Sustained delivery of siRNAs targeting viral infection by cell-degradable multilayered polyelectrolyte films.

Gene silencing by RNA interference (RNAi) has been shown to represent a recently discovered approach for the treatment of human diseases, including viral infection. A major limitation for the success of therapeutic strategies based on RNAi has been the delivery and shortlasting action of synthetic RNA. Multilayered polyelectrolyte films (MPFs), consisting of alternate layer-by-layer deposition of polycations and polyanions, have been shown to represent an original approach for the efficient delivery of DNA and proteins to target cells. Using hepatitis C virus infection (HCV) as a model, we demonstrate that siRNAs targeting the viral genome are efficiently delivered by MPFs. This delivery method resulted in a marked, dose-dependent, specific, and sustained inhibition of HCV replication and infection in hepatocyte-derived cells. Comparative analysis demonstrated that delivery of siRNAs by MPFs was more sustained and durable than siRNA delivery by standard methods, including electroporation or liposomes. The antiviral effect of siRNA-MPFs was reversed by a hyaluronidase inhibitor, suggesting that active degradation of MPFs by cellular enzymes is required for siRNA delivery. In conclusion, our results demonstrate that cell-degradable MPFs represent an efficient and simple approach for sustained siRNA delivery targeting viral infection. Moreover, this MPF-based delivery system may represent a promising previously undescribed perspective for the use of RNAi as a therapeutic strategy for human diseases.

Polyelectrolyte multilayer-mediated gene delivery for semaphorin signaling pathway control.

The capability of multilayered polyelectrolyte films (MPFs) to control the sequential expression of two genes encoding cell receptors involved in a common cell signalling activity is shown, while achieving a fully functional signal transduction. As a functional model system representative of a cell signalling process that proceeds in a top-down manner, cell collapse induced by semaphorin 3A (Sema3A) was chosen as the target. Polyelectrolyte multilayers were sequentially functionalized with two plasmids encoding Neuropilin-1 (NRP-1) and Plexin-A1 (Plx-A1), respectively, acting as co-receptors for Sema3A. By using hyaluronan and chitosan as structural components for the incorporation of plasmid DNA layers onto precursor films made of poly-allylamine hydrochloride and poly-sodium-4-styrenesulfonate, the polyelectrolyte system is established; this systems is capable of delivering both plasmids to Cos-1 cells in a manner that permits control over the timing and the respective order in which the two plasmid DNA constructs are expressed. Importantly, it was observed that, following Sema3A stimulation, COS-1 cells co-expressing Plx-A1 and NRP-1 display a collapse phenotype, which is determined by the multilayer build-up scheme, and that the expression products of both transgenes embedded in MPFs are temporally functional over several days while acting their role of co-receptors for Sema3A.

Filamentous condensation of DNA induced by pegylated poly-L-lysine and transfection efficiency.

In this paper we propose a detailed analysis of structural and morphological properties of two poly-L-lysine (PLL)-based transfection formulations, PLL/DNA and pegylated PLL (PLL-g-PEG)/DNA, by means of atomic force microscopy (AFM) and transmission electron microscopy (TEM). Comparing PLL-g-PEG/DNA with PLL/DNA polyplexes, we demonstrate that, due to the presence of PEG, the particles differ not only in size, shape, and crystalline structure, but also in transfection efficiency. While PLL condensates DNA in large agglomerates, PLL grafted with polyethylene glycol 2000 can condensate DNA in long filaments with diameters of some nanometers (6-20 nm). These structures are dependent on the grafting ratio and are more efficient than compacted ones, showing that DNA uptake and processing by cell is directly related to physicochemical properties of the polyplexes.