Dendrimers as nano-controlled release system of siRNA

ANA PAULA PEREZ; MARIA JOSE MORILLA; EDER LILIA ROMERO

Florianópolis

Congreso; V Encontro da SBPMat; 2006

Sociedade Brasileira de Pesquisas em Materiais

The major 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 utilizing 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.1-2 Dendrimers are three-dimensional nanopolimers that 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. In particular, PAMAM can be used as nano-CRS because of their nearly perfect monodispersity, strict number of superficial groups and perfectly controlled size in the nanorange. Moreover, they are extremely soluble and structurally stable in aqueous medium. Because of those characteristics and their cationic surface charge, they could be potential nano-CRS for nucleic acids3-4 In this work, dendrimers-siRNA complexes were prepared by combining generation 4 (G4) PAMAM dendrimers with anti-green fluorescein protein (GFP) siRNA at different +/- charge ratios, namely: G4/siRNA, 1/1, 5/1, 10/1 and 20/1, in Tris buffer pH 7,5. Analysis of the formation of complexes was made evident by detecting retarded migration of siRNA that started for dendrimer siRNA complexes at charge ratio 5/1, in a polyacrilamide 20% gel electrophoresis. Interaction between PAMAM dendrimers and siRNA was also determined by measuring the ability of dendrimers to displace the intercalating dye ethidium bromide from siRNA (Fig 1). The major displacement was seen also for complexes of charge ratio 5/1, that produced a 15% dye displacement. The complex disruption was observed in the presence of SDS (strong ionic detergent). Size of those complexes was in the range of nanometers as determinated by dinamic light scattering. Cytotoxicity of the complexes after 24 hs incubation, at different concentrations of siRNA and different charge ratios, was determined by methyl thiazolyl tetrazolium (MTT) assay, on Vero cells (endocytic fibroblast ) and J774 cells (macrophages). Viability of both cell lines, in the presence of 50 and 100 nM of siRNA, was reduced to a maximum of 45% when charge ratio was increased, which involved increased dendrimer concentration up to 1,20 µM. Meanwhile, at 200 nM of siRNA, viability was reduced to 55% only in J774 cells at higher charge ratio, whereas viability for the rest of charge ratios was reduced to 30%. Finally, the efficiency of gene silencing was analyzed for G4-siRNA complexes, that showed to be more efficient for silencing GFP than the commercial reagent Lipofectamine 2000. In conclusion, dendrimers are potential nano-controlled release system of siRNA for gene silencing therapy.