**2. Small Interfering RNAs**

Small interfering RNAs (siRNAs) were first described about three decades ago in plants [28], and then in *Caenorhabtidis elegans* [29]. Their therapeutic potential was first reported in 2001 [30], when it was shown that a short, double-stranded RNA duplex (about 21 nucleotide long) can induce target mRNA degradation, thus suppressing gene expression in human cells. The mechanism of action (Figure 3) begins with the uptake of one of the two RNA filaments (antisense strand) of the siRNA by a cellular protein complex termed RISC (RNA-induced silencing complex). While the other filament (sense strand) is discarded, the antisense strand drives RISC to a target RNA via a perfect sequence complementarity. This, in turn, allows RISC to induce the degradation of the target RNA, leading to gene expression inhibition. It is possible to design siRNAs targeted against virtually any deleterious (m)RNA, including viral RNA [31,32]. Moreover, their chemical synthesis is neither complex not particularly expensive. The above characteristics make the therapeutic applicability of siRNA potentially very broad [2,33–36].

**Figure 3.** Short interfering RNA (siRNA) mechanism of action.

#### *2.1. Delivery Barriers for Systemic Administration*

Made of RNA, siRNAs are characterized by a negative electric charge and have poor stability in the biological environment. Thus, siRNAs need to be embedded into specific carriers for protection and to efficiently reach the target cells. It should be considered that for systemic administration, as could be the case in treating ovarian cancer, siRNAs encounter a number of obstacles that can dramatically reduce the possibility of their reaching the target cancer cells. Once in the blood stream, siRNA can: (1) be degraded by blood nucleases, (2) be eliminated by the phagocytic system, (3) be cleared from blood via kidney filtration and/or sequestered by the liver [37] and activate the innate immune response [38] (Figure 4). Once the siRNAs reach the target tissue, additional obstacles remain, like (4) crossing the vessel wall (extravasation), (5) the migration through the extracellular matrix (ECM) and then (6) crossing the cellular membrane (Figure 4). This last step is particularly inefficient for naked siRNA as their global negative charge, derived from the phosphate groups of their backbone, induces the repulsion of siRNA from the negatively charged molecules present on the outer side of the cell membrane. Moreover, the hydrophilic nature of siRNA substantially prevents its crossing through the hydrophobic inner layer of the cell membrane. Only a reduced amount of siRNA can be internalized via endocytosis. Finally, once into the target cell, siRNAs can be entrapped in endosomes (7), an event that can further reduce the amount of siRNA able to reach the target.

**Figure 4.** Obstacles for systemic siRNA delivery.
