*3.1. Cytotoxicity of siRNA-Loaded NPs on MCF-7 and 4T1 Cells*

To explore the efficacy of carbonate apatite in the delivery of siRNAs into the cells and also evaluate the effect of single gene knock down on the viability of cancer cells, carbonate apatite nanoparticles harboring single siRNA were tested on MCF-7 and 4T1 cells. Here, 4 mM of CaCl2 was applied in the presence of various amounts of single siRNAs to produce treatment formulations.

Binding of siRNA to NPs, in the presence of increasing concentrations of calcium and fixed siRNA amounts, is saturable and reaches a plateau level, in spite of the escalating pattern of particle size. Higher amounts of calcium together with limited endogenous phosphate (0.9 mM) accounts for an increased particle size due to aggregation and not larger individual particles. Limited surface area of the particles secondary to aggregation results in restricted siRNA binding [14].

Remarkably, CA capacity in the efficient delivery of siRNAs into their action site is displayed by the considerable enhancement in the cytotoxic effects for almost all amounts of siRNAs complexed with CA compared to free siRNA. The most killing effect on MCF-7 and 4T1 seems to be achieved by muting the HER2 or ROS1 cascade (Table 4) (Supplementary Figures S1 and S2), indicating the strong impact of ERBB2 or ROS signaling on the survival of both cell lines.


**Table 4.** Cytotoxicity (%) enhancement on 4T1 and MCF-7 cells treated with various siRNAs (1 pM, 10 pM, 100 pM, 1 nM, and 10 nM) incorporated into an apatite structure formed with 3 mM of CaCl2. The data is presented as mean ± SD compared to free siRNA.

In another research, carbonate apatite nanoparticles have been confirmed as efficient carriers for electrostatically associated siRNA inside the cells, releasing bound siRNA from endosomes to the cytosol through pH-responsive self-dissolution. This was concluded based on the significant increase in the fluorescence intensity of the intracellular components from the cells treated with CA/fluorescent siRNA compared to untreated and CA-treated cells [14].

Moreover, application of different concentrations of 'Allstars Negative Control siRNA' (1 pM to 10 nM) loaded into a carbonate apatite structure resulted in no alteration of breast cancer cells' viability [11].

However, the duration of response of the cancer cells to single-oncogene inhibitors, even the most potent oncogenes, is mostly short due to the development of resistance or upregulation of compensatory mechanisms in survival and proliferation signaling. Therefore, sustained and significant outcomes are mainly achieved with combinations of various classes of anti-cancer therapies. In case of no initial response to treatment or development of drug resistance, combinatory regimens are of great benefit to induce therapeutic response. In fact, putting off the causal cascades of resistance by one agent might result in re-sensitization of the cells to other therapeutics in the combination.

Markedly, pharmacokinetics and pharmacodynamics interactions between the agents in combination therapy might develop in case of a shared action site, uptake, and metabolism or elimination routes.

Delivery of drugs and siRNAs by means of carbonate apatite nanoparticles into cells was further studied. The aim was to evaluate the impact of silencing different signaling pathways on the response of cancer cells to the cytotoxic effects of drugs. Different concentrations of paclitaxel, docetaxel, mitomycin C, and topotecan (10 pM, 100 pM, and 1 nM) were used together with 1 pM of siRNAs against AKT, ERBB2, MAPK, and ROS1 and 3 mM Ca. Based on extensive experiments and with the aim of exploring possible synergistic effects, the lowest effective doses of therapeutics were applied. This was to perhaps achieve a therapeutic effect with lower doses and hence less side effects.

Notably, application of anti HER2 siRNA together with any of the four drugs caused positive changes in cytotoxicity against 4T1 cells (Supplementary Figures S3–S6). Additionally, co-delivery of Pac and ROS1 siRNA or Doc and MAPK siRNA seemed to sensitize 4T1 cells to the drug to a great extent.

Response of MDA-MB-231 cells to chemotherapy in case of single pathway silencing was also explored. Paclitaxel and docetaxel were used in complexes of carbonate apatite using 3 mM Ca and 10 pM, 100 pM, and 1 nM of the drug together with 1 nM of each siRNA. As the cell viability data reveals, Pac efficacy benefits from silencing ERBB2 or ROS1 pathways in terms of a highest increase in cytotoxicity on MDA-MB-231 cells (Supplementary Figure S7). Whereas, AKT or ERBB2 pathways display a significant role in response to docetaxel in MDA-MB-231 cells, as with knock down of these two oncogenes, all concentrations of Doc exert a substantial higher cytotoxicity (Supplementary Figure S8).

As the next step, simultaneous silencing of AKT and ERBB2 oncogenes was implemented to check if these pathways have any impact on the response to classical anti-cancer drugs.

