A flow cytometric analysis of macrophage–nanoparticle interactions in vitro: induction of altered Toll-like receptor expression

Abstract

Joyce M Njoroge, Jeffrey J Yourick, Mary Ann PrincipatoDivision of Toxicology, Office of Applied Research and Safety Assessment, Center for Food Safety and Applied Nutrition, US Food and Drug Administration, Laurel, MD 20708, USA Background: Nanoparticles exhibit unique physiochemical characteristics that provide the basis for their utilization. The diversity of potential and actual applications compels a thorough understanding regarding the consequences of their containment within the cellular environment.Purpose: This paper presents a flow cytometric examination of the biologic effects associated with the internalization of citrate-buffered silver (Ag) nanoparticles (NP) by the murine macrophage cell line, RAW264.7.Materials and methods: Cells were cultured with varying concentrations of citrate-buffered Ag nanoparticle and analyzed for changes in cellular volume, fluorescence emissions, and surface receptor expression.Results: Notable changes in side scatter (SSC) signal occurred following the phagocytosis of citrate-buffered Ag NP representative of the 10 nm, 50 nm, and 100 nm particle size by cultured RAW 264.7 cells. A characteristic associated with the internalization of all the citrated Ag NP sizes tested, was the detection of emitted infra-red and near-infrared wavelength emissions. This characteristic consistently permitted the detection of 10 nm, 50 nm, and 100 nm Ag NP particles internalized within the RAW cells by flow cytometry. A functional distinction between monocyte subsets within the RAW 264.7 cell line was noted as Ag NP are taken up by the F4/80+ subset of cells within the culture. Further, the internalization of Ag NP by the cells resulted in an increased cell surface expression of the Toll-like Receptor (TLR) 3, but not TLR4.Conclusion: Taken together, these results implicate the more mature macrophage in the ingestion of Ag NP; and an influence upon at least one of the Toll receptors present in macrophages following exposure to Ag NP. Further, our flow cytometric approach presents a potentially viable detection method for the identification of occult Ag NP material using an indicator cell line. Keywords: silver nanoparticle, flow cytometry, innate immunity, macrophage, Toll-like recepto