i An update to this article is included at the end Carbon 176 (2021) 458e469 Contents lists available at ScienceDirect Carbon journal homepage: www.elsevier.com/locate/carbon Shedding plasma membrane vesicles induced by graphene oxide nanoflakes in brain cultured astrocytes Mattia Musto a, 1, Pietro Parisse b, Maria Pachetti b, Christian Memo a, Giuseppe Di Mauro a, Belen Ballesteros c, Neus Lozano c, Kostas Kostarelos c, d, Loredana Casalis b, **, Laura Ballerini a, * a International School for Advanced Studies (SISSA), 34136, Trieste, Italy b ELETTRA Synchrotron Light Source, 34149, Basovizza, Italy c Catalan Institute of Nanoscience and Nanotechnology (ICN2), Campus UAB, Bellaterra, 08193, Barcelona, Spain d Nanomedicine Lab, National Graphene Institute and Faculty of Biology, Medicine & Health, The University of Manchester, Manchester, M13 9PT, United Kingdom a r t i c l e i n f o a b s t r a c t Article history: Microvesicles (MVs) generated and released by astrocytes, the brain prevalent cells, crucially contribute Received 4 December 2020 to intercellular communication, representing key vectorized systems able to spread and actively transfer Received in revised form signaling molecules from astrocytes to neurons, ultimately modulating target cell functions. The 25 January 2021 increasing clinical relevance of these signaling systems requires a deeper understanding of MV features, Accepted 27 January 2021 Available online 8 February 2021 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) Keywords: Graphene oxide nanoflakes are used to boost MVs shedding from astrocytes in cultures and s-GO generated MVs are Extracellular vesicles compared with those generated by a natural stimulant, namely ATP, by atomic force microscopy, light Atomic force microscopy and spectroscopy scattering, attenuated total reflectionefourier transform infra-red and ultraviolet resonance Raman FTIR-ATR and UVRR spectroscopy spectroscopy. We also report the ability of both types of MVs, upon acute and transient exposure of patch Synaptic activity clamped cultured neurons, to modulate basal synaptic transmission, inducing a stable increase in syn- Cortical neuronal cultures aptic activity accompanied by changes in neuronal plasma membrane elastic features. © 2021 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). 1. Introduction embedded in their plasma membrane, modulate relevant processes in the development, physiology and pathology of CNS target cells In biology, newly described forms of intercellular communica- [3e6]. The signaling system based on release of extracellular vesi- tion comprise the release of vesicles, named extracellular vesicles, cles comprises shedding microvesicles (MVs) and exosomes, char- from virtually all cell types, including resident glial cells of the acterized by different size, membrane composition, cargo and central nervous system (CNS), such as astrocytes and microglia origin [7,8]. [1,2]. In particular, the shedding of membrane vesicles is a recog- MVs are nanovesicles able to interact specifically with cells at nized form of cross talk in the multidimensional signaling between local or distant sites [9]. In maintaining CNS functions, glial cells astrocytes, (i.e. the majority of cells in the mammalian CNS), and intensely communicate with neurons, also via the release of MVs, neurons in physiology, but also in neurodegenerative and neuro- which represents a highly versatile tool to functionally impact the inflammatory diseases as well as in brain tumors. Extracellular CNS [10e12]. MVs are considered a “vectorized” signaling system vesicle signaling molecules, either stored within their cargo or able to bind their target cells to transmit specific information. The * Corresponding author. ** Corresponding author. E-mail addresses: loredana.casalis@elettra.eu (L. Casalis), laura.ballerini@sissa.it (L. Ballerini). 1 Present Addresses: Center for Synaptic Neuroscience, Istituto Italiano di Tecnologia (IIT), 16132, Genoa, Italy. https://doi.org/10.1016/j.carbon.2021.01.142 0008-6223/© 2021 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/ ). M. Musto, P. Parisse, M. Pachetti et al. Carbon 176 (2021) 458e469 reported spreading ability of MVs has suggested their potential of DMEM (Invitrogen), supplemented with 10% fetal bovine serum exploitation as biomarkers or as engineered therapeutic carriers (FBS; Thermo Fisher), 100 IU/mL penicillin, and 10 mg/mL [13]. A comprehensive correlation between conditions used to streptomycin. release and harvest MVs from the same cell type, i.e. astrocytes, and Cortical neurons were isolated from neonatal rat cortices their signaling ability, will impact our understanding of MVs (Wistar) at postnatal day 0e1 (P0eP1). Dissociated cells were then physiology and the design of MV-based biomedical applications in plated on poly-L-ornithine (Sigma) coated coverslips (Kindler, EU) the CNS [14,15]. Particular attention has to be conveyed to devise at a concentration of 150000 cells in a volume of 200 mL and novel, cost-effective ways in generating MVs, in particular incubated at 37  C; 5% CO2 in a culture medium composed of enhancing constitutive release. Neurobasal-A (Thermo Fischer) containing 2% B27 (Gibco), 10 mM Here, we concentrate on graphene oxide (GO), the most com- Glutamax and 0.5 mM Gentamycin (Gibco) for 8e10 days in vitro mon derivative of graphene, whose properties can be tailored to (DIV) before performing electrophysiological experiments. adapt to new physical and biological applications [16,17]. GO flakes have been successfully designed for drug delivery applications in 2.3. MV isolation biomedicine [18]. In the CNS, small GO nano-flakes (s-GO) were shown to induce constitutive MV release from cultured astrocytes MV shedding and isolation was performed as previously and to potentiate evoked MV release induced upon exposure to described [19]. One pool of MVs were collected from 21 to 24 DIV bzATP [19]. s-GO flakes, due to their physical features at the glial cultures previously treated with graphene oxide nanoflakes (s- nanoscale, were reported to interfere with cellular membrane dy- GO) (10 mg/mL [19]), added to culture medium once and left for 6 namics [19,20]. In addition, via adhesion to the plasma membrane, days. At the end of 6-days