Carbonate apatite was formed with 3 mM of CaCl2 and 1 pM of AKT and ERBB2 siRNA together with 10 pM, 100 pM, and 1 nM of paclitaxel, docetaxel, mitomycin C, and topotecan. Based on cell viability results (Supplementary Figure S9), knock down of AKT and ERBB2 seems to have the greatest impact on the response to paclitaxel or docetaxel in 4T1 cells. The highest enhancement in cytotoxicity on 4T1 is equal to 19.97 ± 1.73% for Pac and 15.16 ± 3.55% for Doc, resulting from simultaneous blockade of AKT and ERBB2 signaling. Thus, these combinations were applied for in vivo studies since the animal tumor model was developed by local injection of 4T1 cells in mice.

On MDA-MB-231 cells, application of siRNAs for simultaneous silencing of two pathways led to augmented cytotoxicity of paclitaxel as well. This effect was higher with usage of AKT and ROS1 siRNA or co-delivery of AKT and MAPK siRNAs in the presence of Pac (Supplementary Figure S10).

Tables 5–7 demonstrate the enhancement of the cytotoxic effect of classical anti-cancer drugs resulting from the silencing of various pathways in MCF-7, 4T1, and MDA-MB-231 cells, respectively.


**Table 5.** Effect of silencing various pathways on cytotoxicity of classical anti-cancer drugs in MCF-7 cells. Data is presented as mean ± SD compared to CA/drug.

**Table 6.** Effect of silencing various pathways on cytotoxicity of classical anti-cancer drugs in 4T1 cells. Data is presented as mean ± SD compared to CA/drug.



**Table 6.** *Cont.*

**Table 7.** Effect of silencing various pathways on cytotoxicity of classical anti-cancer drugs in MDA-MB-231 cells. Data is presented as mean ± SD compared to CA/drug.


According to the cell viability data displayed in Table 5, silencing AKT or ERBB2 regulates the response of MCF-7 cells to paclitaxel or docetaxel substantially. Thus, the effect of these therapeutic agents on the protein expression was further evaluated via Western blotting.

Induction of cell death followed by application of therapeutically loaded carbonate apatite could be associated with involvement of the apoptotic pathway, based on escalation of caspase-7-mediated signaling [12].

Silencing of AKT or HER2 signaling to expedite treatment outcomes in cancer is backed up by a solid body of knowledge. Expression of HER2 in MCF7 cells has been shown to increase under certain conditions [15,16].

According to recent research, docetaxel induces either apoptosis accompanied by survivin upregulation or necrosis and a lower rate of survivin upregulation in various breast cancer cell lines, based on cells receptor expression profile and the molecular phenotype. Additionally, inhibition of p-AKT was shown to revert survivin upregulation and also induce docetaxel-dependent apoptosis [17].

In another study, inhibition of NRF2 in ERBB2-overexpressing ovarian carcinoma cells was shown to suppress ERBB2 expression, which led to a decrease in phospho-AKT and enhanced p27 protein together with increased sensitivity of these cells to docetaxel cytotoxicity and apoptosis [18].

There are also other published data associating increased paclitaxel-induced apoptosis with inhibition of AKT in ovarian cancer cells. While paclitaxel-resistant cells demonstrated higher levels of p-AKT compared to paclitaxel-sensitive cells, inhibition of AKT increased paclitaxel therapeutic efficacy in both cell lines [19].

Moreover, combination of shRNA against AKT1 with paclitaxel exerted synergistic anti-cancer effects, thus inhibiting the growth of human breast cancer MDA-MB-231 and MCF-7 cells, and breast cancer MDA-MB-231 cell xenografts in mice as well. The combination therapy demonstrated enhanced anti-cancer effects through inhibition of AKT1 signaling and induction of apoptosis [20].

Extensive in vitro and in vivo research has associated ERBB2 overexpression with resistance of breast cancer cells to certain chemotherapeutic agents, namely docetaxel and paclitaxel. Furthermore, in clinical studies, herceptin, the anti-ERBB2 antibody, enhanced the antitumor activity of paclitaxel and doxorubicin against ERBB2-overexpressing human breast cancer xenografts, and the paclitaxel response rate of patients with ERBB2-overexpressing breast cancers was significantly higher among patients receiving paclitaxel plus herceptin than those receiving paclitaxel alone [21]. Additionally, co-administration of a PI3K inhibitor with cytotoxic or targeted anticancer agents, such as carboplatin, paclitaxel, or erlotinib, led to increased tumor growth inhibition over the corresponding single agents [22].

Moreover, high AKT activity has been shown to be responsible for the enhanced resistance of ERBB2-overexpressing cancer cells toward chemotherapeutic agents, and inhibition of AKT activation by peptide aptamer resulted in the restoration of regular sensitivity of breast cancer MCF7 cells towards paclitaxel [23].

In another research, siRNA-based knockdown of HER-2 conferred increased sensitivity to paclitaxel in endometrial cancer cells, attenuating the induction of p-AKT on paclitaxel stimulation, which was cancelled by inactivating AKT by the introduction of a dominant-negative form [24].
