Upregulation of the secretory pathway Ca2+/Mn2+-ATPase isoform 1 in LPS-stimulated microglia and its involvement in Mn2+-induced Golgi fragmentation.

Microglia play an important protective role in the healthy nervous tissue, being able to react to a variety of stimuli that induce different intracellular cascades for specific tasks. Ca2+ signaling can modulate these pathways, and we recently reported that microglial functions depend on the endoplasmic reticulum as a Ca2+ store, which involves the Ca2+ transporter SERCA2b. Here, we investigated whether microglial functions may also rely on the Golgi, another intracellular Ca2+ store that depends on the secretory pathway Ca2+/Mn2+-transport ATPase isoform 1 (SPCA1). We found upregulation of SPCA1 upon lipopolysaccharide stimulation of microglia BV2 cells and primary microglia, where alterations of the Golgi ribbon were also observed. Silencing and overexpression experiments revealed that SPCA1 affects cell morphology, Golgi apparatus integrity, and phagocytic functions. Since SPCA1 is also an efficient Mn2+ transporter and considering that Mn2+ excess causes manganism in the brain, we addressed the role of microglial SPCA1 in Mn2+ toxicity. Our results revealed a clear effect of Mn2+ excess on the viability and morphology of microglia. Subcellular analysis showed Golgi fragmentation and subsequent alteration of SPCA1 distribution from early stages of toxicity. Removal of Mn2+ by washing improved the culture viability, although it did not effectively reverse Golgi fragmentation. Interestingly, pretreatment with curcumin maintained microglia cultures viable, prevented Mn2+-induced Golgi fragmentation, and preserved SPCA Ca2+-dependent activity, suggesting curcumin as a potential protective agent against Mn2+-induced Golgi alterations in microglia.

The potential of graphene coatings as neural interfaces.

Recent advances in nanotechnology design and fabrication have shaped the landscape for the development of ideal cell interfaces based on biomaterials. A holistic evaluation of the requirements for a cell interface is a highly complex task. Biocompatibility is a crucial requirement which is affected by the interface's properties, including elemental composition, morphology, and surface chemistry. This review explores the current state-of-the-art on graphene coatings produced by chemical vapor deposition (CVD) and applied as neural interfaces, detailing the key properties required to design an interface capable of physiologically interacting with neural cells. The interfaces are classified into substrates and scaffolds to differentiate the planar and three-dimensional environments where the cells can adhere and proliferate. The role of specific features such as mechanical properties, porosity and wettability are investigated. We further report on the specific brain-interface applications where CVD graphene paved the way to revolutionary advances in biomedicine. Future studies on the long-term effects of graphene-based materials in vivo will unlock even more potentially disruptive neuro-applications.

Interactions of Graphene Oxide and Few-Layer Graphene with the Blood-Brain Barrier.

Thanks to their biocompatibility and high cargo capability, graphene-based materials (GRMs) might represent an ideal brain delivery system. The capability of GRMs to reach the brain has mainly been investigated in vivo and has highlighted some controversy. Herein, we employed two in vitro BBB models of increasing complexity to investigate the bionano interactions with graphene oxide (GO) and few-layer graphene (FLG): a 2D murine Transwell model, followed by a 3D human multicellular assembloid, to mimic the complexity of the in vivo architecture and intercellular crosstalk. We developed specific methodologies to assess the translocation of GO and FLG in a label-free fashion and a platform applicable to any nanomaterial. Overall, our results show good biocompatibility of the two GRMs, which did not impact the integrity and functionality of the barrier. Sufficiently dispersed subpopulations of GO and FLG were actively uptaken by endothelial cells; however, the translocation was identified as a rare event.

Graphene Nanoplatelets Render Poly(3-Hydroxybutyrate) a Suitable Scaffold to Promote Neuronal Network Development.

