Determinants of Silver Nanoparticle Toxicity

Abstract

Silver nanoparticles (AgNPs) containing consumer products haveincreasingly emerged in the market because of their potential antibacterialproperty, which might result in increased human exposure and environmentalcontamination. AgNPs are toxic to mammalian and other cells but thedeterminants of this toxicity remain to be fully characterised and the potentialimpact of DNA repair systems has been poorly explored. This study, therefore,examined to what extent the size and shape of synthesised AgNPs determinedAgNP toxicity in DNA repair proficient and deficient (8-oxoguanine DNAglycosylase; WT and OGG1-/-, respectively) mouse embryonic fibroblasts (MEFs)as well as a well-known human cell line used in the toxicity testing, HepG2 cells.Citrate-stabilised spherical- and triangular-shaped AgNPs (S-AgNPs andT-AgNPs, respectively) were synthesised chemically from AgNO3 usingcombinations of NaBH4 and sodium citrate as a reducing and stabilising agent,respectively, and purified by dialysis. Three different sized S-AgNPs wereprepared with diameters of 7.6 ± 1.2, 14.3 ± 4.2, and 52.5 ± 17.9 nm as measuredusing transmission electron microscope (TEM), and their zeta potentials were-36.1±2.7, -39.5±2.7 and -36.7±4.1 mV, respectively. T-AgNPs had an edgelength and thickness of 71.4 ± 11.1 nm and 5.7 ± 0.8 nm, respectively. The sizeand zeta potential of the purified AgNPs were constant in distilled water for atleast 6 months. The uptake of both S- and T-AgNPs by cells resulted in a timeanddose-dependent increase in the number of cellular AgNPs and the amount ofAg+ released intracellularly. These increases were associated with a decrease incell viability (as measured using the MTT assay) and cell survival (the clonogenicassay), and an induction in ROS generation (the DCF assay) and DNA damage(the alkaline Comet assay) for all three cell lines.AgNPs were observed in cells using TEM, suggesting the uptake ofAgNPs via an endocytosis pathway. Results suggested that an increase in cellularAgNP level and intracellular released Ag+ content were associated with a timeanddose-dependent toxicity. Interestingly, cellular AgNP level and intracellularreleased Ag+ content might play an important role in size-dependent AgNPtoxicity, in which exposure to the smaller S-AgNP sizes (7nm and 14nm) resultedin higher levels of both cellular AgNPs and Ag+ released intracellularly, and thento increased toxicity when compared with the larger S-AgNP size (50nm).Moreover, different shaped AgNPs might induce toxicity by differentmechanisms: ROS-mediated toxicity might be induced by both 70nm T-AgNPsand 50nm S-AgNPs and 70nm T-AgNPs might also induce cell membranedamage. AgNP-induced toxicity was different in different cell lines with HepG2cells being more sensitive to AgNPs particularly using the clonogenic assay, andthis toxicity was associated with higher DNA damage observed in HepG2 cellsafter 24 h. OGG1-/- MEFs were more sensitive to intracellular released Ag+,leading to higher ROS formation and DNA damage in OGG1-/- MEFs than thatobserved in WT MEFs. In summary, this study strongly suggests that AgNPsinduce toxicity via a Trojan-horse type mechanism, and not only Ag+ releasedintracellularly but also cellular AgNPs take part in this toxicity, and willeventually result in the biological responses of the cells.

Date of Award	1 Aug 2016
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Andrew Povey (Supervisor) & Paul O'Brien (Supervisor)