engleski

Interferences of metal-based nanoparticles with fluorescent probes used for in vitro testings

Accurate in vitro assessment of nanoparticle (NP) cytotoxicity requires a careful selection of the test systems. Commercially available in vitro assays are frequently used to compare the toxicity of different particle types and to generate dose−response data. Due to the unique physicochemical properties of NPs and their unknown reactivity, the possibility of NPs altering the optical properties of fluorometric/colorimetric probes that are used to measure their cyto- and genotoxicity may lead to inaccurate readings. In order to draw valid conclusions from the evaluation of the cellular influences of NPs, we performed comprehensive evaluation on the extent of metal-based NP interferences in common bioassays based on 4 fluorometric probes, i.e. dihydroethidium (DHE) used to monitor superoxide (O2-) production, 2', 7'- dichlorofluorescein diacetate (DCFH-DA) for sensitive and rapid quantitation of oxygen- reactive species in response to oxidative metabolism, monochlorobimane (MCB) for quantifying glutathione levels in cells, and N, N, N-trimethyl-4-(6-phenyl-1, 3, 5-hexatrien-1- yl) phenylammonium-p-toluenesulfonate (TMA-DPH) used as lipid marker for endocytosis. We have selected 3 types of maghemite γ-Fe203 NPs (uncoated, coated with poly-L-lysine and coated with D-manose) and 7 types of silver NPs (coated with citrate, poly-L-lysine, cetyltrimethylammonium, bis(2-ethylhexyl) sulfosuccinate, Tween 20, polyvinylpyrrolidone, and dodecyltrimethylammonium) which differ according to the coating agent applied for surface modifications. Results varied considerably depending on the assay system used. All NPs tested were found to interfere with the DHE and DCFH-DA assay, while no significant effects were observed for TMA-DPH and MCB assays. These findings strongly suggest that each in vitro test system has to be evaluated for each single nanoparticle type to accurately assess the nanoparticle toxicity.

metallic nanoparticles; interferences; fluoro dyes

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