High-resolution synchrotron techniques detect labeled nanoplastics in macrophages

In 2019 the World Health Organization classified air pollution as one of the greatest environmental risks humans are exposed to via different routes (ingestion, inhalation, skin contact), posing a potential risk for health. Among the main alarming pollutants, a significant role is being played by micro- and nano-plastics (MNPs), which are defined as solid particles composed by a mixture of polymers and functional additives, typically smaller than 5 millimeter in diameter (microplastics, MPs) or than 1 micrometer (nanoplastics, NPs).

MNPs can occur in different shapes, such as beads, fibers, fragments, spheroids and are classified into two types: primary MPs, intentionally synthesized for commercial purposes and industrial aspects (i.e., microbeads in personal care products); secondary MPs, resulting from the fragmentation of larger plastics by natural forces in the environment at even smaller sizes (NPs). Thus, plastic doesn’t disappear, it just becomes smaller, and for this reason MNPs can be found in the food we eat and in the water we drink; the average person could be eating over 100.000 MPs a year, that’s like eating a credit card a week.

Although the NPs uptake has been clearly demonstrated for animal models, their epidemiological data in biological matrix is still missing due to the lack of advanced techniques capable to visualize, track, and characterize them. This is due to their small size, low concentration, and mostly co-presence, in biological specimens, of macromolecules (proteins, lipids) that, being composed by similar chemical elements of polymers (carbon, hydrogen), may interfere with the analytical detection when performing for instance elemental analysis. For this purpose, we propose the use of labeled CdSe Quantum Dots (CdSe QDs)-polypropylene (PP) and -polyvinyl chloride (PVC) NPs as a novel model to investigate the NPs-cell interactions. In fact, since nanometric size particles are more prone to enter cells and interact with intracellular organelles, and even with macromolecules present within the cytoplasm, the investigation of NPs accumulation in cells and related toxicological outcomes becomes crucial. To this aim, the research exploited the use of THP-1 human cell line, a model for macrophages in charge of phagocyting non-self-agents and being particularly active in inflammation and infection pathways, and thus the best candidate to phagocyte nanoparticulate material.

For the first time, the nanoplastics tracking across cells and related metabolism impairments have been achieved by a combination of conventional and synchrotron-based analytical techniques. Scanning Transmission and low energy X-ray fluorescence microscopy measurements were carried out at the TwinMic beamline of Elettra and were complemented by Fourier Transform Infrared spectromicroscopy at the Chemical and Life Science branch of SISSI beamline (SISSI-Bio).

The label of CdSe QDs is characterised by a red emitting property (Fig. 1a), which under optical fluorescence microscopy helped in the selection of the cells of interest. STXM imaging allowed to compare the morphology of NPs-treated macrophages with that of the untreated control cells, highlighting the formation of bright vesicles mainly located at the plasma membrane or perinuclearly (Fig. 1b and Fig. 1c). The formation of LDs is associated with the activation of phagocytosis of macrophages internalizing NPs and cellular stress-related response. In addition, the simultaneous detection of low energy X-Ray fluorescence emission provided the visualization of NPs at sub-cellular spatial resolution, by tracking one of two elements labelling NPs (Selenium) which appeared distributed mostly at cytoplasmatic level (Fig. 1d).

Figure 1: Light fluorescence image a), absorption image b) and phase contrast c) of a PVC-NPs exposed macrophage M1 together with the corresponding Se XRF map acquired at 450 nm spatial resolution d). Scale bar is 10 µm.

The samples were also analyzed at SISSI-Bio beamline with Fourier Transform Infrared spectromicroscopy (micro-FTIR), able to evaluate the chemical modifications happening at the macromolecular level when macrophages are in close contact with NPs. The results showed that NPs mostly affect the lipid response. The FTIR fingerprints clearly revealed the effects of PP- and PVC-NPs in macrophages, causing an increased production of Triacylglycerols (TAGs), mainly contained in lipid droplets (LDs), or free fatty acids (FFAs), respectively and showing a preferred localization of TAGs at the cellular outer edge (Figure 2), as confirmed by Scanning Transmission X-ray Microscopy (STXM). In addition, a more in-depth analysis of FTIR absorbance spectra, evidenced subtler differences induced by the different treatments according to the chemical composition of the plastics, such as the structural changes in the protein components.

Figure 2: FTIR images acquired of PP-NPs exposed M1 macrophagic cells. Optical image of a sub-area of the samples a); chemical image of the free fatty acids (FFAs) b); chemical image of the distribution of triacyclglycerols (TAGs) c); RGB composite with a standard scale to better observe the variations of the relative intensities of the three signals d). The scale bar is 50 μm.

Considering the novelty of the reported results, this work will certainly contribute to increase the knowledge of the nanoplastics’ interactions with the immune system and the consequent toxicological impact on long-term exposed humans exploiting the powerful synchrotron-based techniques.

This research was conducted by the following research team:

Federica Zingaro¹, Alessandra Gianoncelli², Giacomo Ceccone³, Giovanni Birarda², Domenico Cassano³, Rita La Spina³, Chiara Agostinis⁴, Valentina Bonanni², Giuseppe Ricci^4,5 and Lorella Pascolo^4
1 Physics Department, University of Trieste, Trieste, Italy
² Elettra - Sincrotrone Trieste S.C.p.A., Trieste, Italy
³ European Commission, Joint Research Centre (JRC), Ispra, Italy
⁴ Institute for Maternal and Child Health, IRCCS Burlo Garofolo, Trieste, Italy
⁵ Department of Medical, Surgical and Health Science, University of Trieste, Trieste, Italy

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