In vitro efficiency of siRNA loaded nanoparticles on cancer cells

RNA interference (RNAi) is a cellular mechanism of selective gene silencing, based on degradation of specific mRNA by small interfering RNAs (siRNAs), and mediated by an RNA-induced silencing complex (RISC). The molecular machinery behind this process has helped in designing therapies for a variety of diseases, including cancer. Silencing of target genes requires that siRNA is delivered into the cell in its active state. To achieve this, an effective vector protecting siRNA that is non-toxic and can be targeted at selected cells is necessary. siRNA molecules can-not readily diffuse across cellular membranes due to their anionic backbone and hydrophilic nature. Thus, delivery vehicles must be used to protect the siRNA within biological fluids, while facilitating its transfection to the cytoplasm of the target cells. Two diffferent approaches for siRNA delivery have been developed: viral and non-viral vectors. Non-viral vectors (also named nanoparticles) have been formulated to associate andto efficiently and safely deliver nucleic acids both in vitro and in vivo, opening many possible applications. Furthermore, they have many advantages and potentialities including large scale manufacture, low toxicity and low immunogenecity and the possibility to customize them to target specific cell types. In this frame, novel nanoparticles (NPs) designed in order to improve the interaction with the cell membrane. In the present study poly(styrene)-graft-poly(linoleic acid) (PS-PLina) and poly(styrene)-graft-poly(linoleic acid)-graft-poly(ethylene glycol) (PS-PLina-PEG) NPs based on polystyrene and polylinoleic acid were prepared using a simple, one step and easily scalable procedure. Therefore, first of all PS-PLina and PS-Plina-PEG graft copolymers were synthesized and characterized. After the PS-PLina and PS-Plina-PEG nanoparticles were produced and modified with poly(L-lysine) (PLL), the siRNA were bound to their surfaces and the nanoparticles optimized and characterized. Optimized NPs formulations in the freeze dried form were assessed with their stability for 1 months of storage at +4 °C. Morphological evaluations were carried out by using SEM and FTIR. Nucleic acid loading and release profiles were investigated. In vitro efficacy of the conjugated with siRNA carrier were investigated using double staining method, cytotocicity test (MTT) and flow cytometry analysis. A549 cancer cell and L929 mice fibloblast cell were employed to evaluate nanocarrier systems.