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Two-dimensional (2D) materials have attracted tremendous interest ever since the isolation of atomically thin sheets of graphene in 2004 due to the specific and versatile properties of these materials. However, the increasing production and use of 2D materials necessitate a thorough evaluation of the potential impact on human health and the environment. Furthermore, harmonized test protocols are needed with which to assess the safety of 2D materials. The Graphene Flagship project (2013-2023), funded by the European Commission, addressed the identification of the possible hazard of graphene-based materials as well as emerging 2D materials including transition metal dichalcogenides, hexagonal boron nitride, and others. Additionally, so-called green chemistry approaches were explored to achieve the goal of a safe and sustainable production and use of this fascinating family of nanomaterials. The present review provides a compact survey of the findings and the lessons learned in the Graphene Flagship.

In neuroinflammation, astrocytes play multifaceted roles that regulate the neuronal environment. Astrocytes sense and respond to pro-inflammatory cytokines (CKs) and, by a repertoire of intracellular Ca 2+ signaling, contribute to disease progression. Therapeutic approaches wish to reduce the overactivation in Ca 2+ signaling in inflammatory-reactive astrocytes to restore dysregulated cellular changes. Cell-targeting therapeutics might take advantage by the use of nanomaterial-multifunctional platforms such as graphene oxide (GO). GO biomedical applications in the nervous system involve therapeutic delivery and sensing, and GO flakes were shown to enable interfacing of neuronal and glial membrane dynamics. We exploit organotypic spinal cord cultures and optical imaging to explore Ca 2+ changes in astrocytes, and we report, when spinal tissue is exposed to CKs, neuroinflammatory-associated modulation of resident glia. We show the efficacy of GO to revert these dynamic changes in astrocytic reactivity to CKs, and we translate this potential in an animal model of immune-mediated neuroinflammatory disease.

We present the design, fabrication, and characterization of an implantable neural interface based on anisotropic magnetoresistive (AMR) magnetic-field sensors that combine reduced size and high performance at body temperature. The sensors are based on La 0.67 Sr 0.33 MnO 3 (LSMO) as a ferromagnetic material, whose epitaxial growth has been suitably engineered to get uniaxial anisotropy and large AMR output together with low noise even at low frequencies. The performance of LSMO sensors of different film thickness and at different temperatures close to 37 °C has to be explored to find an optimum sensitivity of ∼400%/T (with typical detectivity values of 2 nT·Hz -1/2 at a frequency of 1 Hz and 0.3 nT·Hz -1/2 at 1 kHz), fitted for the detection of low magnetic signals coming from neural activity. Biocompatibility tests of devices consisting of submillimeter-size LSMO sensors coated by a thin poly(dimethyl siloxane) polymeric layer, both in vitro and in vivo , support their high suitability as implantable detectors of low-frequency biological magnetic signals emerging from heterogeneous electrically active tissues.

In the past two decades, important results have been obtained on the application of carbon nanotubes (CNTs) as components of smart interfaces promoting neuronal growth and differentiation. Different forms of CNTs have been employed as scaffolds, including raw CNTs and functionalized CNTs, characterized by a different number of walls, mainly single-walled CNTs (SWCNTs) or multiwalled CNTs (MWCNTs). However, double-walled carbon nanotubes (DWCNTs), which present interesting electronic and transport properties, have barely been studied in the field. Apart from the electrical conductivity, the morphology, shape, porosity, and corresponding mechanical properties of the scaffold material are important parameters when dealing with neuronal cells. Thus, the presence of open porous and interconnected networks is essential for cell growth and differentiation. Here, we present an easy methodology to prepare porous self-standing and electrically conductive DWCNT-based scaffolds and study the growth of neuro/glial networks and their synaptic activity. A cross-linking approach with triethylene glycol (TEG) derivatives is applied to improve the tensile performance of the scaffolds while neuronal growth and differentiation are promoted. By testing different DWCNT-based constructs, we confirm that the manufactured structures guarantee a biocompatible scaffold, while favoring the design of artificial networks with high complexity.

