The release of metal nanoparticles (NPs) in ecosystems is constantly growing. NPs are massively manufactured for their useful properties such as UV-filter, anti-bacterial agents, remediation agent, fertilizers, or pesticides. Sampling from lakes, rivers, seas and even tap water indicated the presence of NPs, as well as in soils and in the air of densely urbanized and industrial areas. Whether animals living in these ecosystems are contaminated by NPs remains unknown.With iron oxide (Fe2O3) NPs, titanium dioxide (TiO2)-NPs is one of the most manufactured nanomaterial. Very emergent literature suggests an actual contamination in humans by TiO2-NPs in placental tissue, newborn stools, blood and colon tumor tissues. Whether animals, especially those raised for human consumption may be contaminated is unknown. Scarce studies in lactating rodents exposed to some NPs suggested the passage of NPs in milk and in offsprings whose development and survival were affected. In this context, NANOMILK proposes to evaluate the existence of an actual contamination by NPs in milk (WP1). Using combination of cutting-edge biophysical approaches, we will analyse the presence of NPs in milk from animals raised in urban farms with a known metal pollution in soils and vegetables. This will be compared to milk from animals raised in unpolluted arctic farms in Norway. To understand the mechanisms by which NPs may be released in milk, we will run a comprehensive analysis to characterize the molecular mechanisms underlying the secretion and intercellular transfer of NPs from mammary cells (WP2). To that end, we will investigate NP secretion in extracellular vesicles (EVs) which are an ensemble of membrane-limited carriers playing key roles in cell-to-cell communication and in physio-pathological processes such as immune response or cancer progression. Very recently, EVs have been detected in milk (milk-EVs). Secreted by mammary cells, they may transport information to offspring and influence their immunity and development. Whether milk-EVs secreted by mammary cells may also transport NPs is unknown but partners of NANOMILK have previously shown that Fe2O3-NPs and TiO2-NPs were transferred between non-mammary cells in a process involving EVs. Thorough analysis of the metal and protein content of EVs deriving from NPs-exposed mammary cells will be combined to transfer assays, indirect or direct in co-culture, coupled to silencing of genes involved in cell-cell communication. Furthermore the impact of NPs transferred from mammary cells to recipient cells will be evaluated on genome-wide expression profiles by transcriptomics in recipient cells. Finally, we propose to investigate in vivo the route of NPs within the entire mother-to-offpsring continuum (WP3). After a dose escalation study, we will analyse the behavior of NPs administrated by drinking water in lactating female rabbits. Biodistribution of NPs will be analysed in mother and offspring tissues and in milk-EVs collected at different lactation time. In addition to monitoring the effect of NPs on offspring growth and survival, we will also analyse their effect on milk-EV proteome to uncover potential effect on EVs biogenesis, origin and abundance. From on-field, to in vitro and to in vivo, NANOMILK investigates the spreading of contaminant NPs from mother-to-progeny. NANOMILK may reveal an actual contamination in milk samples from urban farms, influencing this agricultural trend of producing locally when location is polluted. Expected results will generate knowledge on mother-to-offspring communication by milk, and how this can be highjacked by pollutants. NANOMILK may provide new grounds for the use of NPs, for dairy industry, for nanoagriculture and breastfeeding in polluted areas.