CE34 - Contaminants, écosystèmes et santé

Metal nanoparticles contamination of milk: mother-to-young transfer and role of extracellular vesicles – NanoMilk

Metal nanoparticles contamination of milk

Metal nanoparticles are massively released in air, water and soils. Given their suggested toxicity, it is crucial to evaluate their presence in organisms living in ecosystems contaminated by these nanomaterials, characterize the underlying cellular and molecular mechanisms, as well as the impact of exposure on the entire mother-milk-child continuum.

Evaluation of the existence of an actual contamination of milk by metallic nanoparticles, role of mammary extracellular vesicles and impact on the mother-offspring continuum

Metal nanoparticles (NPs) are used in all industrial sectors. Titanium dioxide and iron oxide NPs are widely manufactured for daily life products such as toothpaste, food, medicines and paints but also in agriculture, where they are developed as pesticides or fertilizers. Despite studies revealing their massive release in air, water and soils and their potential toxicity, very few have evaluated their presence in organisms living in contaminated ecosystems. Experimental studies on lactating rodents exposed to some NPs have suggested the transfer of nanoparticles from mother to milk, their transfer to offspring and alteration of offspring organ development and function. Not only NanoMilk evaluates the existence of a contamination of milk by metallic NP in milk from animals living outdoors in more or less exposed settings, it also examines the underlying mechanisms of secretion from mammary cells by focusing on the role of extracellular vesicles (EVs), which are well-known vectors of bioactive molecules that could also carry pollutants. The project also aims at understanding the impact of NP exposure on the overall mother-milk-offspring continuum in vivo.

Cutting-edge approaches including X-ray fluorescence (XRF) and X-ray absorption near edge structure (XANES) under synchrotron illumination combined to RAMAN spectroscopy and inductively coupled plasma mass spectrometry (ICP-MS) are used to identify and characterize the presence of NPs in milk of animals living in regions more or less exposed to known sources of pollution. In vitro, NanoMilk employs biophysical and cell biology approaches to study the mechanisms underlying NP secretion from mammary cells, and in particular the role of EVs, structures known for their role as vectors of bioactive molecules. State-of-the-art approaches including asymmetric flow field fractionation (AF4) to purify mammary and milk EVs are under development, as well as coculture assays for direct visualization of EVs/NPs transfer between cells. NPs will be detected using Raman imaging and confocal fluorescence microscopy coupled with reflectance. The mechanisms of secretion will be analyzed by invalidating the expression of genes of interest and the impact of NPs on recipient cells will be analyzed by transcriptomics. Finally, NanoMilk will study the in vivo transfer of NPs along the mother-milk-offpsring continuum where not only NP biodistribution will be analyzed but also the impact of maternal exposure on milk content, offspring growth and survival.

We have analysed milk samples from different species and locations. By ICP-MS combined to XRF and XANES at SOLEIL synchrotron, metal elements have been detected, quantified and their chemical state and size analyzed. Results show they are metal oxides, accumulated in aggregates of which a part were nanosized. In vitro, to address the role of milk EVs as NP vectors, we have developed approaches to study the NP load of EVs. We used cell line models that we manipulated to express fluorescent EV markers and that we further exposed to NPs. We showed that confocal reflectance/fluorescence microscopy and Raman spectroscopy are suitable to analyse EV metal load. By adapting our previous coculture approach, we show the cotransfer of EVs with a reflectance signal between cells. In vivo, lactating rabbits were chronically and orally exposed to low doses of NP. Milk and milk EVs were sampled on days 2, 5, 9 and 19, and NP biodistribution was assessed in milk and more generally in the mother-offspring continuum along with the impact on milk, EVs and animal health.

From in situ to in vitro and in vivo studies, NanoMilk evaluates and characterizes the presence of NP pollutants in milk and their transfer from mother to offspring, providing crucial insights on their impact on animal health as well as on the basic cellular and molecular mechanisms underlying their secretion by mammary cells and the role of EVs in this process. NanoMilk results may provide new grounds for the use of NPs in industrial sectors, in particular in products used by breastfeeding women or provide recommendations concerning the risks and safety of breastfeeding in exposed areas.

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.

Project coordination

Anne Burtey (Génétique Animale et Biologie Intégrative)

The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.


UiB University of Bergen / Department of Clinical Dentistry
SayFood Paris-Saclay Food and Bioproduct Engineering Research Unit
GABI Génétique Animale et Biologie Intégrative
MSC Laboratoire Matière et Systèmes Complexes

Help of the ANR 493,347 euros
Beginning and duration of the scientific project: December 2021 - 42 Months

Useful links

Explorez notre base de projets financés



ANR makes available its datasets on funded projects, click here to find more.

Sign up for the latest news:
Subscribe to our newsletter