The use of composite biomaterials as innovative bio-friendly neuronal interfaces has been poorly developed so far. Smart strategies to target neuro-pathologies are currently exploiting the mixed and complementary characteristics of composite materials to better design future neural interfaces. Here we present a polymer-based scaffold that has been rendered suitable for primary neurons by embedding graphene nanoplatelets (GnP). In particular, the growth, network formation, and functionality of primary neurons on poly(3-hydroxybutyrate) [P(3HB)] polymer supports functionalized with various concentrations of GnP were explored. After growing primary cortical neurons onto the supports for 14 days, all specimens were found to be biocompatible, revealing physiological growth and maturation of the neuronal network. When network functionality was investigated by whole patch-clamp measurements, pure P(3HB) led to changes in the action potential waveform and reduction in firing frequency, resulting in decreased neuronal excitability. However, the addition of GnP to the polymer matrix restored the electrophysiological parameters to physiological values. Interestingly, a low concentration of graphene was able to promote firing activity at a low level of injected current. The results indicate that the P(3HB)/GnP composites show great potential for electrical interfacing with primary neurons to eventually target central nervous system disorders.

Hydrogenated Graphene Improves Neuronal Network Maturation and Excitatory Transmission.

Graphene is regarded as a viable bio-interface for neuroscience due to its biocompatibility and electrical conductivity, which would contribute to efficient neuronal network signaling. Here, monolayer graphene grown via chemical vapor deposition is treated with remote hydrogen plasma to demonstrate that hydrogenated graphene (HGr) fosters improved cell-to-cell communication with respect to pristine graphene in primary cortical neurons. When transferred to polyethylene terephthalate, HGr exhibits higher wettability than graphene (water contact angle of 83.7° vs 40.7°), while preserving electrical conductivity (≈3 kΩ â–¡-1 ). A rich and mature network is observed to develop onto HGr. The intrinsic excitability and firing properties of neurons plated onto HGr appears unaltered, while the basic passive and active membrane properties are fully preserved. The formation of excitatory synaptic connections increases in HGr with respect to pristine graphene, leading to a doubled miniature excitatory postsynaptic current frequency. This study supports the use of hydrogenation for tailoring graphene into an improved neuronal interface, indicating that wettability, more than electrical conductivity, is the key parameter to be controlled. The use of HGr can bring about a deeper understanding of neuronal behavior on artificial bio-interfaces and provide new insight for graphene-based biomedical applications.

Isobaric Labeling Proteomics Allows a High-Throughput Investigation of Protein Corona Orientation.

The formation of the biomolecular corona represents a crucial factor in controlling the biological interactions and trafficking of nanomaterials. In this context, the availability of key epitopes exposed on the surface of the corona, and able to engage the biological machinery, is important to define the biological fate of the material. While the full biomolecular corona composition can be investigated by conventional bottom-up proteomics, the assessment of the spatial orientation of proteins in the corona in a high-throughput fashion is still challenging. In this work, we show that labeling corona proteins with isobaric tags in their native conditions and analyzing the MS/MS spectra of tryptic peptides allow an easy and high-throughput assessment of the inner/outer orientation of the corresponding proteins in the original corona. We put our results in the context of what is currently known of the protein corona of graphene-based nanomaterials. Our conclusions are in line with previous data and were confirmed by in silico calculations.

2D materials in electrochemical sensors for in vitro or in vivo use.

Individual cells and cell populations are at the present time investigated with a myriad of analytical tools. While most of them are commercially available, some of these analytical tools are just emerging from research laboratories and are in the developmental phase. Electrochemical sensors which allow the monitoring of low molecular weight compounds released (and / or uptaken) by cells are among these emerging tools. Such sensors are increasingly built using 2D materials (e.g. graphene-based materials, transition metal dichalcogenides, etc.) with the aim of conferring better analytical performances to these devices. The present work critically reviews studies published during the last 10 years describing electrochemical sensors made with 2D materials and exploited to monitor small compounds (e.g. H2O2, ·NO, glucose, etc.) in living biological systems. It also discusses the very few 2D material-based electrochemical sensors which are wearable or usable in vivo. Finally, the present work includes a specific section about 2D material biocompatibility, a fundamental requirement for 2D material-based sensor applications in vitro and in vivo. As such, the review provides a critical view on the state of the art of electrochemical sensors made with 2D materials and used at cellular level and it evaluates the possibility that such sensors will be used on / in the human body on a wider scale.