exposure, the medium was removed and s-GO may alter the mechanical features of the lipid bilayer [21] substituted with physiological saline solution, with the following triggering genuine biological responses, such as MVs signaling. composition: 152 mM NaCl, 4 mM KCl, 1 mM MgCl2, 2 mM CaCl2, Thus s-GOs may represent a tool to exploit mechanical signaling at 10 mM HEPES and 10 mM Glucose (pH adjusted to 7.4), at 37  C and the nanoscale to activate membrane release of MVs. Drug delivery 5% CO2 for 60 min prior to MVs collection and purification. The MVs applications where vesicle release from genetically engineered cells pool was isolated from cultures treated (30 min) with benzoyl-ATP is required, may take advantage of the mechanical modulation of (bzATP; 100 mM) diluted in physiological saline solution. The 6-days vesicle release brought about by graphene-based nanomaterials, exposure timepoint was chosen on the basis of previous Western representing a safer and cheaper alternative to pharmacological blot experiments, and confirmed by our current experiments tools. Prolonged exposure to biomolecules able to induce MVs testing MVs release after 3 days of s-GO exposure (supplementary release, such as ATP, could in fact negatively affect cell physiology Figure S2) [19]. Negative controls were incubated with physiolog- by promoting astrogliosis and inducing microglia-mediated neu- ical solution without the presence of bzATP or s-GO. After the in- roinflammatory responses [22]. cubation period, cell medium was collected and centrifuged for We use the ability of s-GO to substantially increase the pro- 15 min at a speed of 300g in order to remove cell debris. Super- duction of MVs from astrocytes to provide, for the first time, a natant was then collected and MVs were pelleted by centrifugation robust and comparative vesicle characterization by means of ultra- at 20000g for 2 h. For Ca2þ deprivation experiments, prior to microscopy, attenuated total reflectionefourier transform infra-red supernatant collection and MVs pellet centrifugation, cultures were (FTIR-ATR) and UV Resonant Raman (UVRR) spectroscopy. We pre-incubated for 45 min in a saline solution identical to the additionally explore by single cell patch-clamp recordings the physiological saline solution except for 0 mM CaCl2, 3 mM MgCl2 impact of acute, local and transient delivery of MVs on neuronal and 1 mM EGTA to allow the depletion of intracellular calcium basal synaptic activity and by atomic force microscopy (AFM) the storage. Upon this pre-treatment, we harvest the MVs from con- accompanying changes in neuronal plasma membrane elastic trols, s-GO treated and bzATP (30 min in Ca2þ deprived solution; features. supplementary Figure S3). 2. Material and Methods 2.4. Western blot analysis 2.1. Graphene oxide nanosheets synthesis MVs were prepared as previously reported, briefly they were re- suspended in lysis buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% GO was manufactured under endotoxin-free conditions through NP40, 0.1% SDS), sonicated for 30 s, and then boiled at 95  C for our modified Hummers’ method as previously described [19]. The 5 min [19]. Samples were run on a 10% polyacrylamide gel and complete characterization of the material used is shown and blotted onto nitrocellulose membranes (Millipore, Italy). Mem- summarized in the Supplementary experimental section and branes were then blocked in PBS-Tween-20 (0.1%) plus 5% nonfat Supplementary Figure S1 and Table S1. dry milk and incubated with the primary antibody antiflotillin-1 (dilution 1:1000) for 16 h at 4  C. Membranes were then washed 2.2. Cell cultures with PBS-Tween and incubated with peroxidase-conjugated anti- mouse secondary antibody (dilution 1:1000). Detection of immu- All experiments were performed in accordance with the EU nolabeled ECL-exposed protein bands was measured with UVI-1D guidelines (Directive 2010/63/EU) and Italian law (decree 26/14) software over three independent experiments. and were approved by the local authority veterinary service and by our institution (SISSA-ISAS) ethical committee. All efforts were 2.5. Immunofluorescence and confocal microscopy made to minimize animal suffering and to reduce the number of animals used. Animal use was approved by the Italian Ministry of Primary glial and cortical neurons cultures were fixed in 4% Health, in agreement with the EU Recommendation 2007/526/EC. formaldehyde (PFA, prepared from fresh paraformaldehyde) in PBS Primary glial cultures were obtained from cortices isolated from for 20 min at room temperature (RT) and then washed in PBS. Free neonatal rats (Wistar) at postnatal day 2e3 (P2eP3), as previously aldehyde groups were quenched in 0.1 M glycine solution for 5 min. described [19,23]. Dissociated cells were plated into plastic 150 cm2 The samples were permeabilized in 5% fetal bovine serum (FBS), flasks and incubated at 37  C; 5% CO2 in culture medium composed 0.3% Triton-X 100 in phosphate buffer solution (PBS) for 30 min at 459 M. Musto, P. Parisse, M. Pachetti et al. Carbon 176 (2021) 458e469 RT. Samples were then incubated with primary antibodies (mouse processing was performed using Gwyddion freeware AFM analysis monoclonal anti-GFAP, Invitrogen, 1:500 dilution; rabbit polyclonal software, version 2.40. Diameter and height of each vesicle were anti-b-tubulin III, Sigma-Aldrich, 1:500 dilution) diluted in PBS evaluated from cross-line profiles, and results were statistically with 5% FBS at 4  C for 1 h. Samples were then incubated with analyzed using Prism (Graphpad software). secondary antibodies (Alexa 488 goat anti-mouse, Invitrogen, 1:500 dilution; Alexa 594 goat anti-rabbit, Invitrogen, 1:500 dilution), and 2.9. Neuronal stiffness DAPI (Invitrogen, dilution 1:200) to stain the nuclei, for 45 min at RT and finally mounted on 1 mm thick glass coverslips using Flu- 9-10 DIV cortical neurons were exposed to MVs obtained by glial oromount mounting medium (Sigma-Andrich). Images were ac- cultures treated with bzATP or s-GO and neuronal rigidity was quired using a Nikon C2 Confocal, equipped with Ar/Kr, He/Ne and assessed with AFM, 24 h after MVs exposure. Force spectroscopy UV lasers with a 40  or 60  (1.4 NA) oil-objective (using oil measurements were performed with a commercial Smena AFM mounting medium, 1.515 refractive index) to acquire glial cultures (NT-MDT, RU) mounted on an inverted microscope (Nikon Eclipse images and cortical neurons images respectively. 200  200 mm TieU). AFM cantilever deflection was measured when pushed fields were acquired for cortical neurons images and 300  300 mm against cortical neurons plated on a glass coverslip. Deflection fields were acquired for glial cells images. Confocal sections were values were subsequently converted into a force versus indentation acquired every 0.25 mm for both the cultures. curve based on cantilever spring constant and its displacement. Neuronal rigidity was evaluated in 50 randomly chosen neurons for 2.6. Glial cell viability assay each condition (from 3 independent experiments), acquiring three force spectroscopy curves in the center of each cell soma. The AFM Primary rat astrocytes (21e24 DIV) were exposed to s-GO 10 mg/ tip was positioned by using an inverted microscope in bright field mL or to equivalent volumes of the vehicle for 6 days. Cells were mode. stained with propidium iodide (PI, 1 mg/mL; 15 min) for cell death AFM micro-cantilevers with an elastic constant of about quantification and subsequently fixed in PFA and labeled for DAPI 0.03 nN/nm and a resonance frequency of about 10 kHz (CSG01 for nuclei visualization and GFAP for visualizing astrocytes. The red tipless cantilevers from NT-MDT, RU) were used. A borosilicate (PI positive) fluorescent nuclei indicating dead cells were quanti- glass microsphere of about 18 mm in diameter (18.2 ± 1.0 mm from fied at 40  (1.4 NA) magnification using a Nikon C2 Confocal mi- Duke Standards, CA, USA) was manually glued at the end of each croscope, equipped with Ar/Kr, He/Ne and UV lasers, with random cantilevers using a UV curable glue (Norland Optical Adhesive 61 sampling of 10 fields per sample (n ¼ 3 coverslips/sample, from 3 from Norland Products Inc., NJ, USA). Force spectroscopy mea- independent culture preparations). The average percentage of dead surements were performed at a constant indentation speed of cells was calculated counting visual fields selected. 1 mm/s with a maximum value of indentation deepness set at 500 nm. Elastic modulus values (E), expressed in kPa, were deter- 2.7. FM1-43 staining mined by fitting obtained forceeindentation curves with a Hertzian model for the tip, using AtomicJ (v. 1.7.3) analysis software [24]. Glial cells were incubated with the fluorescent styryl dye FM1- 43 (2 mM) for 2 min in order to completely stain plasma membrane, 2.10. Nanoparticle tracking analysis (NTA) then extensively washed with PBS and exposed for 30 min to bzATP (100 mM) or to standard saline solution [19]. Samples were placed Measurement and analysis of MVs size distribution by NTA was in a recording chamber mounted on an inverted microscope (Nikon performed on a NanoSight LM10 system (Malvern) using approxi- Eclipse TieU) and observed with a 40 objective (0.6 NA, PlanFluor, mately 500 mL of MVs of both conditions (bzATP-derived and s-GO- Nikon). Images (512  512 px) were acquired for 10 min with an derived) diluited 1:20 in MilliQ H2O. Individual videos of 60 s exposure time of 150 ms (6.6 Hz) by a Hamamatsu Orca-Flash 4.0 (recorded at 25 FPS; 3 videos per group) for each sample were digital camera controlled by an integrating imaging software acquired at RT using the maximum camera gain, a detector package (HCImage, Hamamatsu). Recorded images were analyzed threshold equal to 8 and analyzed by the NanoSight particle offline with the Clampfit software (pClamp suite, 10.2 version; Axon tracking software to calculate size and vesicle concentration. Instruments). Image time stacks were analyzed in selected regions of interest (ROI) to measure the variations in FM1-43 fluorescence 2.11. FTIR-ATR spectroscopy and UV Resonant Raman (UVRR) intensity over time. Natural sample bleaching over time, due to measurements prolonged light exposure, has the same time-course and intensity in all the three groups, as previously described [19]. MVs were isolated from 21 to 24 DIV glial cultures by centrifu- gation as described above. MVs pellet was successively washed 2.8. Atomic force microscopy analysis with NaCl solution (150 mM) and finally re-suspended in 50 mL of the same solution in order to avoid contribution of phosphate and AFM characterization was performed as previously described sugar groups to the IR absorbance spectra. The IR measurements [19]. Briefly, the pellet of MVs was re-suspended in PBS solution were carried out at the BL10.2-IUVS beamline at Elettra synchro- after isolation from cell cultures and a 15 mL drop of sample solution tron Trieste. The spectra were collected in ATR mode using a MIR was placed and left to adsorb (30 min) onto a freshly peeled mica DLaTGS detector and a KBr-broadband beam-splitter. For each IR substrate. Vesicles were then fixed with 1% formaldehyde for 1 h measurement, 2 mL of sample solution were spread over the whole (RT) in order to prevent their collapse during AFM acquisition. MVs area of a monolithic diamond ATR plate and left to dry forming a were then washed with PBS and dried under a gentle stream of thin film. For each sample, 20 spectra were collected in the range nitrogen. AFM analysis was performed in air at RT, using the sem- 4000e800 cm1, accumulating 256 scans for each spectrum icontact mode of a commercial instrument (Solver Pro, NT-MDT, reaching a resolution of 4 cm1. Each spectrum was corrected for RU). Silicon tips (NSC36/CR-AU, MikroMash, USA) with a typical the background, aqueous vapor, CO2 and not normalized to any force constant of 0.6 nN/nm and a resonance frequency of about absorbance band. 65 kHz were employed. Topographic height and phase images were UVRR measurements were performed at the BL10.2-IUVS recorded at 512  512 pixels at a scan rate of 0.5 Hz. Image beamline at Elettra synchrotron Trieste using the experimental 460 M. Musto, P. Parisse, M. Pachetti et al. Carbon 176 (2021) 458e469 set-up reported [25]. 