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Hydrodynamic chromatography coupled to single-particle ICP-MS to elucidate the fate of silver nanoparticles in human plasma
Marco Roman1, Chiara Rigo1, Ilaria Tocco Trussardi2, Hiram Castillo-Michel3, Carlo Barbante4, Warren R.L. Cairns4
Materials containing silver nanoparticles (AgNPs) are increasingly used in a wide variety of commercial products, as they are innovative carriers of the metal and optimize its antibacterial action. Over the last few years, our research group has demonstrated that when AgNPs are used for the treatment of skin burns, they can be massively released, penetrate deeply into the dermis, are taken up by the cells and probably interact with mitochondria. However, no data are currently available on the subsequent chemical transformations and fate of AgNPs in the human body, which are key factors to establish their possible toxicity.Here we present a new method for the simultaneous determination of dissolved silver and characterization of AgNPs in human serum based on hydrodynamic chromatography (HDC) hyphenated to ICP-quadrupole MS in single particle (SP) detection mode. While HDC separates NPs based on their hydrodynamic diameter and SP provides information on their mass distribution, both techniques have the potential to physically and statistically (respectively) discriminate the signal of NPs from that of dissolved Ag, but this advantage has not yet been exploited. We combined the two techniques and a home-made software based on a new algorithm able to process the raw ICP-MS data (signal intensity vs time), and deconvolute for the first time the signal of dissolved Ag from that of AgNPs when HDC is coupled to SP. With a 15-minutes analysis and a single calculation run, our method provides the chromatogram of dissolved Ag, the concentrations of which can be found by external calibration, as well as the distribution of NPs in terms of hydrodynamic diameter, mass-derived diameter, size-dependent number of detected NPs, total numerical concentration, and size-dependent total mass concentration. A variety of 3D chromatograms can be obtained from the results to achieve a comprehensive characterization of AgNPs. Standards of citrate-stabilized AgNPs suspensions with a diameter of 100 to 10 nm were used for the external calibration of the elution time, signal intensity as function of the size, number of detected NPs as function of the numerical concentration and NPs transport efficiency with a R2>0.98 in all cases. Our method allows to us detect AgNPs in solution with limits of 10-20 nm diameter and at a ppt concentration range (depending on the diameter). It is a powerful approach for the detailed characterization of the dynamics of AgNPs in solution, and has proved to be robust and reproducible also for the direct analysis of diluted human plasma.The method was applied to the analysis of pure AgNPs and AgNPs-coated dressing incubated in whole human plasma. The results are discussed in the context of compatible experiments carried out using synchrotron radiation µXANES (unpublished data). In combination with our recent studies, these results provide new and complete insights into the metallomics of AgNPs in humans in real hospital conditions.
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