Suchergebnisse

Abstract
Advances in in-vitro culture systems have stimulated an interest in more reliable testing approaches that predict nanomaterial toxicity. Lung organoids are one example, and display functions consistent with their in-vivo counterparts. Organoids have been used in a variety of applications, but studies have yet to interrogate their suitability for nanomaterial testing. Here, we report the step-wise development of multilineage lung organoids, and validate their potential as tools for predicting pulmonary toxicity of carbon-based nanomaterials. After 50-days of differentiation, the lung organoids exhibited the six major proximal (goblet/basal/club/ciliated) and distal (AECI/II) epithelial cell types of the adult lung. Using microinjection, we developed an exposure model to exploit the airspace-like lumen of the organoids for cargo delivery. We compared the impact of small nanometric (50-950nm, sGO) and large micrometric (4-41µm, lGO) graphene oxide sheets, with multi-walled carbon nanotubes (1-20µm, MWCNT) on lung organoids after 1/7/28d exposure. Consistent with in-vivo data, sGO and lGO exhibited a transient interaction with the alveolar epithelium, and did not induce any histoarchitectural changes indicative of fibrosis. Organoids did however produce 2.5-fold more mucins in response to lGO than sGO exposure, suggesting that a thicker mucin barrier is required to prevent lGO-induced damage. In contrast, MWCNT caused detrimental effects on organoid architecture. Its persistent interaction with the alveolar epithelium led to a pro-fibrotic phenotype, where MWCNT caused AECII to undergo epithelial-mesenchymal transition. With further development, our model may reduce the need for rodent inhalation studies in the assessment of lung toxicity.

Abstract
The growing production of graphene oxide (GO) and GO-based products has led to an increased risk of human exposure and caused scientific and public concern about human health and safety. Even though human exposure to GO can occur via different pathways, the main exposure route for airborn GO is through the lungs, thus placing the respiratory tract at particular risk. Occupational exposure to GO might occur repeatedly over a prolonged period of time but insights on lung toxicity of long-term repeated GO administration is largely lacking. Moreover, there is a risk that GO co-exposure with prevalent lung pathogens (e.g. Streptococcus pneumoniae (SP)) could increase the susceptibility to lung infections. In this study, we aimed to investigate the impact of repeated and prolonged GO exposure in healthy and SP-infected lungs using well-characterized materials with distinct properties and reconstituted primary human bronchial epithelial cultures. Our results revealed that GO had no major toxicity effects on healthy airway epithelium even upon prolonged exposure, but alterations in inflammatory/immune signaling was apparent and showed a correlation with the lateral particle size. GO pre-exposure slightly altered the response of the airway epithelium towards subsequent SP infection as evidenced by a decreased mitochondrial activity, increased bacterial translocation and altered inflammatory/immune responses. Whole genome transcriptomic profiling is ongoing to gain insights in deregulated genes and pathways following chronic GO administration. Our findings on the health risks of GO inhalation in healthy and infected human lung is important to risk assessment and management of this promising material.

Engineered small graphene oxide (s-GO) sheets were previously shown to reversibly down-regulate glutamatergic synapses in the hippocampus of juvenile rats, disclosing an unexpected translational potential of these nanomaterials to target selective synapses in vivo. Synapses are anatomical specializations acting in the Central Nervous System (CNS) as functional interfaces among neurons. Dynamic changes in synaptic function, named synaptic plasticity, are crucial to learning and memory. More recently, pathological mechanisms involving dysfunctional synaptic plasticity were implicated in several brain diseases, from dementia to anxiety disorders. Hyper-excitability of glutamatergic neurons in the lateral nucleus of the amygdala complex (LA) is substantially involved in the storage of aversive memory induced by stressful events enabling post-traumatic stress disorder (PTSD). Here we translated in PTSD animal model the ability of s-GO, when stereotaxically administered to hamper LA glutamatergic transmission and to prevent the behavioral response featured in long-term aversive memory. We propose that s-GO, by interference with glutamatergic plasticity, impair LA-dependent memory retrieval related to PTSD. ; This work has received funding from the European Union Horizon 2020 Research and Innovation Programme under Grant Agreement numbers GrapheneCore2 (785219) and GrapheneCore3 (881603) and from the São Paulo Research Foundation (FAPESP) number 2016/18218–0. We would like to acknowledge the staff of the Electron Microscopy Unit at ICN2 for their expertise and assistance on the SEM. The ICN2 is funded by the CERCA programme, Generalitat de Catalunya, and is supported by the Severo Ochoa Centres of Excellence programme by the Spanish Research Agency (AEI, grant no. SEV-2017-0706).

