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.
Perez
A P
AP
Departamento de Ciencia y Tecnología, Universidad Nacional de Quilmes, Buenos Aires, Argentina.
Romero
E L
EL
Morilla
M J
MJ
D002461:Cell Line, Transformed; D002470:Cell Survival / Q000187:drug effects; D050091:Dendrimers / Q000138:chemical synthesis* / Q000737:chemistry; D004337:Drug Carriers / Q000138:chemical synthesis* / Q000737:chemistry / Q000493:pharmacokinetics; D004339:Drug Compounding / Q000379:methods*; D004355:Drug Stability; D005029:Ethylenediamines / Q000737:chemistry*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D009994:Osmolar Concentration; D011073:Polyamines / Q000737:chemistry*; D034622:RNA Interference / Q000187:drug effects*; D034741:RNA, Small Interfering / Q000737:chemistry / Q000493:pharmacokinetics / Q000494:pharmacology
D050091:Dendrimers; D004337:Drug Carriers; D005029:Ethylenediamines; C104700:PAMAM Starburst; D011073:Polyamines; D034741:RNA, Small Interfering; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins