Elettra | TwinMic Nanoparticles in the lungs: a new study reveals the molecular mechanisms at play Tiny particles — whether from natural air pollution or engineered for industrial use — are so small they can penetrate deep into our lungs and further into the bloodstream. These nanoparticles, less than 100 nanometers in size (around 1,000 times thinner than a human hair), can linger in the body and potentially trigger serious health problems, such as inflammation, tissue damage, fibrosis, and even cancer. To better understand the mechanisms, the risks and the adverse outcomes of persistent exposure, we need to examine what happens at the moment these particles come into contact with our lung cells (schematics in Fig. 1 a). But at such a small scale, this is no simple task. It requires a set of highly advanced imaging tools that can reveal both the structure and chemical behavior of these microscopic and nanoscopic interactions. One such tool is Correlated Light and Electron Microscopy (CLEM) — a methodology that combines different types of microscopes to provide a fuller picture of how biological systems behave at the micron and nanoscale. Despite the continuous development of new CLEM approaches in nanomaterial toxicity research to elucidate the interaction at the nano-bio interface and the subsequent impact of nanomaterials and/or elements on biological matter, the full potential of multimodalities and their combinations has not yet been fully exploited, with many opportunities and challenges. In an international study, the team or researchers developed an enhanced CLEM approach and a novel correlative microscopy pipeline, combining multiple state-of-the-art imaging methods (Fig. 1 b) to investigate how lung epithelial cells and immune-activated macrophages respond to a specific type of engineered particle: titanium dioxide nanotubes (TiO2 NTs). These worldwide and one of the most widely used industrial nanomaterials are known to cause inflammation and are considered potentially hazardous to human health and are likely to be carcinogenic for humans, especially in the form of nanotubes with high surface area. Using the integrated and complementary imaging strategy, which was clearly demonstrated in the paper, the team examined the interface between lung epithelial cells and nanoparticles across multiple scales — from entire cells down to individual molecules: • Live-cell imaging techniques: Confocal Laser-Scanning Microscopy (CLSM), Fluorescence Lifetime Imaging (FLIM), and Hyperspectral Fluorescence Imaging (fHSI) to visualize organelles and cellular activity down to molecular level. • Surface imaging tools: Scanning Electron Microscopy (SEM) and Helium Ion Microscopy (HIM) for ultra-detailed, nanoscopic views of the cell surface. • Elemental mapping: Synchrotronbased X-ray Fluorescence (SR µXRF) and Scanning Transmission X-ray Microscopy (STXM), performed at the TwinMic beamline at Elettra Sincrotrone, to detect and map the chemical elements involved in the interaction at submicron lengths. Among these, x-ray fluorescence at Elettra played a key role by revealing how elements like iron (Fe) and phosphorus (P) accumulate near the particles — providing clues about the underlying biological responses. The findings from the study show that lung epithelial cells respond to titanium dioxide nanotubes in complex and layered ways with different inflammatory and anti-inflammatory outcomes. The study reveals new insights into the DNA binding to nanoparticles and their transport in apoptotic cells, suggesting a possible downstream inflammation and disturbing intracellular balance that might cause genotoxic effects. Immobilization of nanomaterial and biological matter (nano-bio) clusters on the cell surface intertwined with fibrous structures, could impact normal clearance of apoptotic cells and hence inducing pathological condition. The observed local accumulation of sodium (Na+) and iron (Fe2+) ions accompanied with distinct morphological features on nanoscale (Fig. 1 c) indicated on either cell inflammation or cellprotective mechanism. The formed nano-bio clusters on the cell surface, composed of essential minerals and intrinsic biomolecules may further act as biological "triggers," setting off immune responses. This is revealed by the early formation of fibrin-like fibrous networks (Fig. 1 d), which are a sign of acute cellular reaction through the local pro-inflammatory stimuli accompanied by plausible secretion of fibrinogen and TNFα cytokines. In this discovery, scientists find one of the key plausible molecular initiating events that trigger inflammation in cells when exposed to such nanomaterial. Despite the technical challenges of combining so many advanced methods to preserve the nanoscale morphology at the interface — including both live-cell and fixed-sample techniques — the integrated data provided to the community a remarkably detailed view of how nanomaterials interact with lung cells and tissue. This work demonstrates that correlative microscopy — uniting light, electron, and ion-based imaging — is a powerful tool for future studies of nanoparticle toxicity. By seeing what happens at the molecular level, we move closer to understanding how to protect human health in an increasingly nano-engineered world. As exposure to airborne nanoparticles continues to rise in urban and industrial areas, the study underscores the need for more stringent air quality monitoring and further research into the long-term effects of nanoparticle inhalation. Elettra Highlights 2024–2025 66
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