The winding road for carbon nanotubes in nanomedicine

Carbon nanotubes (CNTs) are recognized as promising nanomaterials for technological advancement. However, the stigma of structural similarity with asbestos fibers has slowed down progress of CNTs in nanomedicine. Nevertheless, it also prompted thorough studies that have revealed that functionalized CNTs ( f CNTs) can biologically behave in a very different and safer manner. Here we review pristine and f CNT fate in biological settings, focusing on the importance of protein interaction, formation of the protein corona, and modulation of immune response. The emerging consensus on the desirable f CNT properties to achieve immunological neutrality, and even biodegradation, shows great promise for CNT adoption in medicine

Functioning human lung organoids model pulmonary tissue response from carbon nanomaterial exposures

Human lung organoids (HLOs) are increasingly used to model development and infectious diseases, however their ability to recapitulate functional pulmonary tissue response to nanomaterial (NM) exposures has yet to be demonstrated. Here, we established a lung organoid exposure model that utilises microinjection to present NMs into the lumen of organoids. Our model assures efficient, reproducible and controllable exposure of the apical pulmonary epithelium, emulating real-life human exposure scenario. By comparing the impact of two well studied carbon-based NMs, graphene oxide sheets (GO) and multi-walled carbon nanotubes (MWCNT), we validated lung organoids as tools for predicting pulmonary NM-driven responses. In agreement with established in vivo data, we demonstrate that MWCNT, but not GO, elicit adverse effects on lung organoids, leading to a pro-fibrotic phenotype. Our findings reveal the capacity and suitability of HLOs for hazard assessment of NMs, aligned with the much sought-out 3Rs (animal research replacement, reduction, refinement) framework

Primary microglia maintain capacity to function despite internalisation and intracellular loading with carbon nanotubes

Biomedical applications of functionalised carbon nanotubes (f-CNTs) for imaging as well as drug or gene delivery in the brain have recently gained interest. Several studies have demonstrated the potential of f-CNTs to offer treatment options for neurological conditions with success. However, there is also evidence that f-CNTs accumulate preferentially within microglial cells when introduced in the brain. Considering the key immunological role of these cells in the brain and the limited knowledge regarding the interaction of CNTs with microglial cells, it is imperative to understand whether accumulation of CNTs in microglial cells can alter their physiological functions or trigger pro-inflammatory signalling. The aim of the present study was to investigate the basic physiological functions of isolated primary microglial cells over time, following their exposure to multi-walled carbon nanotubes functionalized via different surface chemistries. We rationalized that some chemical strategies may be more deleterious for microglial cell functions than others. We used rat primary microglial cells that can be maintained in cell culture for a long period of time without undergoing cell division. Cell viability, phagocytosis, migration, and pro-inflammatory factor release were studied over one month, following a single exposure to a non-cytotoxic concentration of three different f-CNTs (carboxylated, aminated and both carboxylated and aminated). We show that f-CNTs do not induce inflammation and do not affect the basic functions of microglial cells under these conditions. Importantly, this was the case from day 1 to day 28 post-exposure, even though microglial cells had internalized f-CNTs in subcellular compartments and retained the nanotube load in their cytoplasm over time.This work was supported by the European Commission, under the FP-7 Marie Curie actions (Career Development Intra-European Fellowship, PIEF-GA-2010-276051, project NANONEUROHOP). AB wishes to thank the CNRS financial support from PICS (Project for International Scientific Cooperation). MP, as the recipient of the AXA Chair, is grateful to the AXA Research Fund for financial support. MP was also supported by the Spanish Ministry of Economy and Competitiveness MINECO (project CTQ2016-76721-R), by the University of Trieste and by Diputación Foral de Gipuzkoa program Red (101/16)

