**1. Introduction**

Combinations of individual therapeutics indicated against malignancies might result in superior outcomes in resolving the complexity of cancer cells. Among the tackled complexities is an acquired or intrinsic resistance of the cancer cells to treatment. In fact, combining different therapeutic strategies might provide various benefits as follows: (a) Maximized therapeutic efficacy without increased overall toxicity to the host due to different mechanisms of action; (b) prevention of the development of resistance to single agents; (c) covering the heterogeneous tumor cell population with different drug sensitivity profiles; and (d) possible synergy between therapeutics, resulting in increased anticancer efficacy [1].

The underlying mechanisms of resistance might involve genetic alterations, which could be depleted via RNA interference technologies, such as small interfering RNAs (siRNAs). Phenotype modifications through suppression of mRNA transcripts by the usage of siRNA, might render the cancer cells more responsive to the accompanying chemotherapeutic agents. In the co-delivery of nucleic acids and small molecule drugs, protective vectors would assist in boosted cellular uptake, lysosomal escape, and protection against serum nucleases together with limited off-target effects.

Nano-based formulations have the potential to be versatile and multifunctional, enabling the co-delivery of multiple agents entrapped in the nanoparticles' structure to gain synergistic anticancer effects or multi-functions.

Co-delivery of drugs and siRNAs has been documented via application of different carrier systems [2–4]. In one study, doxorubicin and Bcl-2-targeted siRNA were applied via a polyethylenimine-coated graphene oxide (PEI-GO) vehicle. Here, the GO moiety adsorbs the therapeutics while PEI enhances cell membrane penetration [2]. In another example, a triblock polymer loaded with VEGF siRNA and paclitaxel delivered augmented efficacy against paclitaxel [3]. *N*-succinyl chitosan-poly-l-lysine-palmitic acid (NSC-PLL-PA) has been utilized in the synthesis of a triblock copolymer for co-delivery of P-glycoprotein-targeted siRNA and doxorubicin at pH 7.4. These therapeutics are released upon degradation of the carrier in the acidic pH of lysosomes [4].

In fact, numerous examples of synthetic dual siRNA and drug-delivery vehicles have been reported in the literature, with varying formulations and results [5–8]. However, carriers consisting of protein or peptide origins have demonstrated superiority due to biodegradability, complex structure details and functional groups, feasible adjustments in the synthesis process, and also compatibility in the construction of multifunctional hybrid materials [9].

As published data demonstrate, a lipoproteoplex has been developed for the purpose of dual delivery of siRNA and doxorubicin, with the ability to condense siRNA and encapsulate the small-molecule chemotherapeutic, doxorubicin. The lipoproteoplex demonstrates improved doxorubicin loading, resulting in a substantial decrease in MCF-7 cell viability, plus effective transfection of GAPDH (60% knockdown) in MCF-7 breast cancer cells [9].

Inorganic carbonate apatite is a recently developed nanocarrier synthesized via calcium phosphate precipitation in the presence of bicarbonate. The controlled crystal growth dynamic leads to the formation of particles with a size ranging between 50 and 300 nm. These carriers encompass optimal features of efficient endocytosis, fast dissolution rate in endosomal acidic pH, and effective release of loaded therapeutics. In more detail, addition of 3 mM of Ca in the synthesis of carbonate apatite results in the formation of nanoparticles with a size less than 50 nM. In presence of 10 nM of paclitaxel, NPs reach a maximum size of around 170 nm. Markedly, paclitaxel-loaded carbonate apatite demonstrates a 20.71 ± 4.34% loading efficiency [10]. Carbonate apatite nanoparticles have been utilized for co-delivery of anti-cancer drugs and various siRNAs, resulting in improved outcomes [11,12].

Members of the human epidermal growth factor receptor (HER) family are among the key factors in regulating the response of breast cancer cells to chemotherapy. As another mediator in the complex pathway of oncogenesis, AKT, a serine–threonine protein kinase, has been heavily studied.

AKT signaling has been shown to be regulated by enhanced HER signals, mutational activation of Ras leading to AKT activation via the PI3K pathway, or the mutational inactivation of the PTEN phosphatase resulting in diminished AKT activity. Secondary to these alterations, AKT kinase activity would be augmented. Thereafter, increased AKT kinase activity on its own and in the absence of any upregulation in AKT protein concentrations may have a broader effect on oncogenesis as well as the cellular response to cancer therapy [13].

Thus, various components of these inter-related signaling cascades have been targeted via siRNA application in this study to obtain an accurate assessment of their role in the response of the cancer cells to chemotherapy agents.

#### **2. Materials and Methods**

#### *2.1. Materials*

Dulbecco's modified eagle medium (DMEM), calcium chloride dehydrate (CaCl2.2H2O), sodium bicarbonate (NaHCO3), dimethyl sulphoxide (DMSO), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), phosphoric acid solution (H3PO4), trifluroacetice acid (TFA; CF3COOH), Ethylene Diamine Tetraacetic Acid (EDTA), and anti-cancer drugs docetaxel (Doc) and paclitaxel (Pac) were purchased from Sigma-Aldrich (St Louis, MO, USA). DMEM powder, fetal bovine serum (FBS), trypsin ethylene diamine tetraacetate (trypsin-EDTA), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), and penicillin-streptomycin were obtained from Gibco BRL (Carlsbad, CA, USA). All functionally validated siRNAs used in this study (listed in Table 1) were obtained from Qiagen and dissolved in RNase-free water provided by the company to obtain 10-μM stock solution. MCF-7, 4T1 and MDA-MB-231 cells were originally from ATCC.