Referred to by: Musto, Pietro Parisse, Maria Pachetti, Christian Memo, Giuseppe Di Mauro, Belen Ballesteros, Neus Lozano, Kostas Kostarelos, Loredana Casalis, Laura Ballerini. Corrigendum to Shedding plasma membrane vesicles induced by graphene oxide nanoflakes in brain cultured astrocytes [CARBON 176 (2021) 458–469] Carbon, Volume 178, 30 June 2021, Pages 824. ; Microvesicles (MVs) generated and released by astrocytes, the brain prevalent cells, crucially contribute to intercellular communication, representing key vectorized systems able to spread and actively transfer signaling molecules from astrocytes to neurons, ultimately modulating target cell functions. The increasing clinical relevance of these signaling systems requires a deeper understanding of MV features, currently limited by both their nanoscale dimensions and the low rate of their constituent release. Hence, to investigate the features of such glial signals, nanotechnology-based approaches and the applications of unconventional, cost-effective tools in generating MVs are needed. Here, small graphene oxide (s-GO) nanoflakes are used to boost MVs shedding from astrocytes in cultures and s-GO generated MVs are compared with those generated by a natural stimulant, namely ATP, by atomic force microscopy, light scattering, attenuated total reflection–fourier transform infra-red and ultraviolet resonance Raman spectroscopy. We also report the ability of both types of MVs, upon acute and transient exposure of patch clamped cultured neurons, to modulate basal synaptic transmission, inducing a stable increase in synaptic activity accompanied by changes in neuronal plasma membrane elastic features. ; We acknowledge the financial support from the European Union's Horizon 2020 Research and Innovation Programme under grant agreement no. 785219 and no. 881603 Graphene Flagship. MM, PP and LC acknowledge CERIC-ERIC proposal grant n. 20167063 for the IR measurements, performed at the SISSI-Bio beamline of Elettra Sincrotrone Trieste. PP and LC acknowledge also the European Regional Development Fund and Interreg V-A Italia-Austria 2014–2020 project EXOTHERA (ITAT1036). ; Peer reviewed

Carbon nanomaterials, including 2D graphene-based materials, have shown promising applicability to drug delivery, tissue engineering, diagnostics, and various other biomedical areas. However, to exploit the benefits of these materials in some of the areas mentioned, it is necessary to understand their possible toxicological implications and long-term fate in vivo. We previously demonstrated that following intravenous administration, 2D graphene oxide (GO) nanosheets were largely excreted via the kidneys; however, a small but significant portion of the material was sequestered in the spleen. Herein, we interrogate the potential consequences of this accumulation and the fate of the spleen-residing GO over a period of nine months. We show that our thoroughly characterized GO materials are not associated with any detectable pathological consequences in the spleen. Using confocal Raman mapping of tissue sections, we determine the sub-organ biodistribution of GO at various time points after administration. The cells largely responsible for taking up the material are confirmed using immunohistochemistry coupled with Raman spectroscopy, and transmission electron microscopy (TEM). This combination of techniques identified cells of the splenic marginal zone as the main site of GO bioaccumulation. In addition, through analyses using both bright-field TEM coupled with electron diffraction and Raman spectroscopy, we reveal direct evidence of in vivo intracellular biodegradation of GO sheets with ultrastructural precision. This work offers critical information about biological processing and degradation of thin GO sheets by normal mammalian tissue, indicating that further development and exploitation of GO in biomedicine would be possible. ; This work was partially supported by the EPSRC NOWNANO DTC program and grants EP/K016946/1 and EP/M010619/1. This work has received funding from the European Union's Horizon 2020 Research and Innovation Programme under Grant Agreement numbers GrapheneCore1 (696656) and GrapheneCore2 (785219). D.J. and K.K. would like to acknowledge the support by the UKRI, EPSRC from the Programme Grant 2D-Health (EP/P00119X/1). The DTRA, USA (grant HDTRA1-12-1-0013), is also acknowledged for partial support. ; Peer reviewed