Converging mechanisms of epileptogenesis and their insight in glioblastoma

Glioblastoma (GBM) is the most common and advanced form of primary malignant tumor occurring in the adult central nervous system, and it is frequently associated with epilepsy, a debilitating comorbidity. Seizures are observed both pre- and post-surgical resection, indicating that several pathophysiological mechanisms are shared but also prompting questions about how the process of epileptogenesis evolves throughout GBM progression. Molecular mutations commonly seen in primary GBM, i.e., in PTEN and p53, and their associated downstream effects are known to influence seizure likelihood. Similarly, various intratumoral mechanisms, such as GBM-induced blood-brain barrier breakdown and glioma-immune cell interactions within the tumor microenvironment are also cited as contributing to network hyperexcitability. Substantial alterations to peri-tumoral glutamate and chloride transporter expressions, as well as widespread dysregulation of GABAergic signaling are known to confer increased epileptogenicity and excitotoxicity. The abnormal characteristics of GBM alter neuronal network function to result in metabolically vulnerable and hyperexcitable peri-tumoral tissue, properties the tumor then exploits to favor its own growth even post-resection. It is evident that there is a complex, dynamic interplay between GBM and epilepsy that promotes the progression of both pathologies. This interaction is only more complicated by the concomitant presence of spreading depolarization (SD). The spontaneous, high-frequency nature of GBM-associated epileptiform activity and SD-associated direct current (DC) shifts require technologies capable of recording brain signals over a wide bandwidth, presenting major challenges for comprehensive electrophysiological investigations. This review will initially provide a detailed examination of the underlying mechanisms that promote network hyperexcitability in GBM. We will then discuss how an investigation of these pathologies from a network level, and utilization of novel electrophysiological tools, will yield a more-effective, clinically-relevant understanding of GBM-related epileptogenesis. Further to this, we will evaluate the clinical relevance of current preclinical research and consider how future therapeutic advancements may impact the bidirectional relationship between GBM, SDs, and seizures

Thin graphene oxide nanoflakes modulate glutamatergic synapses in the amygdala cultured circuits: exploiting synaptic approaches to anxiety disorders

Anxiety disorders (ADs) are nervous system maladies involving changes in the amygdala synaptic circuitry, such as an upregulation of excitatory neurotransmission at glutamatergic synapses. In the field of nanotechnology, thin graphene oxide flakes with nanoscale lateral size (s-GO) have shown outstanding promise for the manipulation of excitatory neuronal transmission with high temporal and spatial precision, thus they were considered as ideal candidates for modulating amygdalar glutamatergic transmission. Here, we validated an in vitro model of amygdala circuitry as a screening tool to target synapses, towards development of future ADs treatments. After one week in vitro, dissociated amygdalar neurons reconnected forming functional networks, whose development recapitulated that of the tissue of origin. When acutely applied to these cultures, s-GO flakes induced a selective modification of excitatory activity. This type of interaction between s-GO and amygdalar neurons may form the basis for the exploitation of alternative approaches in the treatment of ADs

A method for the measurement of mass and number of graphene oxide sheets in suspension based on non-spherical approximations

Currently, particle analysis of 2D materials in suspension is commonly restricted to microscopic techniques in the dry state, and thus does not permit an accurate investigation of colloidal suspensions. Colloids in bulk can be assessed by light scattering and diffraction to investigate features such as their hydrodynamic size, charge and concentration. However, the main drawback of such techniques lies in the application of analytical and computational methods based on models assuming particle sphericity which are not representative for 2D materials. Resonance mass measurement (RMM) is a technique which can enable the analysis of 2D materials in suspension without the assumptions of spherical models. Here, we report the application of RMM to measure particle mass and concentration for three types of graphene oxide (GO) aqueous dispersions. Using micro- and nano-suspended resonating sensors, we were able to decipher gravimetric differences between GO and graphitic materials. Our results support the urge for proper definitions and standardisations of graphene based materials, and offer a new method of characterisation for 2D material colloids in liquid suspension

Nano-scavengers for blood biomarker discovery in ovarian carcinoma

The development and implementation of biomarker-based screening tools for ovarian cancer require novel analytical platforms to enable the discovery of biomarker panels that will overcome the limitations associated with the clinically used CA-125.The systematic discovery of protein biomarkers directly from human plasma using proteomics remains extremely challenging, due to the wide concentration range of plasma proteins. Here, we describe the use of lipid-based nanoparticles (NPs) as an 'omics' enrichment tool to amplify cancer signals in the blood and to uncover disease specific signatures. We aimed to exploit the spontaneous interaction of clinically-used liposomes (Caelyx®) with plasma proteins, also known as' protein corona' formation, in order to facilitate the discovery of previously unreported differentially abundant molecules. Caelyx® liposomes were incubated with plasma samples obtained from advanced ovarian carcinoma patients and healthy donors and corona-coated liposomes were subsequently recovered. Comprehensive comparison between 'healthy' and 'diseased' corona samples by label-free proteomics resulted in the identification of multiple differentially abundant proteins. Moreover, immunoassay-based validation of selected proteins demonstrated the potential of nanoparticle-platform proposed to discover novel molecules with great diagnostic potential. This study proposes a nanoparticle-enabled workflow for plasma proteomic analysis in healthy and diseased states and paves the way for further work needed to discover and validate panels of novel biomarkers for disease diagnosis and monitoring

