Detection and characterization of silver nanoparticles and dissolved species of silver in culture medium and cells by AsFlFFF-UV-Vis-ICPMS: application to nanotoxicity tests

Abstract

A methodology based on Asymmetric Flow Field-Flow Fractionation (AsFlFFF) coupled with UV-Vis absorption spectrometry and ICP mass spectrometry (ICPMS) has been developed and applied to the study of silver nanoparticles (AgNPs) and dissolved species of silver in culture media and cells used in cytotoxicity tests. The effect of a nano-silver based product (protein stabilized silver nanoparticles ca. 15 nm average diameter) on human hepatoma (HepG2) cell viability has been studied. UV-Vis absorption spectrometry provided information about the nature (organic vs. nanoparticle) of the eluted species, whereas the silver was monitored by ICPMS. A shift towards larger hydrodynamic diameters was observed in the AgNPs after a 24 hour incubation period in the culture medium, which suggests a "protein corona" effect. Silver(I) associated with proteins present in the culture medium has also been detected, as a consequence of the oxidation process experimented by the AgNPs. However, the Ag(I) released into the culture medium did not justify the toxicity levels observed. AgNPs associated with the cultured HepG2 cells were also identified by AsFlFFF, after applying a solubilisation process based on the use of tetramethylammonium hydroxide (TMAH) and Triton X-100. These results have been confirmed by transmission electronic microscopy (TEM) analysis of the fractions collected from the AsFlFFF. The effect of AgNPs on HepG2 cells has been compared to that caused by silver(I) as AgNO3 under the same conditions. The determination of the total content of silver in the cells confirms that a much larger mass of silver as AgNPs with respect to AgNO3 (16 to 1) is needed to observe a similar toxicity.

Exploring influences on the cellular uptake of medium-sized silver nanoparticles into THP-1 cells

“…Within this study, the abundance of ionic Ag in the original nanoparticle suspensions or in mixed culture media was not further studied. It is known that especially for bio-available Ag ENPs and if these ENPs are additionally of smaller sizes (b 20 nm), this might be of influence [13]. Obtained fractograms showed one resp.…”

“…A large range of human cell lines is available for in vitro testing. Human liver cells like HepG2 and colon Caco2 cells are well studied Microchemical Journal 120 (2015) 45-50 cell lines in relation to nanosilver [12,13]. As ENPs tend to end up in phagocytizing cells, a human acute monocytic leukemia cell line (THP-1) can be used for exposure testing.…”

“…Commonly, Ag ENPs in less complex matrices like aqueous matrices can be separated by AF4 [18,19]. To the best of our knowledge, only one publication is dealing about the analytical method of nanosilver in relation to cell line; in this case smaller (15 nm) ENPs and Ag(I) were studied with HepG2 cells by AF4-ICPMS [13].…”

Separation and Determination of Silver Nanoparticles

“…The AF4 fractogram revealed that the size of AgNPs increased from 17 nm to 32 nm after AgNPs were incubated in the culture medium for 24 h. UV-vis spectra showed no differences at the absorbance maximum wavelength before and after the incubation, indicating no obvious aggregation occurred. The increment in the size distribution probably due to the"protein corona" effect [25]. Compare with other techniques like TEM, AF4 can get similar results but is much faster and simpler [21,23].…”

“…However, KOH and NaOH may induce the aggregation of AgNPs [28], and TMAH, which does not change the integrity of AgNPs in short times, appears more suitable for digesting complex samples. AgNPs associated with the cultured HepG2 cells were readily identified by AF4-ICP-MS after a digestion process based on TMAH and Triton X-100 [25]. FFF can also find their way in analyzing AgNPs in consumer products [20], dietary supplements [20], and untreated wastewater [29], showing promises to study the environmental fate of AgNPs in the complex systems.…”