Human primary monocytes as model for evaluating the effects and safety of engineered nanoparticles.

Monocytes/macrophages have a central role in initiating defensive responses to external or endogenous challenges. Thus, monocyte reactivity is paradigmatic of the immediate host reaction to engineered nanoparticles (NPs), and exploitable both for evaluating their safety and for identifying possible use in nanomedicine, in particular vaccination. The interaction of NPs with human monocytes/macrophages can be modelled in vitro in a series of relevant and representative systems. First, the direct toxicity/capacity to induce inflammation can be assessed by exposing blood-derived human monocytes to NPs and measuring different parameters of cell activation. In prototypical assays, the expression/production of the inflammatory cytokine IL-1? and its antagonist IL-1Ra served as biomarkers of monocyte activation in response to engineered Au NPs (diameter 10 nm). In this context, we found that the presence of LPS, a ubiquitous bioactive contaminant that can easily adsorb on the NP surface, triggers inflammatory monocyte activation, while clean NPs do not. This finding suggests that we need to measure the possible LPS contamination of NPs, to avoid mistaking LPS-induced activation/toxicity for NP-induced effects. We have optimised the assay conditions of commercial LAL tests, so as to adapt them to LPS detection in NP suspensions. The use of such optimised LPS detection procedures has allowed us to design NP synthesis protocols that minimise LPS contamination. We have therefore found that LPS-free Au or FexOy NPs are unable to trigger toxic/inflammatory effects in human isolated monocytes, as well as in human peripheral blood mononuclear cells, and even in whole human blood. As a second level of investigation, we have examined the possibility that NPs, even in the absence of direct toxicity, may interfere with the normal development of the innate defensive response. In such a situation, the presence of NPs may cause inadequate immunity and eventual pathological consequences. To test the possible interference of NPs with the normal development of the innate/inflammatory response, we have exposed human monocytes to LPS-free Au NPs (1.28 ?g/ml, 0.16 cm2/million cells) within an in vitro model, which reproduces the full course of the human inflammatory response. We modelled the course of the physiological inflammatory reaction, from initiation and development to eventual resolution, by exposing CD14+ human blood monocytes to a sequence of different microenvironmental conditions (CCL2, LPS, TNF?, IFN-?, 37°C vs. 39°C). Gene expression and protein production were analysed at different time points for a series of inflammatory factors (IL-1?, IL-1?, IL-1RI, IL-6, IL-18, IL-36?, CCL5) and anti-inflammatory molecules (IL-1Ra, IL-1RII, IL-18BP). Results show that Au NPs are unable to affect in a significant fashion the course of the in vitro physiological inflammatory reaction, suggesting that these NPs do not alter the innate defensive ability of the human body. We conclude that the use of a valid and representative in vitro model of human inflammation may allow us to realistically investigating the effects of NPs on inflammatory/innate immune responses, thereby being useful to predicting the immunological risk posed by engineered nanomaterials as well as for testing the possible immunostimulating effects of NP-based adjuvants.