Subretinally injected semiconducting polymer nanoparticles rescue vision in a rat model of retinal dystrophy.

Inherited retinal dystrophies and late-stage age-related macular degeneration, for which treatments remain limited, are among the most prevalent causes of legal blindness. Retinal prostheses have been developed to stimulate the inner retinal network; however, lack of sensitivity and resolution, and the need for wiring or external cameras, have limited their application. Here we show that conjugated polymer nanoparticles (P3HT NPs) mediate light-evoked stimulation of retinal neurons and persistently rescue visual functions when subretinally injected in a rat model of retinitis pigmentosa. P3HT NPs spread out over the entire subretinal space and promote light-dependent activation of spared inner retinal neurons, recovering subcortical, cortical and behavioural visual responses in the absence of trophic effects or retinal inflammation. By conferring sustained light sensitivity to degenerate retinas after a single injection, and with the potential for high spatial resolution, P3HT NPs provide a new avenue in retinal prosthetics with potential applications not only in retinitis pigmentosa, but also in age-related macular degeneration.

Membrane Environment Enables Ultrafast Isomerization of Amphiphilic Azobenzene.

The non-covalent affinity of photoresponsive molecules to biotargets represents an attractive tool for achieving effective cell photo-stimulation. Here, an amphiphilic azobenzene that preferentially dwells within the plasma membrane is studied. In particular, its isomerization dynamics in different media is investigated. It is found that in molecular aggregates formed in water, the isomerization reaction is hindered, while radiative deactivation is favored. However, once protected by a lipid shell, the photochromic molecule reacquires its ultrafast photoisomerization capacity. This behavior is explained considering collective excited states that may form in aggregates, locking the conformational dynamics and redistributing the oscillator strength. By applying the pump probe technique in different media, an isomerization time in the order of 10 ps is identified and the deactivation in the aggregate in water is also characterized. Finally, it is demonstrated that the reversible modulation of membrane potential of HEK293 cells via illumination with visible light can be indeed related to the recovered trans→cis photoreaction in lipid membrane. These data fully account for the recently reported experiments in neurons, showing that the amphiphilic azobenzenes, once partitioned in the cell membrane, are effective light actuators for the modification of the electrical state of the membrane.

Beyond graphene oxide acidity: Novel insights into graphene related materials effects on the sexual reproduction of seed plants.

Graphene related materials (GRMs) are currently being used in products and devices of everyday life and this strongly increases the possibility of their ultimate release into the environment as waste items. GRMs have several effects on plants, and graphene oxide (GO) in particular, can affect pollen germination and tube growth due to its acidic properties. Despite the socio-economic importance of sexual reproduction in seed plants, the effect of GRMs on this process is still largely unknown. Here, Corylus avellana L. (common Hazel) pollen was germinated in-vitro with and without 1-100 µg mL-1 few-layer graphene (FLG), GO and reduced GO (rGO) to identify GRMs effects alternative to the acidification damage caused by GO. At 100 µg mL-1 both FLG and GO decreased pollen germination, however only GO negatively affected pollen tube growth. Furthermore, GO adsorbed about 10 % of the initial Ca2+ from germination media accounting for a further decrease in germination of 13 % at the pH created by GO. In addition, both FLG and GO altered the normal tip-focused reactive oxygen species (ROS) distribution along the pollen tube. The results provided here help to understand GRMs effect on the sexual reproduction of seed plants and to address future in-vivo studies.

Polymer/enzyme-modified HF-etched carbon nanoelectrodes for single-cell analysis.