Nanoscale graphene-based materials (GBMs) enable targeting subcellular structures of the nervous system, a feature crucial for the successful engineering of alternative nanocarriers to deliver drugs and to treat neurodisorders. Among GBMs, graphene oxide (GO) nanoflakes, showing good dispersibility in water solution and being rich of functionalizable oxygen groups, are ideal core structures for carrying biological active molecules to the brain, such as the neuropeptide Y (NPY). In addition, when unconjugated, these nanomaterials have been reported to modulate neuronal function per se . Although some GBM-based nanocarriers have been tested both in vitro and in vivo, a thorough characterization of covalent binding impact on the biological properties of the carried molecule and/or of the nanomaterial is still missing. Here, a copper(I)-catalyzed alkyne-azide cycloaddition strategy was employed to synthesize the GO-NPY complex. By investigating through electrophysiology the impact of these conjugates on the activity of hippocampal neurons, we show that the covalent modification of the nanomaterial, while making GO an inert platform for the vectorized delivery, enhances the duration of NPY pharmacological activity. These findings support the future use of GO for the development of smart platforms for nervous system drug delivery., Competing Interests: The authors declare no competing financial interest., (© 2022 The Authors. Published by American Chemical Society.)

Peptides constituted of backbone homologated α-amino acids combined with carbon materials offer interesting possibilities in the modulation of cellular functions. In this work, we have prepared diphenylalanine β- and γ-peptides and conjugated them to carbon nanotubes (CNTs). These hybrids were able to self-assemble into fibrillar dendritic structures enabling the growth of primary hippocampal cells and the modulation of their neuronal functions. In particular, following the deposition of the different nanomaterials on glass substrates, we have evaluated their effects on circuit function and geometry. The geometrical restrictions due to CNT nucleated nodes allowed growth of neuronal networks with control over network geometry, and exploring its functional impact. In diverse applications from basic neuroscience, the presence of CNT nodes may be exploited in brain interfaces able to convey highly localized electrical stimuli.

In recent years, the use of free-standing carbon nanotube (CNT) films for neural tissue engineering has attracted tremendous attention. CNT films show large surface area and high electrical conductivity that combined with flexibility and biocompatibility may promote neuron growth and differentiation while stimulating neural activity. In addition, adhesion, survival, and growth of neurons can be modulated through chemical modification of CNTs. Axonal and synaptic signaling can also be positively tuned by these materials. Here we describe the ability of free-standing CNT films to influence neuronal activity. We demonstrate that the degree of cross-linking between the CNTs has a strong impact on the electrical conductivity of the substrate, which, in turn, regulates neural circuit outputs.

Synapses compute and transmit information to connect neural circuits and are at the basis of brain operations. Alterations in their function contribute to a vast range of neuropsychiatric and neurodegenerative disorders and synapse-based therapeutic intervention, such as selective inhibition of synaptic transmission, may significantly help against serious pathologies. Graphene is a two-dimensional nanomaterial largely exploited in multiple domains of science and technology, including biomedical applications. In hippocampal neurons in culture, small graphene oxide nanosheets (s-GO) selectively depress glutamatergic activity without altering cell viability. Glutamate is the main excitatory neurotransmitter in the central nervous system and growing evidence suggests its involvement in neuropsychiatric disorders. Here we demonstrate that s-GO directly targets the release of presynaptic vesicle. We propose that s-GO flakes reduce the availability of transmitter, via promoting its fast release and subsequent depletion, leading to a decline ofglutamatergic neurotransmission. We injected s-GO in the hippocampus in vivo, and 48 h after surgery ex vivo patch-clamp recordings from brain slices show a significant reduction in glutamatergic synaptic activity in respect to saline injections.

Graphene and its derivatives are heralded as "miracle" materials with manifold applications in different sectors of society from electronics to energy storage to medicine. The increasing exploitation of graphene-based materials (GBMs) necessitates a comprehensive evaluation of the potential impact of these materials on human health and the environment. Here, we discuss synthesis and characterization of GBMs as well as human and environmental hazard assessment of GBMs using in vitro and in vivo model systems with the aim to understand the properties that underlie the biological effects of these materials; not all GBMs are alike, and it is essential that we disentangle the structure-activity relationships for this class of materials.

The design and use of materials in the nanoscale size range for addressing medical and health-related issues continues to receive increasing interest. Research in nanomedicine spans a multitude of areas, including drug delivery, vaccine development, antibacterial, diagnosis and imaging tools, wearable devices, implants, high-throughput screening platforms, etc. using biological, nonbiological, biomimetic, or hybrid materials. Many of these developments are starting to be translated into viable clinical products. Here, we provide an overview of recent developments in nanomedicine and highlight the current challenges and upcoming opportunities for the field and translation to the clinic.

Graphene offers promising advantages for biomedical applications. However, adoption of graphene technology in biomedicine also poses important challenges in terms of understanding cell responses, cellular uptake, or the intracellular fate of soluble graphene derivatives. In the biological microenvironment, graphene nanosheets might interact with exposed cellular and subcellular structures, resulting in unexpected regulation of sophisticated biological signaling. More broadly, biomedical devices based on the design of these 2D planar nanostructures for interventions in the central nervous system require an accurate understanding of their interactions with the neuronal milieu. Here, we describe the ability of graphene oxide nanosheets to down-regulate neuronal signaling without affecting cell viability.

Neural-interfaces rely on the ability of electrodes to transduce stimuli into electrical patterns delivered to the brain. In addition to sensitivity to the stimuli, stability in the operating conditions and efficient charge transfer to neurons, the electrodes should not alter the physiological properties of the target tissue. Graphene is emerging as a promising material for neuro-interfacing applications, given its outstanding physico-chemical properties. Here, we use graphene-based substrates (GBSs) to interface neuronal growth. We test our GBSs on brain cell cultures by measuring functional and synaptic integrity of the emerging neuronal networks. We show that GBSs are permissive interfaces, even when uncoated by cell adhesion layers, retaining unaltered neuronal signaling properties, thus being suitable for carbon-based neural prosthetic devices.

Myocardial tissue engineering currently represents one of the most realistic strategies for cardiac repair. We have recently discovered the ability of carbon nanotube scaffolds to promote cell division and maturation in cardiomyocytes. Here, we test the hypothesis that carbon nanotube scaffolds promote cardiomyocyte growth and maturation by altering the gene expression program, implementing the cell electrophysiological properties and improving networking and maturation of functional syncytia. In our study, we combine microscopy, biological and electrophysiological methodologies, and calcium imaging, to verify whether neonatal rat ventricular myocytes cultured on substrates of multiwall carbon nanotubes acquire a physiologically more mature phenotype compared to control (gelatin). We show that the carbon nanotube substrate stimulates the induction of a gene expression profile characteristic of terminal differentiation and physiological growth, with a 2-fold increase of α-myosin heavy chain (P < 0.001) and upregulation of sarcoplasmic reticulum Ca(2+) ATPase 2a. In contrast, markers of pathological hypertrophy remain unchanged (β-myosin heavy chain, skeletal α-actin, atrial natriuretic peptide). These modifications are paralleled by an increase of connexin-43 gene expression, gap junctions and functional syncytia. Moreover, carbon nanotubes appear to exert a protective effect against the pathologic stimulus of phenylephrine. Finally, cardiomyocytes on carbon nanotubes demonstrate a more mature electrophysiological phenotype of syncytia and intracellular calcium signaling. Thus, carbon nanotubes interacting with cardiomyocytes have the ability to promote physiological growth and functional maturation. These properties are unique in the current vexing field of tissue engineering, and offer unprecedented perspectives in the development of innovative therapies for cardiac repair.