5 mL of vesicles solution were drop-casted homogenous s-GO dispersions containing s-GO nanosheets with onto an aluminum foil, allowed to dry and kept under nitrogen lateral dimensions predominantly between 50 and 500 nm [19,20]. purging. All the measurements were performed at RT, using an We treated pure glial cell cultures with s-GO (10 mg/mL) for 6 days excitation wavelength of 244 nm and tuning the power of the [19]. Immunofluorescence labeling by antigen against glial- incoming radiation to 50 mW. The outcoming radiation was fibrillary acidic protein (GFAP), an intermediate filament protein collected in backscattering geometry by using a triple stage spec- that is highly specific for cells of the astroglial lineage, was used to trometer (Trivista, Princeton Instrument) with a spectral resolution visualize control and s-GO-treated neuroglial cultures (GFAP, in of 8 cm1. Samples were continuously oscillated horizontally in green; Fig. 1a) [26]. At the low concentrations used, s-GO treatment order to avoid photodamaging. did not impair astrocyte morphology and cell density when compared with matched control cultures (box-plot in Fig. 1b) 2.12. Electrophysiological recordings [19,27]. Viability of glial cells was confirmed by propidium iodide (PI) cell death assay. Control and s-GO treated cultures were incu- Patch-clamp recordings (whole-cell, voltage clamp mode) were bated with PI, which stain dead cells nuclei (Fig. 1c, in red) and the performed from visually identified (under differential interference percentage of PI-positive nuclei was calculated (bar plot in Fig. 1d). contrast - DIC - microscopy) cortical neurons (DIV 8e10) placed in a Graphene nanosheets cytotoxicity is a largely debated issue, due to recording chamber, mounted on an inverted microscope (Eclipse the variable impact on cell toxicity of several material’s features, TieU, Nikon, Japan) and superfused with control physiological saline related either on the GO physical-chemical properties (thickness, solution of the following composition (in mM): 152 NaCl, 4 KCl, 1 size, surface functionalization, aggregation state and concentra- MgCl2, 2 CaCl2, 10 HEPES and 10 Glucose (pH adjusted to 7.4 by tion) or on the synthesis method [28,29], the experimental condi- NaOH 1 M; osmolarity 300 mOsm). Cells were patched with glass tions adopted here and in our previous works [19,20] exclude any pipettes (4e7 MU) containing (in mM): 120 K gluconate, 20 KCl, 10 cytotoxic effect on glial cells or neurons, both in vitro and in vivo. HEPES, 10 EGTA, 2 MgCl2 and 2 Na2ATP (pH adjusted to 7.35 by KOH; MVs are released into the extracellular space by direct budding osmolarity 298 mOsm). All electrophysiological recordings were from the plasma membrane of astrocytes [30]. To explore the dy- performed at RT and the spontaneous, basal synaptic activity was namics of MVs release in control, in s-GO treated and in ATP treated recorded by clamping the membrane voltage at 70 mV (corrected (see below) astrocytes, we measured the presence of changes in for liquid junction potential, which was 14 mV). To investigate the membrane trafficking by briefly incubating cultures with the acute effect on synaptic activity of glia-derived MVs, an injection fluorescent styryl dye FM1-43 and then quantifying the astrocyte- pipette (patch pipette with resistance of 1e4 MU) filled with MVs membrane fluorescence decay to provide a cumulative measure previously isolated glial cultures as described above and re- of exocytosis in the different growth conditions [19,30]. suspended in 100 mL of extracellular saline solution was positioned FM dyes are fluorescent probes that reversibly stain membranes, at 200 mm from the cell soma and connected to a pico-spritzer and are largely used for optical real-time measurements of mem- (PDES-02DX, npi Electronics) with 0.3 psi in-line pressure [19]. On brane dynamics and secretory processes [31e33]. Incubation with the basis of MVs quantification obtained by NTA measurements, and the FM dye (2 mM, 2 min) resulted in clear surface membrane considering that MVs isolation was performed using the same pro- staining of control, bzATP, an agent known to evoke massive MVs tocol in all the experiments described, we calculated that the con- release (100 mM, 30 min) and s-GO treated cultures (10 mg/mL, 6 centration of MVs used for these tests was approximately of days), highlighted in Fig. 1e (left panels) [30]. Brighter spots were 6.64  109 for MVs obtained by bzATP stimulation and 1.64  1010 for considered as adherent debris and were excluded from the analysis. MVs isolated from s-GO treated cultures. Baseline spontaneous Besides these, both bright and weak FM-stained plasma membrane synaptic activity was recorded for the 10 min prior delivering the domains were present along the whole cytoplasmic surface and puff (500 ms duration) of MVs and followed up for 20 min to verify became visible within 2 min incubation (Fig. 1e). Due to this initial changes in post synaptic current (PSC) frequency and amplitude variability in the intensity of the membrane staining, all FM de- induced by the fusion of MVs with neuronal membranes. staining measures were normalized to the relative time 0. Once Data were collected by Multiclamp 700B patch amplifier (Axon astrocyte membranes were labeled by the fluorescent dye FM1-43 CNS, Molecular Devices) and digitized at 10 kHz with the pClamp we measured the plasma membrane de-staining over a fixed time 10.2 software (Molecular Devices LLC, USA). All recorded events (10 min) in control, in s-GO treated cultures, or during acute were analyzed offline with the AxoGraph 1.4.4 (Axon Instrument) exposure to bzATP, under the same culturing conditions [19]. event detection software (Axon CNS, Molecular Devices). Representative fluorescence intensity traces are shown in Fig. 1f (top plot); the dynamic of the fluorescence decay observed in 2.13. Statistical analysis control cultures, indicates the presence of physiological bleaching of fluorophore over the acquisition time course, however s-GO and Data sets found to follow a non-normal distribution, were rep- bzATP groups, despite the same bleaching-induced loss of fluo- resented as box plot. The central thick horizontal bar in the box rescence, showed a faster de-staining rate in respect to controls. plots indicates the median value, while the boxed area extends This is also visualized by the time-lapse images framed at time 0 s, from the 25th to 75th percentiles with the whiskers ranging from 300 s and 600 s of the crude recordings (Fig. 1e middle and right