Ethylendiamine core PAMAM dendrimers/siRNA complexes as in vitro silencing agents

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 150 mM 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 containing medium (30-130 nm, +25 mV zeta potential vs. several microm-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 medium were 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 50 nM 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.