*3.2.2* In vitro *studies*

Many researchers have demonstrated the cytotoxic effects of AgNPs *in vitro*, however there is still a lack of consistent and reliable data amongst publications. For example, in a recent review, Kim and Ryu (2013) attributed oxidative stress, apoptosis and genotoxicity to be the main *in vitro* outcome of AgNP exposure [120]. Later, Gliga *et al*. identified a major drawback of this review, highlighting that the AgNPs were different in each study, *i.e.* synthesised by different techniques, of varying size distributions and coatings, tested on different cell lines under different cell culture conditions and often without the use of appropriate controls [121]. Additionally, Hackenberg *et al*. reported cytotoxicity of human mesenchymal stem cells at a concentration of 10 μg ml−1 AgNPs (˂50 nm), whereas Samberg *et al*. reported no toxicity of progenitor human adipose-derived stem cells at concentrations up to 100 μg ml−1 AgNPs (10–20 nm) [122, 123]. To determine the effect of size on cytotoxicity, Liu *et al*. compared the cytotoxicity of AgNPs ranging in size from 5 to 50 nm on four different cell lines (A549, HepG2, MCF-7 and CGC-7901) and reported that 5 nm AgNPs were most toxic [124]. On the contrary, Kim *et al*. reported the enhanced release of lactate dehydrogenase (LDH) and reduced cell viability in the presence of 100 nm sized AgNPs when compared to smaller AgNPs (10–50 nm) [125]. It can be noted that the variation in parameters in these studies makes it difficult to observe trends and come to accurate assumptions. To achieve some consensus in this regard, Gliga *et al*. studied the cytotoxic effect of varying sized AgNPs capped by various agents on the normal bronchial epithelial cell line (BEAS-2B). They reported that 10 nm sized AgNPs induced cytotoxicity

irrespective of the capping agents, at high concentrations (20–50 μg ml−1), whilst larger AgNPs did not display significant cytotoxic effects at all tested concentrations. The group additionally reported that at non-cytotoxic concentrations (10 μg ml−1), significant DNA damage was observed for all AgNPs independent of size and coating. In contrast, panda *et al*. reported no genotoxicity of AgNPs capped with protein at 20–80 μg ml−1 for 24–55 nm sized particles [126].

Overall, it is difficult to establish the cytotoxic effect of AgNPs due to the differences in nanoparticle synthetic methods, their various sizes and capping agents and lastly the diverse evaluation tests used to determine toxicity. In fact, by using different organisms and/or culture cells there is no conclusive evaluation of AgNP toxicity [127]. However, bearing in mind the results presented in this review, it can be tentatively suggested that smaller sized AgNPs are more cytotoxic than larger sized particles at higher concentrations.