3D Organotypic Spinal Cultures: Exploring Neuron and Neuroglia Responses Upon Prolonged Exposure to Graphene Oxide

Graphene-based nanomaterials are increasingly engineered as components of biosensors, interfaces or drug delivery platforms in neuro-repair strategies. In these developments, the mostly used derivative of graphene is graphene oxide (GO). To tailor the safe development of GO nanosheets, we need to model in vitro tissue responses, and in particular the reactivity of microglia, a sub-population of neuroglia that acts as the first active immune response, when challenged by GO. Here, we investigated central nervous system (CNS) tissue reactivity upon long-term exposure to GO nanosheets in 3D culture models. We used the mouse organotypic spinal cord cultures, ideally suited for studying long-term interference with cues delivered at controlled times and concentrations. In cultured spinal segments, the normal presence, distribution and maturation of anatomically distinct classes of neurons and resident neuroglial cells are preserved. Organotypic explants were developed for 2 weeks embedded in fibrin glue alone or presenting GO nanosheets at 10, 25 and 50 \u3bcg/mL. We addressed the impact of such treatments on premotor synaptic activity monitored by patch clamp recordings of ventral interneurons. We investigated by immunofluorescence and confocal microscopy the accompanying glial responses to GO exposure, focusing on resident microglia, tested in organotypic spinal slices and in isolated neuroglia cultures. Our results suggest that microglia reactivity to accumulation of GO flakes, maybe due to active phagocytosis, may trim down synaptic activity, although in the absence of an effective activation of inflammatory response and in the absence of neuronal cell death

Graphene oxide elicits microbiome-dependent type 2 immune responses via the aryl hydrocarbon receptor

The gut microbiome produces metabolites that interact with the aryl hydrocarbon receptor (AhR), a key regulator of immune homoeostasis in the gut(1,2). Here we show that oral exposure to graphene oxide (GO) modulates the composition of the gut microbiome in adult zebrafish, with significant differences in wild-type versus ahr2-deficient animals. Furthermore, GO was found to elicit AhR-dependent induction of cyp1a and homing of lck(+) cells to the gut in germ-free zebrafish larvae when combined with the short-chain fatty acid butyrate. To obtain further insights into the immune responses to GO, we used single-cell RNA sequencing to profile cells from whole germ-free embryos as well as cells enriched for lck. These studies provided evidence for the existence of innate lymphoid cell (ILC)-like cells(3) in germ-free zebrafish. Moreover, GO endowed with a 'corona' of microbial butyrate triggered the induction of ILC2-like cells with attributes of regulatory cells. Taken together, this study shows that a nanomaterial can influence the crosstalk between the microbiome and immune system in an AhR-dependent manner.Peer reviewe

Functionalized Carbon Nanotubes in the Brain: Cellular Internalization and Neuroinflammatory Responses

The potential use of functionalized carbon nanotubes (f-CNTs) for drug and gene delivery to the central nervous system (CNS) and as neural substrates makes the understanding of their in vivo interactions with the neural tissue essential. The aim of this study was to investigate the interactions between chemically functionalized multi-walled carbon nanotubes (f-MWNTs) and the neural tissue following cortical stereotactic administration. Two different f-MWNT constructs were used in these studies: shortened (by oxidation) amino-functionalized MWNT (oxMWNT-NH3+) and amino-functionalized MWNT (MWNT-NH3+). Parenchymal distribution of the stereotactically injected f-MWNTs was assessed by histological examination. Both f-MWNT were uptaken by different types of neural tissue cells (microglia, astrocytes and neurons), however different patterns of cellular internalization were observed between the nanotubes. Furthermore, immunohistochemical staining for specific markers of glial cell activation (GFAP and CD11b) was performed and secretion of inflammatory cytokines was investigated using real-time PCR (qRT-PCR). Injections of both f-MWNT constructs led to a local and transient induction of inflammatory cytokines at early time points. Oxidation of nanotubes seemed to induce significant levels of GFAP and CD11b over-expression in areas peripheral to the f-MWNT injection site. These results highlight the importance of nanotube functionalization on their interaction with brain tissue that is deemed critical for the development nanotube-based vector systems for CNS application