A new study reveals that the shape, not just the substance, of nanofibers plays a key role in lung toxicity. The research uncovers protein-level clues that could help design safer, smarter nanomaterials.
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A new paper in Nano Today provides information on a critical but often overlooked factor in nanomaterial safety: morphology. Researchers have shown that the shape and size of nanofibers (NFs), specifically their length, diameter, and rigidity, can dramatically influence how lung cells respond to exposure. The team identified molecular markers using advanced proteomic profiling of rat alveolar macrophages, which may one day help predict the toxicity of different fiber types.
This study shifts the conversation of nanotoxicology from chemical composition to structure, showing how fiber shape alone can drive inflammation and cell damage. These findings could be a step towards designing safer nanomaterials and reducing reliance on animal testing.
Nanofiber Morphology
Nanofibers are increasingly used in energy storage, water purification, medicine, and more, thanks to their high surface area and tailored physical properties. But their thin, elongated structure can cause problems, particularly when inhaled. The World Health Organization defines “critical fibers” as those thinner than three microns, longer than five microns, and with aspect ratios over 3:1, dimensions closely associated with diseases like lung fibrosis and mesothelioma.
The Fiber Pathogenicity Paradigm links fiber durability and shape to toxicity, but a major variable factor, rigidity, has often been ignored. This rigidity is a problem when macrophages in the lungs encounter long, stiff fibers; they can't fully engulf them. This failed immune response, known as frustrated phagocytosis, leads to persistent inflammation. The new study dives deep into this cellular battleground, aiming to develop tools for toxicity prediction without animal testing.
Disentangling Shape From Substance
To explore how morphology alone affects toxicity, the researchers tested silicon carbide (SiC) and titanium dioxide (TiO2) in both their intact and mechanically ground forms. These two widely used nanofibers were ground to shorten them and change their aspect ratios, helping to isolate the effects of shape from chemical composition.
They exposed rat alveolar macrophage cells (NR8383) to each type and used a combination of cellular assays and liquid chromatography-tandem mass spectrometry (LC-MS/MS) to track responses. The assays measured key indicators of cell stress: lactate dehydrogenase (cell membrane damage), hydrogen peroxide (oxidative stress), β-glucuronidase (lysosomal leakage), and TNF-α (inflammation).
Scanning electron microscopy confirmed that intact SiC fibers averaged 9.5 microns in length and 156 nanometers in diameter, while TiO2 fibers were shorter and thinner. Grinding dramatically reduced fiber length, allowing full internalization by cells and fewer toxic effects.
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Proteomics Reveal a Toxic Fingerprint
Morphology had a direct impact on toxicology. Intact nanofibers led to membrane piercing, frustrated phagocytosis, and elevated inflammatory signals. Ground fibers, in contrast, were more easily internalized and caused minimal disruption, even at higher concentrations.
Proteomic analysis showed that exposure to intact SiC fibers altered the abundance of over 1,000 proteins after 18 hours. Ground SiC fibers only affected 10. Intact TiO2 fibers altered 266 proteins; their ground counterparts altered five. Principal component analysis clustered ground samples together, further confirming morphology as the dominant factor.
Several inflammation-related proteins, like arginase-1 and interleukin-1 receptor antagonist, were demonstrably upregulated in response to intact fibers. Methionine sulfoxide, a marker of oxidative stress, was elevated particularly in SiC-treated cells. Meanwhile, lysosomal proteins were found in the surrounding medium rather than inside the cells, confirming lysosomal rupture. Markers of necrosis were more prominent than those for programmed cell death.
A Universal Fingerprint for Nanotoxicity
Based on these results, the team proposed a panel of 58 proteins as a morphology-driven toxicity fingerprint. This set covers pathways related to inflammation, lysosomal integrity, metabolism, and cell death. Crucially, the fingerprint appears to be material-independent, meaning it could be used to predict toxicity across different types of nanofibers.
The implications are significant. Rather than relying solely on animal testing, researchers could use in vitro assays backed by this proteomic fingerprint to screen nanomaterials early in the design process. This approach supports the 3R principle (Replacement, Reduction, Refinement) in toxicology and aligns with growing regulatory and industry interest in alternative testing methods.
Toward Safer Nanotechnology
This research offers a new perspective on nanofiber safety. As nanofibers become more common in consumer goods, industrial applications, and even healthcare, understanding their biological interactions is more urgent than ever.
By focusing on molecular-level responses, the study clarifies the mechanisms behind nanofiber toxicity and points the way toward safer material engineering and smarter regulatory frameworks.
The team calls for further validation of their protein fingerprint in primary human cells and in studies mimicking long-term exposure. Expanding the method across various nanomaterials and biological models will help refine its predictive power.
Journal Reference
Stobernack, T., et al. (2025). Predicting the morphology-driven pathogenicity of nanofibers through proteomic profiling. nanotoday, 102812 (65). DOI: 10.1016/j.nantod.2025.102812, https://www.sciencedirect.com/science/article/pii/S1748013225001847
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