Carbon-based nanoelectrodes fabricated by means of pyrolysis of an alkane precursor gas purged through a glass capillary and subsequently etched with HF were modified with redox polymer/enzyme films for the detection of glucose at the single-cell level. Glucose oxidase (GOx) was immobilized and electrically wired by means of an Os-complex-modified redox polymer in a sequential dip coating process. For the synthesis of the redox polymer matrix, a poly(1-vinylimidazole-co-acrylamide)-based backbone was used that was first modified with the electron transfer mediator [Os(bpy)2Cl]+ (bpy = 2,2'-bipyridine) followed by the conversion of the amide groups within the acrylamide monomer into hydrazide groups in a polymer-analogue reaction. The hydrazide groups react readily with bifunctional epoxide-based crosslinkers ensuring high film stability. Insertion of the nanometre-sized polymer/enzyme modified electrodes into adherently growing single NG108-15 cells resulted in a positive current response correlating with the intracellular glucose concentration. Moreover, the nanosensors showed a stable current output without significant loss in performance after intracellular measurements.

Neuronal firing modulation by a membrane-targeted photoswitch.

Optical technologies allowing modulation of neuronal activity at high spatio-temporal resolution are becoming paramount in neuroscience. In this respect, azobenzene-based photoswitches are promising nanoscale tools for neuronal photostimulation. Here we engineered a light-sensitive azobenzene compound (Ziapin2) that stably partitions into the plasma membrane and causes its thinning through trans-dimerization in the dark, resulting in an increased membrane capacitance at steady state. We demonstrated that in neurons loaded with the compound, millisecond pulses of visible light induce a transient hyperpolarization followed by a delayed depolarization that triggers action potential firing. These effects are persistent and can be evoked in vivo up to 7 days, proving the potential of Ziapin2 for the modulation of membrane capacitance in the millisecond timescale, without directly affecting ion channels or local temperature.

Graphene-based materials do not impair physiology, gene expression and growth dynamics of the aeroterrestrial microalga Trebouxia gelatinosa.

The effects of two graphene-based materials (GBMs), few-layers graphene (FLG) and graphene oxide (GO), were studied in the aeroterrestrial green microalga Trebouxia gelatinosa. Algae were subjected to short- and long-term exposure to GBMs at 0.01, 1 and 50 µg mL - 1. GBMs internalization after short-term exposures was investigated with confocal microscopy, Raman spectroscopy and TEM. Potential negative effects of GBMs, compared to the oxidative stress induced by H2O2, were verified by analyzing chlorophyl a fluorescence (ChlaF), expression of stress-related genes and membrane integrity. Effects of up to 4-week-long exposures were assessed analyzing growth dynamics, ChlaF and photosynthetic pigments. GBMs were not observed in cells but FLG was detected at the interface between the cell wall and plasma membrane, whereas GO was observed adherent to the external wall surface. FLG caused the down-regulation of the HSP70-1 gene, with the protein levels remaining stable, whereas GO had no effect. In comparison, H2O2 produced dose- and time-dependent effects on ChlaF, gene expression and HSP70 protein level. Long-term exposures to GBMs did not affect growth dynamics, ChlaF or photosynthetic pigment contents, indicating that the few observed short-term effects were not dangerous on the long-term. Results suggest that interactions between FLG and plasma membrane were harmless, activating a down-regulation of the HSP70-1 gene similar to that induced by H2O2. Our work shows that studying GBMs effects on non-model organisms is important since the results of model green microalgae are not representative of the whole taxonomic group.

Neuronal Cultures and Nanomaterials.

