradable inorganic NPs of CA with ability of effective intracellular delivery and release of associated siRNA, and plasmid DNA from endosomes to cytosol, leading to specific cleavage of a particular mRNA transcript, or expression of a desirable protein, respectively [26]. At low pH 7.4, in bicarbonate buffered medium, addition of Ca\({}^{2+}\) salt and plasmid DNA or siRNA generates nano-sized carbonate apatite/siRNA/DNA complexes, following incubation at 37 \({}^{\circ}\)C for 30 min. Next, FBS is added to stop further nanoparticle complex formation [27]. In Field Emission Scanning Electron Microscope (FE-SEM) images, CA NPs displayed spherical shape, rough texture, and an average diameter of 50-300 nm [28; 29; 30]. With molecular formula Ca\({}_{10}\) (PO\({}_{4}\))\({}_{6-\mathrm{x}}\) (CO\({}_{3}\))\({}_{\mathrm{x}}\) (OH)\({}_{2}\), the small sized nanoparticles have durable binding affinity towards the siRNA/DNA; the positively charged Ca\({}^{2+}\) present in the CA NPs can electrostatically combine with the negatively charged siRNA/DNA molecules and come close to the cell membrane through ionic interaction, ultimately transporting the siRNA/DNA into cytoplasmic acidic compartments (endosomes) by endocytosis [27]. Upon cellular internalization by endocytosis, particles are quickly dissolved into component ions such as Ca\({}^{2+}\), PO\({}_{4}\)\({}^{3-}\) and HCO\({}_{3}\)\({}^{-}\) in endosomal acidic pH, resulting in particle dissolution, swelling and breakage of the endosomes following development of osmotic pressure across the endosomal membrane, and release of the siRNA/DN A in cytosol [27; 28]. In the cytoplasm, the released siRNA is merged into a RISC (RNA-induced silencing complex) and relaxed into single-stranded RNAs by Argonaute-2.