the 2.5th to the 97.5th percentiles. Statistically significant differ- panels). We quantified the fluorescence decay time constant (t) ences between two non-parametric data sets were assessed by values (box plot of Fig. 1f) and detected shorter decay values in both Mann-Whitney’s test, while to assess statistically significant dif- bzATP and s-GO groups (mediancontrol ¼ 219 s; ferences among three data-set we used the Kruskal-Wallis test and medianbzATP ¼ 184.2 s; medians-GO ¼ 163.9 s). This result suggested Dunn’s post hoc test. P < 0.05 was considered at a statistically that the membrane de-staining was actually related to MVs release, significant. as expected in bzATP treated cells, more than to other membrane turnover activities. Such a release was comparable between bzATP 3. Results and discussion and s-GO, both significantly faster than controls (PbzATP < 0.001; Ps- GO < 0.001). Astrocytes were isolated from postnatal (2e3 days) rat (Wistar) The release of MVs suggested by FM1-43 measures, was cortices, as previously described [19,20,23]. We used visually confirmed by immunoblot analysis for the biomarker flotillin-1 of 461 M. Musto, P. Parisse, M. Pachetti et al. Carbon 176 (2021) 458e469 Fig. 1. Graphene oxide nanosheets does not affect astrocytes vitality. Cultured astrocytes release microvesicles (MVs) upon ATP or s-GO stimulation. a) Confocal images visualize cultured astrocytes in control and after s-GO (10 mg/mL; 6 days) treatment; anti-GFAP, in green, and DAPI (to visualize nuclei), in blue; scale bar 50 mm. b) Box plot summarizes the cell density measures; note the similar values in both groups. c) Confocal images visualize cultured astrocytes in control and after s-GO (10 mg/mL; 6 days) treatment. Cultures were treated with propidium iodide (PI) to visualize death cells. Anti-GFAP, in green, DAPI (to visualize nuclei), in blue and PI in red; scale bar 50 mm. d) Histogram summarizes the percentage of death cells followed the s-GO exposure and in control condition; note that there are no significant difference between two groups e) Surface membrane staining and activity dependent de-staining of FM1-43 in cultured astrocytes, scale bar 25 mm. f) Normalized FM1-43 de-staining traces (top) in control astrocytes (light grey), in bzATP treated once (orange) and in s-GO treated once (black). The box plot (bottom) summarizes the decay time constant t of FM1-43 de-staining in the three conditions (mediancontrol ¼ 219.2 s; medianbzATP ¼ 184.2 s; medians-GO ¼ 163.9 s). Thick horizontal bars in the box plots indicate median value; boxed area extends from the 25th to 75th percentiles, whiskers from 2.5th to the 97.5th percentiles. Significance: **P < 0.01 ***P < 0.001, Kruskal-Wallis test, Dunn’s post hoc test). (A colour version of this figure can be viewed online.) the supernatant collected from control and treated cultures with bzATP (s-GORinger is quantified as 100% more than Con- (Fig. 2a). As expected, bzATP stimulation and s-GO incubation trolRinger; bzATPRinger is quantified as 360% more than ControlRinger; induced the appearance of a thick band corresponding to flotillin-1 bzATPs-GO is quantified as 2900% more than ControlRinger. Calcu- (Fig. 2a), a signature of MVs release by astrocytes, with an additive lated over three independent experiments) [19,30,34]. In control effect between s-GO exposure and pharmacological stimulation conditions only a weak band was perceived, indicating that MVs 462 M. Musto, P. Parisse, M. Pachetti et al. Carbon 176 (2021) 458e469 Fig. 2. Graphene oxide nanosheets and bzATP induce MVs release in Astrocytes. Microvesicle released by glial cells via bzATP or s-GO stimulations characterized by ultra- resolution approaches. a) Western blotting of the pellets (top row) and cell lysates (bottom row) for the MV marker flotillin-1 (N ¼ 3). Pellets were obtained from the medium of glial cultures treated or untreated (control) with s-GO under two different conditions: not stimulated (ringer) or stimulated (bzATP) by 100 mM bzATP. s-GORinger is quantified as 100% more than ControlRinger; bzATPRinger is quantified as 360% more than ControlRinger; bzATPs-GO is quantified as 2900% more than ControlRinger. Calculated over three independent experiments. b) AFM topographic reconstruction of MVs isolated from cultured primary astrocytes treated with bzATP (100 mM) and s-GO (10 mg/mL) and performed in air (semi- contact mode). Scale bar 500 nm. c) Lateral size values distribution and median values for both groups, note that mvG lateral size is significantly smaller than that of mvA (medianmvA ¼ 479 nm; medianmvA ¼ 244.1 nm; ***P > 0.001, Mann-Whitney test). d) AFM measures of lateral size are plotted against AFM measures of height of MVs isolated from glial cells treated by bzATP (100 mM; mvA; in orange) or by s-GO (10 mg/mL; mvG; in black). e) Size distribution of MVs isolated from glial cells treated by bzATP (100 mM; in orange) or by s-GO (10 mg/mL; in black) measured by nanoparticle tracking analysis (NTA). Values of the peaks are expressed in nm. f) Lateral size values distribution and median values for both groups, obtained by NTA measurement. (A colour version of this figure can be viewed online.) constitutive release in culture was poorly detectable. Atomic force control condition, a thick band appeared upon calcium removal, microscopy (AFM) topographic reconstruction of re-suspended suggestive of an increase in constitutive release (supplementary MVs pellet (Fig. 2b) confirmed the presence of MVs detected by Fig. S3). These preliminary results hint at release mechanisms the immunoblot in both bzATP (mvA) and s-GO (mvG) groups. differently tuned by calcium among control, bzATP and s-GO and When investigating the effect of shorter (3 days) exposure to s-GO, require further studies. Western blot experiments (supplementary Fig. S2) showed the We systematically investigated and compared the MV size dis- absence of a significant increase in MVs constitutive release when tribution by means of AFM and nanoparticle tracking analysis (NTA) compared to control. Yet, bzATP release of MVs was potentiated by measurements. AFM images show the presence of roundish pro- 3 days s-GO (supplementary Fig. S2) suggesting that s-GO already trusions of dimensions compatible with the size