In recent years, the scientific community has witnessed an exponential increase in the use of nanomaterials for biomedical applications. In particular, the interest of graphene and graphene-based materials has rapidly risen in the neuroscience field due to the properties of this material, such as high conductivity, transparency and flexibility. As for any new material that aims to play a role in the biomedical area, a fundamental aspect is the evaluation of its toxicity, which strongly depends on material composition, chemical functionalization and dimensions. Furthermore, a wide variety of three-dimensional scaffolds have also started to be exploited as a substrate for tissue engineering. In this application, the topography is probably the most relevant amongst the various properties of the different materials, as it may allow to instruct and interrogate neural networks, as well as to drive neural growth and differentiation.This chapter discusses the in vitro approaches, ranging from microscopy analysis to physiology measurements, to investigate the interaction of graphene with the central nervous system. Moreover, the in vitro use of three-dimensional scaffolds is described and commented.

An Increase in Membrane Cholesterol by Graphene Oxide Disrupts Calcium Homeostasis in Primary Astrocytes.

The use of graphene nanomaterials (GNMs) for biomedical applications targeted to the central nervous system is exponentially increasing, although precise information on their effects on brain cells is lacking. In this work, the molecular changes induced in cortical astrocytes by few-layer graphene (FLG) and graphene oxide (GO) flakes are addressed. The results show that exposure to FLG/GO does not affect cell viability or proliferation. However, proteomic and lipidomic analyses unveil alterations in several cellular processes, including intracellular Ca2+ ([Ca2+ ]i ) homeostasis and cholesterol metabolism, which are particularly intense in cells exposed to GO. Indeed, GO exposure impairs spontaneous and evoked astrocyte [Ca2+ ]i signals and induces a marked increase in membrane cholesterol levels. Importantly, cholesterol depletion fully rescues [Ca2+ ]i dynamics in GO-treated cells, indicating a causal relationship between these GO-mediated effects. The results indicate that exposure to GNMs alters intracellular signaling in astrocytes and may impact astrocyte-neuron interactions.

Ecotoxicological effects of polystyrene microbeads in a battery of marine organisms belonging to different trophic levels.

The aim of this study was to detect ecotoxicological effects of 0.1 µm polystyrene microbeads in marine organisms belonging to different trophic levels. MP build up, lethal and sub-lethal responses were investigated in the bacterium Vibrio anguillarum (culturability), in the green microalga Dunaliella tertiolecta (growth inhibition), in the rotifer Brachionus plicatilis (mortality and swimming speed alteration) and in the sea urchin Paracentrotus lividus (immobility and swimming speed alteration) exposed to a wide range of microplastic (MP) concentrations (from 0.001 to 10 mg L-1). Survival was not affected in all organisms up to 10 mg L-1, while algal growth inhibition, rotifer and sea urchin larvae swimming behaviour alterations were observed after exposure to MPs. Ingestion was only observed in rotifers and it was directly correlated with sub-lethal effects. These results account for the ecotoxicological risk associated to the polystyrene microbeads, which are able to affect different endpoints in primary producers and consumers (rotifers and sea urchins) since no effects were observed in decomposers. This study points out the importance of using a battery of marine organisms belonging to different trophic levels by studying acute toxicity of MPs at low and high contamination levels, and investigating sub-lethal responses. Further investigations aimed at studying the transfer of these materials through the web are particularly recommended.

Graphene Oxide Upregulates the Homeostatic Functions of Primary Astrocytes and Modulates Astrocyte-to-Neuron Communication.

Graphene-based materials are the focus of intense research efforts to devise novel theranostic strategies for targeting the central nervous system. In this work, we have investigated the consequences of long-term exposure of primary rat astrocytes to pristine graphene (GR) and graphene oxide (GO) flakes. We demonstrate that GR/GO interfere with a variety of intracellular processes as a result of their internalization through the endolysosomal pathway. Graphene-exposed astrocytes acquire a more differentiated morphological phenotype associated with extensive cytoskeletal rearrangements. Profound functional alterations are induced by GO internalization, including the upregulation of inward-rectifying K+ channels and of Na+-dependent glutamate uptake, which are linked to the astrocyte capacity to control the extracellular homeostasis. Interestingly, GO-pretreated astrocytes promote the functional maturation of cocultured primary neurons by inducing an increase in intrinsic excitability and in the density of GABAergic synapses. The results indicate that graphene nanomaterials profoundly affect astrocyte physiology in vitro with consequences for neuronal network activity. This work supports the view that GO-based materials could be of great interest to address pathologies of the central nervous system associated with astrocyte dysfunctions.

