INVESTIGADORES
MORILLA Maria Jose
artículos
Título:
Complexes between ethylendiamine core PAMAM dendrimers and siRNA revisited as in vitro silencing agents
Autor/es:
ANA PAULA PEREZ; EDER L ROMERO; MARIA JOSE MORILLA
Revista:
INTERNATIONAL JOURNAL OF PHARMACEUTICS
Editorial:
Elsevier
Referencias:
Lugar: Amsterdam; Año: 2009 vol. 380 p. 189 - 200
ISSN:
0378-5173
Resumen:
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 siRNAD complexes (siRNAD) smaller than those obtained in NaCl containing
medium (30130 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
siRNAD size. siRNAD 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 siRNAD 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 siRNAD suitable for silencing activity. The simple use of a low ionic strength preparation
media has been critical to get small siRNAD 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.