of MVs. No other modulated MVs release, but longer time of s-GO exposure are kind of contaminant was present, to indicate that the procedure for needed to enhance basal release in the absence of additional isolating and collecting MVs from the medium was clean and stimuli. In an additional set of Western blot experiments, we tested effective. When analyzed by AFM (Fig. 2c) s-GO-derived MVs (mvG) the sensitivity of bzATP and s-GO MVs release to extracellular cal- lateral size were significantly smaller (n ¼ 72, medianmvG ¼ 244 nm) cium deprivation (supplementary Fig. S3). Differently from bzATP, than bzATP-derived ones (mvA; n ¼ 107, s-GO release was apparently not affected by calcium removal. In medianmvA ¼ 479 nm) (P < 0.001). Conversely, we detected no 463 M. Musto, P. Parisse, M. Pachetti et al. Carbon 176 (2021) 458e469 Fig. 3. MVs characterization by infrared spectroscopy. Microvesicles produced by glial cells via bzATP or s-GO stimulation and characterized by infrared spectroscopy. a) Infrared spectra of microvesicles obtained by bzATP stimulation (mvA, in orange) or by s-GO exposure (mvG, in black) in the region 950-3600 cm1. Contributions arisen from nucleic acids, proteins and lipids characterize the spectra. The two boxes in green and the green dashed lines are used as eye-guides to highlight nucleic acids, CH2eCH3 stretching of lipids and protein amide bands, respectively. b) Fitting procedure applied to mvA (on the left) and mvG (on the right) amide bands spectra. Multicomponent Gaussian curves were used to actually reproduce the experimental data. The centres of the Gaussian curves were chosen as the minimum of the 2nd derivative of the spectrum and kept free to variate within 4 cm1 around its maximum. c) Histograms representing the areas of the Gaussian curves used to reproduce the experimental data of mvA (in orange) and mvG (in black) in the region 1500-1785 cm1. The areas of each band has been weighted respect to the total amide band area which they belongs to (i.e. 1640 cm1 band has been weighted with respect to the total AmeI band area). (A colour version of this figure can be viewed online.) differences in MV height values (medianmvG ¼ 19 nm; lead to a variable collapsing of MVs when measured in air. medianmvA ¼ 22 nm; P ¼ 0.17). Within each group, the distribution of Since AFM measurements are affected by the reduced size of the size values detected was not negatively correlated to the height, as analyzed samples, and might not reflect the entire MVs population, shown in Fig. 2d (left; rmvG ¼ 0.8808 and rmvA ¼ 0.4039; P < 0.001). we decided to use nanoparticle tracking analysis (NTA) to perform However, AFM experiments were performed in air, thus a not specific bulk analysis of vesicles in aqueous suspension. NTA tracks single flattening of MVs caused by vesicle collapsing might have influenced particle Brownian motion within a dark field microscope, derives these measurements. In principle differences in MV elastic proper- mean square vesicles velocity and translates them into size distri- ties, potentially related to diverse membrane components, might bution [35]. NTA revealed a more complex pattern of size 464 M. Musto, P. Parisse, M. Pachetti et al. Carbon 176 (2021) 458e469 Fig. 4. MVs released by astrocytes affect cortical neuron post-synaptic activity and mechanical properties. Potentiation of synaptic activity upon local applications of MVs in cortical neurons. a) Confocal micrograph visualizing cortical primary cultures at 8 days in vitro; anti-class III b-tubulin is used to visualize neurons (in red), anti-GFAP for astrocytes (in green) and DAPI (in blue) to visualize neurons. Scale bar 50 mm. On the right, a representation of the experimental setting for the simultaneous MVs pressure-release (puff) and the cell patch-clamp recording from cultured neurons. b) Box plot summarizes the PSCs frequency and amplitude values in control cortical neurons. c) Top: diagram of the experimental protocol. Bottom: representative current tracings of the spontaneous synaptic activity detected prior and after puff applications of control saline (in light grey) or mvA 465 M. Musto, P. Parisse, M. Pachetti et al. Carbon 176 (2021) 458e469 distribution (Fig. 2e): in the case of mvA we observed three sub- can conclude that mvGs contain proteins with perturbed secondary populations of vesicles at 115 nm, 235 nm and 400 nm respectively structure, characterized by beta structure-based aggregates and while in the case of mvG we found two partially overlapping peaks flexible helices. No significant variations in the DNA phosphate at 135 nm and 168 nm, plus two distinct peaks at 275 nm and bands were measured as well as the nature or localization of the 385 nm (Fig. 2e). The diameter analysis revealed a slight, but not DNA signature detected. It is interesting to note that in previous significant, difference between the two populations with the measures the DNA delivered by EVs has been reported to be stocked diameter of mvG smaller and less distributed, compared to those of either inside the vesicles or on their surface [39]. mvA (medianmvA ¼ 235.4 nm; medianmvG ¼ 183.6 nm) (Fig. 2f). UV Resonant Raman (UVRR) measurements taken using an These results convincingly suggested a comparable size distribu- excitation wavelength of 244 nm (see experimental section and tion in both MV populations and subpopulations. However, we supplementary Fig. S4) confirmed the amide I signal depression in detected a significant difference in the number of vesicles released mvG absorption spectrum. Due to an overlap between the UVRR s- within the same time window (Fig. 2e) to indicate that cultures GO band, we could not confirm or discard the presence of s-GO treated with s-GO produced more MVs when compared to cultures inside the vesicles. Thus, we cannot exclude that the changes in stimulated with bzATP (mvA ¼ ̴3.32  108 vesicle/mL; protein native structure might be due to s-GO altering the MV mvG ¼̴8.19  108 vesicles/mL), consistently with our results ob- micro(nano)environment or the possibility of a general interfer- tained by MV release analysis and immunoblot (but see also Vis- ence with the measurements. The absence of astrocytes cytotox- novitz et al., 2019 [36]). Therefore, even though the overall size of