Interfacing Graphene-Based Materials With Neural Cells.

The scientific community has witnessed an exponential increase in the applications of graphene and graphene-based materials in a wide range of fields, from engineering to electronics to biotechnologies and biomedical applications. For what concerns neuroscience, the interest raised by these materials is two-fold. On one side, nanosheets made of graphene or graphene derivatives (graphene oxide, or its reduced form) can be used as carriers for drug delivery. Here, an important aspect is to evaluate their toxicity, which strongly depends on flake composition, chemical functionalization and dimensions. On the other side, graphene can be exploited as a substrate for tissue engineering. In this case, conductivity is probably the most relevant amongst the various properties of the different graphene materials, as it may allow to instruct and interrogate neural networks, as well as to drive neural growth and differentiation, which holds a great potential in regenerative medicine. In this review, we try to give a comprehensive view of the accomplishments and new challenges of the field, as well as which in our view are the most exciting directions to take in the immediate future. These include the need to engineer multifunctional nanoparticles (NPs) able to cross the blood-brain-barrier to reach neural cells, and to achieve on-demand delivery of specific drugs. We describe the state-of-the-art in the use of graphene materials to engineer three-dimensional scaffolds to drive neuronal growth and regeneration in vivo, and the possibility of using graphene as a component of hybrid composites/multi-layer organic electronics devices. Last but not least, we address the need of an accurate theoretical modeling of the interface between graphene and biological material, by modeling the interaction of graphene with proteins and cell membranes at the nanoscale, and describing the physical mechanism(s) of charge transfer by which the various graphene materials can influence the excitability and physiology of neural cells.

APache Is an AP2-Interacting Protein Involved in Synaptic Vesicle Trafficking and Neuronal Development.

Synaptic transmission is critically dependent on synaptic vesicle (SV) recycling. Although the precise mechanisms of SV retrieval are still debated, it is widely accepted that a fundamental role is played by clathrin-mediated endocytosis, a form of endocytosis that capitalizes on the clathrin/adaptor protein complex 2 (AP2) coat and several accessory factors. Here, we show that the previously uncharacterized protein KIAA1107, predicted by bioinformatics analysis to be involved in the SV cycle, is an AP2-interacting clathrin-endocytosis protein (APache). We found that APache is highly enriched in the CNS and is associated with clathrin-coated vesicles via interaction with AP2. APache-silenced neurons exhibit a severe impairment of maturation at early developmental stages, reduced SV density, enlarged endosome-like structures, and defects in synaptic transmission, consistent with an impaired clathrin/AP2-mediated SV recycling. Our data implicate APache as an actor in the complex regulation of SV trafficking, neuronal development, and synaptic plasticity.

Low uptake of silica nanoparticles in Caco-2 intestinal epithelial barriers.

Cellular barriers, such as the skin, the lung epithelium or the intestinal epithelium, constitute one of the first obstacles facing nanomedicines or other nanoparticles entering organisms. It is thus important to assess the capacity of nanoparticles to enter and transport across such barriers. In this work, Caco-2 intestinal epithelial cells were used as a well-established model for the intestinal barrier, and the uptake, trafficking and translocation of model silica nanoparticles of different sizes were investigated using a combination of imaging, flow cytometry and transport studies. Compared to typical observations in standard cell lines commonly used for in vitro studies, silica nanoparticle uptake into well-developed Caco-2 cellular barriers was found to be very low. Instead, nanoparticle association to the apical outer membrane was substantial and these particles could easily be misinterpreted as internalised in the absence of imaging. Passage of nanoparticles through the barrier was very limited, suggesting that the low amount of internalised nanoparticles was due to reduced uptake into cells, rather than a considerable transport through them.