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

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

INTERNATIONAL JOURNAL OF PHARMACEUTICS

ELSEVIER SCIENCE BV

Año: 2009 p. 189 - 200

0378-5173

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 ionicstrength of the medium (lacking or containing 150mM NaCl), the D generation (G4, G5, G6 and G7) andthe 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 strictlydependent on the ionic strength of the media. The strong electrostatic interactions occurring in NaCllacking 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). Notsurprisingly, both the uptake and inhibition of EGFP expression in cell culture, resulted dependent onsiRNA–D size. siRNA–D prepared in NaCl containing mediumwere poorly captured and presented a basalactivity 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 highestinhibition 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 wayof achieving siRNA–D suitable for silencing activity. The simple use of a low ionic strength preparationmedia has been critical to get small siRNA–D that could be captured by cells and in particular, siRNA-G7but not those formed by lower generation D, possessed structural constraints other than size that couldfavor its silencing activity.