icity, even upon prolonged exposure to s-GO, together with the the MV population produced did not change, s-GO was more effi- functional measures of increased MVs release kinetic obtained by cient in generating MVs from astrocytes. live imaging, are against a mere alteration in protein integrity due In order to analyze the macromolecular composition of MVs we to denaturation of membrane proteins in the presence of s-GO took advantage of two complementary techniques: FTIR-ATR and [40e42]. FTIR-ATR spectroscopy is a powerful tool to assess the UVRR spectroscopy. For these measurements, MVs were isolated by disordered character of proteins, and the absence of a well-defined differential centrifugation as described before (see Material and structure under native conditions is a peculiar property of intrin- Methods). To avoid any spurious effect due to the absorption of sically disordered proteins (IDP) [43,44]. In this framework, IDPs phosphate groups from the buffer, we washed and re-suspended might represent a specific signal vehiculated by mvG and the lack of the MVs pellet with a NaCl solution (150 mM). The infrared (IR) protein structural constraints could facilitate several, yet unknown, absorbance spectra (Fig. 3a) revealed a clear contribution of the CH biological processes [44]. and the phosphate bands linked to DNA in both samples, mvA and It is clearly visible that mvA and mvG vesicles have a different mvG, as well as the lipid signatures at 2900-3000 cm1, arisen from secondary structure: the former is mainly characterized by an the CH2 and CH3 stretching mode. The amide I and amide II bands intense peak at 1645 cm1 (43%), which can be assigned to random between 1500 and 1785 cm1, the two major protein bands in the IR structures/a-helix structure, and at 1678 cm1 (22%) usually spectrum, show more pronounced differences between the two assigned to b-turn and at 1613 cm1 (27%), which could be assigned populations of vesicles. The amide I band, which is primarily related to side chains vibrations; in contrast, mvG are mainly characterized to the C]O stretching in the peptide bonds and modulated by the by anti-parallel b-sheets structure (1632 (15%) and 1693 cm1 (3%) proteins’ secondary structures, displayed similar shapes for mvA bands) with a strong contribution of flexible 3e10 helix (1661 cm1 and mvG. The amide II band, primarily due to CeN stretching and peak (34%)) and of side chain band at 1610 cm1 (37%). NeH in plane bending vibrations, also reflecting the protein sec- Finally, we set up a functional test to compare the impact of mvG ondary structure, was instead clearly depleted in the case of mvG. delivery with that of mvA on synaptic activity, when neuronal To gain insights into the contribution of different protein secondary networks are acutely and transiently exposed to MVs. To this aim, structures in the two families of vesicles, we analyzed the second we isolated cortical neurons and glial cells from postnatal rat derivative of the IR signal in the amide I þ amide II region (Fig. 3b) cortices and cultured them for 10 days. Fig. 4a shows confocal high and used the position of the minima to guide a multicomponent magnification microscopy images of cortical cultures where neu- gaussian fit of the bands in the region 1500 and 1785 cm1. The % rons were visualized by labeling class III b-tubulin (in red), a area contributions obtained by the fit are reported in Fig. 3c. The microtubule component expressed exclusively by neurons, while low-energy flank of amide I of both mvA and mvG is characterized astrocytes were visualized by GFAP labeling (in green) [45]. We by the presence of the 1610-1613 cm1 vibrational peak which patch-clamped visually identified cortical neurons (in voltage could be addressed to a mixed contribution arisen from b-sheets clamp configuration, holding potential e 70 mV), while a second and side-chains vibrations. Additionally, the band at 1660 cm1 pipette for the local delivery of saline solution was positioned at a might also be derived from the presence of RNA in the vesicles [37]. distance of 200 mm (under microscopy visual control) from the Noteworthy, the relative populations of the peaks at approximately recorded cell (sketched in Fig. 4a, right). We estimated that, at this 1645 cm1, 1660 cm1 and 1678 cm1 are inverted between mvA distance, the application of a brief (500 ms) pulse of pressure and mvG. According to the literature [37], the 1645 cm1 peak should result in a local (i.e. on the recorded neuron) and transient (depleted in mvG) might be assigned to random structures and/or delivery of standard saline solution alone or containing mvG or helices; the 1660 cm1 peak (depleted in mvA) to flexible helices mvA (re-suspended in saline). A typical feature of these cultures is (as 310 helices); the 1678 cm1 peak (depleted in mvG) to beta the prominent expression of spontaneous synaptic activity, repre- structures such as b-turns. The bands at 1632 cm1 and at sented by heterogeneous postsynaptic currents (PSCs) of variable 1693 cm1, both present in the mvG only, are ascribed to anti- frequency and amplitude (box plots in Fig. 4b). Baseline PSCs were parallel beta sheet, as found in aggregates in tissues [37,38]. We recorded before (10 min) and after (15 min) the local saline, mvG or (in orange) or mvG (in black). d) Bar plots of pooled data summarize the % of cells displaying PSCs frequency increase upon delivery of pressure ejected saline (light grey), mvA (orange) and mvG (black). Note that in control (saline) the large majority (88%) of neurons did not increase their basal activity. e) The plot summarizes the distribution of the % of increase in PCSs frequency detected within the three groups. f) Elastic moduli of cortical neurons, grown on glass, and exposed to MVs isolated from glial cells previously treated with bzATP (orange) or s-GO flakes (black). Neurons treated with mvA and mvG are significantly less rigid if compared with control (PmvA < 0.001; PmvG <0.01). Thick horizontal bars in the box plots indicate median value; boxed area extends from the 25th to 75th percentiles, whiskers from 5th to the 95th percentiles. Significance: **P < 0.01 ***P < 0.001, Kruskal-Wallis test, Dunn’s post hoc test) g. Indentation curves of cortical neurons previously treated with mvA or mvG. (A colour version of this figure can be viewed online.) 