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  • Influence of the internalization pathway on the efficacy of siRNA delivery by cationic fluorescent nanodiamonds in the Ewing sarcoma cell model.

Influence of the internalization pathway on the efficacy of siRNA delivery by cationic fluorescent nanodiamonds in the Ewing sarcoma cell model.

PloS one (2013-01-04)
Anna Alhaddad, Catherine Durieu, Géraldine Dantelle, Eric Le Cam, Claude Malvy, François Treussart, Jean-Rémi Bertrand
ABSTRACT

Small interfering RNAs (siRNAs) are powerful tools commonly used for the specific inhibition of gene expression. However, vectorization is required to facilitate cell penetration and to prevent siRNA degradation by nucleases. We have shown that diamond nanocrystals coated with cationic polymer can be used to carry siRNAs into Ewing sarcoma cells, in which they remain traceable over long periods, due to their intrinsic stable fluorescence. We tested two cationic polymers, polyallylamine and polyethylenimine. The release of siRNA, accompanied by Ewing sarcoma EWS-Fli1 oncogene silencing, was observed only with polyethylenimine. We investigated cell penetration and found that the underlying mechanisms accounted for these differences in behavior. Using drugs selectively inhibiting particular pathways and a combination of fluorescence and electronic microscopy, we showed that siRNA gene silencing occurred only if the siRNA:cationic nanodiamond complex followed the macropinocytosis route. These results have potential implications for the design of efficient drug-delivery vectors.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Polyethylenimine, branched, average Mw ~800 by LS, average Mn ~600 by GPC
Sigma-Aldrich
Poly(allylamine hydrochloride), average Mw 50,000
Sigma-Aldrich
Poly(allylamine hydrochloride), average Mw ~17,500 (GPC vs. PEG std.)
Sigma-Aldrich
Poly(allylamine) solution, 20 wt. % in H2O
Sigma-Aldrich
Poly(allylamine) solution, average Mw ~65,000, 10 wt. % in H2O