Dual pathway intervention

Co-delivery of small interfering RNA (siRNA) and small molecule anticancer drugs is a very classic case of dual pathway intervention. The siRNA and small molecule drugs have the extreme different physical, chemical, and biological properties, such as the different molecular weight, hydrophobicity, and intra-body stability.

Therefore, designing an effective co-delivery system of these two molecules is a challenge. To keep one thing in mind, the carrier for achieving the co-delivery should be nontoxic and non-immunogenic for systemic administration. Besides, the carriers have to condense the siRNA effectively, either via the electrostatic adsorption or the chemical conjugation. However, for the small molecule drug, it should be encapsulated via the physical encapsulation, inclusion, or direct chemical conjugation. Moreover, the major mission of the co-delivery carrier is to simultaneously deliver the siRNA and small molecule drug to the target cancer cells, meanwhile ensure their intrinsic release kinetics and pharmacological properties. To date, a number of promising nano-carriers have been researches and developed for the siRNA/drug co-delivery (Yadav et al., 2009). Co-delivery system delivers the siRNA and the anticancer drugs at the designated ratio to the cancer cells, thus usually brings a synergistic effects in treatment (Cao et al., 2011, Sun et al., 2011). Some specially designed co-delivery systems can

even deliver multiple drugs to the target cell with a programmed release profiles (Choudhury and He, 2012).

Factors that reduces the efficacy of the treatment are many. Besides the enhanced drug efflux observed in cancer cells, the alteration the apoptotic behaviour also plays a important role in the stubborn cancer therapy (Aliabadi et al., 2013). Apoptosis is a programmed cell death process regulated by many genetic pathways, such as the tumour-suppressor genes (p53), pro-apoptotic genes, and anti-apoptotic genes. The p53 is the most often mutated gene in the human cancers. Many studies have suggested that the lack of the p53 function in cells, which can caused by the mutated p53 genes, induces several oncogenic behaviour like enhanced genomic instability, abnormal cell proliferation, MDR, and suppression of the apoptosis (Guerra-Vladusic et al., 1999). To overcome this issue, the silencing siRNA is used to deal with the mutated p53 for reducing the MDR and enhancing the apoptosis in cancer cells. For example, Zhu et al.

reported the co-delivery system that delivers the p53 targeting siRNA and cisplatin to the human bladder cancer cells in vitro (Zhu et al., 2013b). As the result, it achieves the over 70% knockdown of the p53 mRNA expression and the G2 phase cell cycle arrest.

The reduction of the cell viability of the cells treated with co-delivery system (72.3%) is much higher than the reduction in cells treated with only siRNA (38.7%) or cisplatin (44.9%).

When it comes to the proteins that regulate the apoptosis, the Bcl-2 family of proteins (including both the pro-apoptotic Bax and anti-apoptotic Bcl-2, and Bcl-xL) plays a very important role. The pro-apoptotic proteins serve as a mitochondrial gateway to induce the release of cytochrome c for triggering the programmed cell death. But the over- expressed anti-apoptotic proteins oppositely suppress the apoptosis (Kroemer, 1997).

Besides, there are also evidences suggesting that the over-expressed anti-apoptotic

proteins can cause the resistance to several drugs, such as the DOX, paclitaxel, etoposide, camptothecin, mitoxantrone, and cisplatin (Youle and Strasser, 2008). That is why the knockdown of the anti-apoptotic proteins can effectively enhance the efficacy of the anti-cancer drug. On the basis of this synergistic effect, Zheng et al. have reported the co-delivery of siRNA-Bcl-2 and docetaxel (DTX) by a polymer micelles contracted by the poly (ethylene glycol)-b- poly(^L-lysine)-b-poly(^L-leucine) tri-block copolymers into the NCI- ADR-RES cell (which is a kind of MCF-7 human breast cancer cells with over-expressed Bcl-2 protein) (Zheng et al., 2013). The hydrophobic core is used to load DTX through a hydrophobic interaction. The siRNA-Bcl-2 is absorbed into the polymer micelles through the electrostatic interactions. The higher siRNA-Bcl-2 concentration successfully causes more Bcl-2 mRNA knockdown, which subsequently reduces the cell proliferation due to the demonstrated synergistic effect of the co-delivery of siRNA-Bcl-2 and the DTX.

In addition to the Bcl-2, the survivin is another highly expressed anti-apoptotic proteins in human cancer cells. It facilitates cancer development by inhibiting the caspase activation, such as the reported mitotic regulatory activity that leads to the enhanced cell proliferation. Wang et al. researched the co-delivery of the DOX, paclitaxel, and survivin targeted siRNA with a nano-emulsion carrier. The carrier is made from an amphiphilic block copolymer of methoxy poly(ethylene glycol)–poly(lactide-co-glycolide) (mPEG–PLGA) and e-polylysine (EPL) (Wang et al., 2013). The DOX is thus encapsulated in the hydrophilic core and the paclitaxel is packaged in the hydrophobic layer. At the outer surface of the carrier, the survivin siRNA is adsorbed through the electrostatic interactions. As a result, the powerful synergistic anti-cancer effects of the co-delivery of DOX, paclitaxel, and survivin-siRNA is obtained in a B16-F10 melanoma bearing mice.

Some kinds of transcription factor also play the important role in cancer development.

For example, the nuclear factor-kb (NF-κB) is a one that regulates the expression of a variety of genes to inhibit the apoptosis in cancer cells. Moreover, the expression of the NF-κb is also relevant to the MDR in cancer cells. Hence, it is rational to suppress this transcription factor for better cancer inhibition efficacy. Zhao et al. have reported the co-delivery of DOX and NF-κb p65 siRNA with a spermine-grafted poly-g-benzyl-^L -glutamate (PGS) polyelectrolyte brushes in vitro (Zhao et al., 2013b). The NF-κb p65 siRNA enhanced the bioavailability of the DOX in the cancer cells because of the NF-κB p65 suppression. The total apoptosis of co-delivery nano-medicine treated cell is 170% of that treated with the same dose of DOX alone.