siRNA-Dendrimers: effective in vitro silencing agents

A.P. PEREZ; E.L. ROMERO; M.J. MORILLA

Lisboa

Otro; 2nd European Summer School in Nanomedicine; 2009

Introduction. The main obstacle for small interfering RNAs (siRNAs) to be applied as silencing therapy is the difficulty involved in effective in vivo delivery to target organs and cells. In the last years gene silencing therapy via siRNA has been focused on developing methods for delivering siRNAs to cells and for enhancing siRNA stability in vitro and in vivo, with controversial results. Dendrimers, three-dimensional nanopolimers, consist of a core, branches and end groups. Polyamidoamines polymers with an ethylendiamine core or PAMAM dendrimers are one of the most studied types of dendrimers. PAMAM can be used as nano-controlled release system (nano-CRS) because of their nearly perfect monodispersity, strict number of superficial groups and perfectly controlled size in the nanorange. Moreover, they are soluble and structurally stable in aqueous medium. Because of those characteristics and their cationic surface charge, they could be potential nano-CRS for nucleic acids. Materials and Methods. In this work, siRNA-dendrimers complexes (siRNA-D) were prepared by combining different generations of PAMAM dendrimers (G4, G5, G6 and G7) with anti-green fluorescein protein (eGFP) siRNA at different N/P ratio (nitrogen amines in dendrimer/phosphate in siRNA, PAMAM/siRNA, 1/1, 5/1, 10/1 and 20/1), in Tris buffer pH 7,5 in presence or absence of NaCl. Interaction between PAMAM dendrimers and siRNA was determined by measuring the ability of dendrimers to displace the intercalating dye ethidium bromide (EtBr) from siRNA. Analysis of the formation of siRNA-D complexes, as well as their stability in the presence of RNAse, was made evident by detecting retarded migration of siRNA in a polyacrilamide 20% gel electrophoresis. Complex disruption was observed in the presence of SDS (strong ionic detergent). On the other hand, ternary complexes were formed by combining different polymers (colominic acid, dextran or chitosan) with dendrimers and siRNA. Zeta potential and size of siRNA-D complexes were determined using a nanoZ sizer (Malvern). Complexes were also examined by Transmission Electron Microscopy and Atomic Force Microscopy. Cytotoxicity of siRNA-D complexes after 24 h incubation, at different concentrations of siRNA (50 y 100 nM) and different N/P ratios, was determined by LDH and MTT assay, on T98G cells (endocytic fibroblast ) and J774 cells (macrophages). Cellular uptake of naked siRNA and siRNA-D complexes after 5 h incubation, by T98G and J774 cells, was evaluated using Cy3 labeled siRNA. The emission of Cy3 was monitored with a Confocal Laser Microscopy. Finally, the inhibition of eGFP expression of siRNA-D complexes prepared in presence or absence of NaCl was compared to the inhibition of Lipofectamine 2000. Results and discussion. The formation of siRNA-D complexes was demonstrated by displacement of EtBr as well as electrophoretic retard. While naked siRNA was completed degraded, siRNA-D complexes protected in variable degree the siRNA of degradation by RNases. Zeta Potential was positive in all complexes. It was worth noting that, the effect of ionic strength strongly impacted on the size of complexes, in absence of NaCl presented smaller diameter than in presence of the salt. There was not registered a significant difference of sizes between siRNA-D and ternary complexes in absence of NaCl. The viability, in both cellular types, was reduced only when N/P ratio was increased up to 20/1, which involved the higher dendrimer concentration used. On the other hand, it was found that while naked siRNA-Cy3 did not enter the cells, siRNA-Cy3-D complexes were internalized by cells in different degree depending on dendrimer generation and the ionic strength of the complexation media. Complexes prepared in presence of NaCl were not efficient in the inhibition of eGFP expression, probably due to their large size. On the contrary, inhibition of eGFP expression of siRNA-D complexes in absence of NaCl was similar to lipofectamina 2000, about 40 % in J774 cells. Conclusion. siRNA-D complexes prepared in absence of NaCl had size in the range of nanometers and protected siRNA from degradation by RNAse. In addition, these complexes were not cytotoxic in vitro in low concentrations of dendrimers and produced a 40% inhibition of eGFP expression. On the basis of these findings, dendrimers are potential nano-CRS of siRNA for gene silencing therapy. Key references.  Dung, T. H., Kim, J.S., Juliano, R.L. and Yoo, H., 2008. Preparation and evaluation of cholesteryl PAMAM dendrimers as nano delivery agents for antisense oligonucleotides. Colloids and Surfaces A: Physicochemical and Engineering Aspects 313, 273-377.  Eccleston, A. and Eggleston, A. K., 2004. Introduction RNA interference. Nature 431, 337.  Fattal, E. and Bochot, A., 2008. 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