Nanotechnology can offer a number of options against coronavirus disease 2019 (COVID-19) acting both extracellularly and intracellularly to the host cells. Here, the aim is to explore graphene oxide (GO), the most studied 2D nanomaterial in biomedical applications, as a nanoscale platform for interaction with SARS-CoV-2. Molecular docking analyses of GO sheets on interaction with three different structures: SARS-CoV-2 viral spike (open state – 6VYB or closed state – 6VXX), ACE2 (1R42), and the ACE2-bound spike complex (6M0J) are performed. GO shows high affinity for the surface of all three structures (6M0J, 6VYB and 6VXX). When binding affinities and involved bonding types are compared, GO interacts more strongly with the spike or ACE2, compared to 6M0J. Infection experiments using infectious viral particles from four different clades as classified by Global Initiative on Sharing all Influenza Data (GISAID), are performed for validation purposes. Thin, biological-grade GO nanoscale (few hundred nanometers in lateral dimension) sheets are able to significantly reduce copies for three different viral clades. This data has demonstrated that GO sheets have the capacity to interact with SARS-CoV-2 surface components and disrupt infectivity even in the presence of any mutations on the viral spike. GO nanosheets are proposed to be further explored as a nanoscale platform for development of antiviral strategies against COVID-19. ; M.A.U. and F.B. contributed equally to this work. L.G.D. acknowledges the Starting Grant 2020 from the Department of Biomedical Science, University of Padua. K.K. acknowledges financial support from the UKRI EPSRC Programme Grant 2D-Health (project EP/P00119X/1) and the European Union Horizon 2020 Graphene Flagship project under grant agreement GrapheneCore2 785219. A.Y. is thankful to the Turkish Academy of Sciences (TUBA) for financial support under the young investigator programme. M.A.U., H.N., K.C.A., and A.Y. would like to acknowledge the funding by the Scientific and Technology Council of Turkey (TUBITAK) under the grant 18AG020.

Abstract
Graphene oxide nanomaterials have been developed for wide-ranging applications, but has potential safety concerns for human health. Controlled inhalation exposures in human volunteers have been a vital means to determine the effects and mechanisms of ultrafine particles in air pollution, however, few studies have used this approach to explore the effects of nanomaterials. We conducted a double-blind randomised controlled study to determine whether inhalation of graphene oxide affects pulmonary or cardiovascular function. A high purity graphene oxide was synthesised with a thickness of 1-2 layers in two sizes: 'small' (lateral dimensions: 100-1700 nm) and 'ultrasmall' (30-500 nm). Graphene oxide particles at 200 µg/m3, or filtered air, were inhaled for 2 hours by 14 young healthy volunteers on repeated visits, with measurement of cardiorespiratory parameters before and across 4 hours after exposure. Graphene oxide exposure was well-tolerated with no adverse effects. Heart rate, blood pressure, lung function and inflammatory markers were unaffected by graphene oxide irrespective of particle size. GO did not change blood biomarkers of coagulation, however, there was a mild increase in thrombus formation in an ex vivo model of arterial injury. Proteomics revealed very few differential plasma proteins. Overall, acute inhalation of graphene oxide was not associated with overt detrimental effects in healthy humans. These findings demonstrate the feasibility of carefully controlled human exposures for risk assessment of graphene nanomaterials.