Nanomaterials interact with cells and modify their function and biology. Manufacturing this ability can provide tissue-engineering scaffolds with nanostructures able to influence tissue growth and performance. Carbon nanotube compatibility with biomolecules motivated ongoing interest in the development of biosensors and devices including such materials. More recently, carbon nanotubes have been applied in several areas of nerve tissue engineering to study cell behavior or to instruct the growth and organization of neural networks. To gather further knowledge on the true potential of future constructs, in particular to assess their immune-modulatory action, we evaluate carbon nanotubes interactions with human dendritic cells (DCs). DCs are professional antigen-presenting cells and their behavior can predict immune responses triggered by adhesion-dependent signaling. Here, we incorporate DC cultures to carbon nanotubes and we show by phenotype, microscopy, and transcriptional analysis that in vitro differentiated and activated DCs show when interfaced to carbon nanotubes a lower immunogenic profile.

In the past decade, nanotechnology applications to the nervous system have often involved the study and the use of novel nanomaterials to improve the diagnosis and therapy of neurological diseases. In the field of nanomedicine, carbon nanotubes are evaluated as promising materials for diverse therapeutic and diagnostic applications. Besides, carbon nanotubes are increasingly employed in basic neuroscience approaches, and they have been used in the design of neuronal interfaces or in that of scaffolds promoting neuronal growth in vitro. Ultimately, carbon nanotubes are thought to hold the potential for the development of innovative neurological implants. In this framework, it is particularly relevant to document the impact of interfacing such materials with nerve cells. Carbon nanotubes were shown, when modified with biologically active compounds or functionalized in order to alter their charge, to affect neurite outgrowth and branching. Notably, purified carbon nanotubes used as scaffolds can promote the formation of nanotube-neuron hybrid networks, able per se to affect neuron integrative abilities, network connectivity, and synaptic plasticity. We focus this review on our work over several years directed to investigate the ability of carbon nanotube platforms in providing a new tool for nongenetic manipulations of neuronal performance and network signaling.

Nanoscale manipulations of the extracellular microenvironment are increasingly attracting attention in tissue engineering. Here, combining microscopy, biological, and single-cell electrophysiological methodologies, we demonstrate that neonatal rat ventricular myocytes cultured on substrates of multiwall carbon nanotubes interact with carbon nanotubes by forming tight contacts and show increased viability and proliferation. Furthermore, we observed changes in the electrophysiological properties of cardiomyocytes, suggesting that carbon nanotubes are able to promote cardiomyocyte maturation., (© 2012 American Chemical Society)

New developments in nanotechnology are increasingly designed to modulate relevant interactions between nanomaterials and neurons, with the aim of exploiting the physical properties of synthetic materials to tune desired and specific biological processes. Carbon nanotubes have been applied in several areas of nerve tissue engineering to study cell behavior or to instruct the growth and organization of neural networks. Recent reports show that nanotubes can sustain and promote electrical activity in networks of cultured neurons. However, such results are usually limited to carbon nanotube/neuron hybrids formed on a monolayer of dissociated brain cells. In the present work, we used organotypic spinal slices to model multilayer tissue complexity, and we interfaced such spinal segments to carbon nanotube scaffolds for weeks. By immunofluorescence, scanning and transmission electronic microscopy, and atomic force microscopy, we investigated nerve fiber growth when neuronal processes exit the spinal explant and develop in direct contact to the substrate. By single-cell electrophysiology, we investigated the synaptic activity of visually identified ventral interneurons, within the ventral area of the explant, thus synaptically connected, but located remotely, to the substrate/network interface. Here we show that spinal cord explants interfaced for weeks to purified carbon nanotube scaffolds expand more neuronal fibers, characterized by different mechanical properties and displaying higher growth cones activity. On the other hand, exploring spontaneous and evoked synaptic activity unmasks an increase in synaptic efficacy in neurons located at as far as 5 cell layers from the cell-substrate interactions., (© 2012 American Chemical Society)

We demonstrate the possibility of using carbon nanotubes (CNTs) as potential devices able to improve neural signal transfer while supporting dendrite elongation and cell adhesion. The results strongly suggest that the growth of neuronal circuits on a CNT grid is accompanied by a significant increase in network activity. The increase in the efficacy of neural signal transmission may be related to the specific properties of CNT materials, such as the high electrical conductivity.