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Innate immune memory, the ability of innate cells to react in a more protective way to secondary challenges, is induced by exposure to infectious and other exogeous and endogenous agents. Engineered nanoparticles are particulate exogenous agents that, as such, could trigger an inflammatory reaction in monocytes and macrophages and could therefore be also able to induce innate memory. Here, we have evaluated the capacity of engineered gold nanoparticles (AuNPs) to induce a memory response or to modulate the memory responses induced by microbial agents. Microbial agents used were in soluble vs. particulate form (MDP and the gram-positive bacteria Staphylococcus aureus; β-glucan and the β-glucan-producing fungi C. albicans), and as whole microrganisms that were either killed (S. aureus, C. albicans) or viable (the gram-negative bacteria Helicobacter pylori). The memory response was assessed in vitro, by exposing human primary monocytes from 2-7 individual donors to microbial agents with or without AuNPs (primary response), then resting them for 6 days to allow return to baseline, and eventually challenging them with LPS (secondary memory response). Primary and memory responses were tested as production of the innate/inflammatory cytokine TNFα and other inflammatory and anti-inflammatory factors. While inactive on the response induced by soluble microbial stimuli (muramyl dipeptide -MDP-, β-glucan), AuNPs partially reduced the primary response induced by whole microorganisms. AuNPs were also unable to directly induce a memory response but could modulate stimulus-induced memory in a circumscribed fashion, limited to some agents and some cytokines. Thus, the MDP-induced tolerance in terms of TNFα production was further exacerbated by co-priming with AuNPs, resulting in a less inflammatory memory response. Conversely, the H. pylori-induced tolerance was downregulated by AuNPs only relative to the anti-inflammatory cytokine IL-10, which would lead to an overall more inflammatory memory response. These effects of AuNPs may depend on a differential interaction/association between the reactive particle surfaces and the microbial components and agents, which may lead to a change in the exposure profiles. As a general observation, however, the donor-to-donor variability in memory response profiles and reactivity to AuNPs was substantial, suggesting that innate memory depends on the individual history of exposures.

Engineered gold nanoparticles (AuNPs) find application in several fields related to human activities (i.e., food and cosmetic industry or water purification) including medicine, where they are employed for diagnosis, drug delivery and cancer therapy. As for any material/reagent for human use, the safety of AuNPs needs accurate evaluation. AuNPs are prone to contamination by bacterial endotoxin (lipopolysaccharide, LPS), a potent elicitor of inflammatory responses in mammals. It is therefore important, when assessing AuNP immunosafety and immune-related effects, to discriminate between inflammatory effects intrinsic to the NPs from those caused by an undeliberate and undetected LPS contamination. Detection of LPS contamination in AuNP preparations poses different problems when using the current LPS detection assays, given the general interference of NPs, similar to other particulate agents, with the assay reagents and endpoints. This leads to time-consuming search for optimal assay conditions for every NP batch, with unpredictable results, and to the use in parallel of different assays, each with its weaknesses and unpredictability. Thus, the development of highly sensitive, quantitative and accurate assays able to detect of LPS on AuNPs is very important, in view of their medical applications. Surface-enhanced Raman spectroscopy (SERS) is a label-free, sensitive, chemical-specific, nondestructive and fast technique that can be used to directly obtain molecular fingerprint information and a quantitative analysis of LPS adsorbed on AuNPs. Within this study, we describe the use of SERS for the label-free identification and quantitative evaluation - down to few attograms - of the LPS adsorbed on the surface of 50 nm AuNPs. We thus propose SERS as an efficient tool to detect LPS on the AuNP surface, and as the basis for the development of a new sensitive and specific LPS-detection sensor based on the use of AuNPs and SERS.