Pulmonary toxicity of graphene based materials is governed by size-dependant biodegradation and clearance in alveolar phagocytes

Background: Graphene based materials (GBMs) have drawn a lot of interest in the material community thanks to unique physicochemical properties. Owing to a high surface area to volume ratio, they possess a good mechanical resistance. Additionally, this specific feature confers a huge advantage when designing drug delivery systems, due to a potential increased binding of therapeutic molecules. Nevertheless, their biosafety in lungs has yet to be confirmed in vivo. Their high aspect ratio could remain a source of danger due to potential frustrated phagocytosis, as previously highlighted with carbon nanotubes. Herein, we investigated the biological impact as well as material clearance and degradation of several GBMs after pulmonary administration.

Methods: Mice were exposed by single pharyngeal aspiration to 30 µg of graphene oxide (GO) and few layer graphene (FLG) (micrometric and nanometric sizes). Multiwalled carbon nanotubes (MWCNT) were used as positive control. At day 1, 7 and 28, bronchoalveolar lavage fluids and lungs were collected. The pulmonary adverse effects were evaluated (immune response, histopathology, tissue remodelling) and put in perspective with the biodegradation and clearance of GBMs in lungs. Confocal Raman Microscopy (CRM) was used to assess biodegradation and clearance.

Results: GOs and FLGs caused an acute innate immune response, characterized by an influx of granulocytes in the alveolar space. The inflammation was rapidly reversible and there was no permanent damage in lungs, as highlighted by the absence of tissue remodeling. The GBMs could be detected in lungs by CRM. The GBMs were internalized rapidly by alveolar phagocytic cells (macrophages, neutrophils), resulting in an efficient, but size-dependent clearance from the lungs. Degradation over time of GO and FLG materials was found. GO sheets with nanometric lateral size were degraded and eliminated significantly more rapidly than micrometric GOs. We revealed the important role played by neutrophils in this faster process. The persistence of micrometric GO sheets in the lungs was associated to an increase in multinucleated macrophages and the presence of small granulomatous-like structures made of laden macrophages. In contrast, MWCNTs caused a strong and chronic inflammatory response, characterized by a sustained inflammation up to 28 days after exposure as well as the activation of both the innate and adaptive immunity. A clear allergy-like response was detected and was materialized by an increase in lymphocytes, a chronic eosinophilia and the presence of eosinophilic crystals. Additionally, we observed permanent tissue remodeling in the lungs exposed to nanotubes.

Conclusion: We revealed the safer toxicological profile of GBMs in vivo in comparisons to nanotubes. We demonstrated the importance of some physicochemical characteristics of GBMs, in particular dimensions, for the internalisation by alveolar phagocytes and the degradation and clearance from the lungs.