CONICET | Buscador de Institutos y Recursos Humanos

We have screened the formation of complexes between ethylendiamine (EDA) core polyamidoamine (PAMAM) dendrimers (D) and a short interfering RNA (siRNA) as a function of three variables: the ionic strength of the medium (lacking or containing 150mM NaCl), the D generation (G4, G5, G6 and G7) and the N/P ratio (nitrogen amines in D/phosphate in siRNA). It was observed that all D formed complexes with siRNA, being the size of the complexes strictly dependent on the ionic strength of the media. The strong electrostatic interactions occurring in NaCl lacking medium made siRNA?D complexes (siRNA?D) smaller than those obtained in NaCl containingmedium (30?130 nm, +25mV zeta potential vs. several m-800 nm, 0 zeta potential, respectively). Not surprisingly, both the uptake and inhibition of EGFP expression in cell culture, resulted dependent on siRNA?D size. siRNA?D prepared in NaCl containing mediumwere poorly captured and presented a basal activity on phagocytic (J-774-EGFP) cells, being inactive on non-phagocytic cells (T98G-EGFP). However, the smaller siRNA?D prepared in NaCl lacking medium were massively captured, exhibiting the highest inhibition of EGFP expression at 50nM siRNA (non-cytotoxic concentration). Remarkably, siRNA-G7 produced the highest inhibition of EGFP expression both in T98G-EGFP (35%) and J-774-EGFP (45%) cells, in spite of inducing a lower protection of siRNA against RNase A degradation. Taken together, our results showed that modifying the chemical structure of D is not the only way of achieving siRNA?D suitable for silencing activity. The simple use of a low ionic strength preparation media has been critical to get small siRNA?D that could be captured by cells and in particular, siRNA-G7 but not those formed by lower generation D, possessed structural constraints other than size that could favor its silencing activity

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