466 M. Musto, P. Parisse, M. Pachetti et al. Carbon 176 (2021) 458e469 mvA ejection. Fig. 4c shows representative current tracings where in facilitating synaptic vesicles release by changing the membrane standard saline was pressure ejected (light grey, top), or where mvA composition at pre-synaptic level [48,53]. This intrinsic capacity of solution (orange, middle) and mvG (black, bottom) were MVs to participate in membrane lipid metabolism may therefore administered. modulate the contribution of plasma membrane to neuronal ri- Since spontaneous fluctuations in PSCs frequency 15% of base- gidity. In this context, the slight difference of stiffness values re- line values were frequently detected, we took this as the threshold ported among mvA and mvG-treated neurons, although not value to estimate changes when comparing PSCs before and after statistically significant (PmvA-mvG> 0.05) should not be under- pressure ejections of saline. In the large majority (88%, n ¼ 16/18 estimated [54,55]. It is tempting to speculate that this difference neurons; histograms in Fig. 4d) of neurons exposed to saline solution can be explained by the presence of specific proteins in the vesicles, alone, spontaneous PSCs frequency did not change. On the contrary, that are unstable in mvG. Based on our measures, we cannot within 5e8 min from the acute mvA and mvG ejections, PSCs fre- exclude the presence of residual s-GO flakes either inside or on the quency was stably increased in 64% (n ¼ 16/25 neurons, mvA) and vesicle surface. It is known that GO is reportedly able to interact 54% (n ¼ 13/24 neurons, mvG; summarized in the histograms of with the hydrophobic region of biological and model lipid mem- Fig. 4d) of recorded neurons. Fig. 4e shows the increases in PSCs branes, even though the nature of the interaction is strictly frequency in individual experiments and highlights the variability of dependent to its size and degree of surface oxygenation [56,57]. such changes when administering MVs, with increased frequencies Assuming that the GO nanosheets, once added to the culture me- ranging from 25% to 200%. Since we could not experimentally control dium, can adsorb on plasma membrane or pierce it and being the amount of MVs collected by primary astrocytes and delivered by embedded in the lipid bilayer, there is a concrete possibility that pressure ejection, neurons were exposed to different amounts of MVs, which directly originate from plasma membrane, can include MVs and this can in part explain the detected variability. PSCs fre- those flakes in their structure [30]. The horizontal transfer of s-GO quency increases due to MVs exposures were not reversible upon from glial cells to cortical neurons, mediated by MVs, may therefore 20 min washout. From such functional investigation, glial-signaling affect plasma membrane rigidity of targeted neurons. Regardless of generated by ATP or s-GO affected similarly neuronal synapses this, we can exclude a direct effect of s-GO in synaptic transmission, upon transient, direct exposure. reported to be transient and reversible upon acute exposure, in To our knowledge, this is the first time that the functional effects view of the persistent modulation of synaptic current frequency of MVs generated by astrocytes on synaptic activity upon local brought about by MVs [19,20]. delivery have been electrophysiologically documented. Neuroglia extracellular vesicles have been described to provide support on 4. Conclusion synaptic activity, with the majority of studies focused on microglia and inflammation, apparently regulating neural transmission at the Astrocyte-derived MVs may play significant roles in propagating pre-synaptic level [5,46e48]. Astrocytic MVs have been proposed signaling molecules, in CNS physiology and disease. Despite the to exert neuroprotective effects in neuropathology and in physi- increasing knowledge on extracellular vesicles (in particular exo- ology, however the role of astrocytes or of discrete astrocyte pop- somes) ability to promote inflammation or contribute in spreading ulations in delivering different messages via MV release has yet to of pathogenic proteins in neurodegenerative disorders (from be elucidated [49,50]. Amyotrophic Lateral Sclerosis to Alzheimer disease [58,59]) little is After assessment of the ability of MVs released by glial cells to currently known on their properties (mechanical, biochemical, affect cortical neuron physiology within min after their interaction lipid membrane composition, cargo nature etc.), in particular when with the targeted neuron, we asked if the presumed fusion of focusing on the smaller class of such natural vectors, the MVs. vesicles with neuronal plasma membrane could also affect their Nevertheless, MVs emerge as key players in neuronal and synaptic mechanical properties. To investigate this aspect, we delivered MVs physiology, able to influence neurotransmission, or to support obtained from glial cultures previously treated with bzATP or s-GO neurons [48,60e62]. In our study, we report the ability of artificially to cortical neurons by re-suspending the isolated MVs in 100 mL of generating MVs by s-GO transient exposure. MVs generated by s- neuronal culture medium and adding them to neuronal cultures. GO were apparently characterized by altered protein content when 24 h after the exposure, force spectroscopy measurement on compared to the ATP-driven ones. Intriguingly, the tuning of syn- treated neuronal cultures were performed with AFM by positioning aptic activity by mvG or mvA was similar, supposedly being related the tipless cantilever with a borosilicate glass bead previously glued to features diverse from MV protein content. The ability of s-GO to on it [51], at the center of randomly chosen neurons. As showed by interfere with exo-endocytotic membrane dynamics is not sur- the boxplot in Fig. 4f, the exposure to mvA caused a significant prising, indeed we have described the ability of s-GO nanoflakes to softening of neuronal soma, when compared to controls interfere with presynaptic vesicle release in vitro and in vivo [19,20]. (medianmvA ¼ 0.22 kPa, mediancontrol ¼ 0.59 kPa; PmvA < 0.001). A In the current work, we describe the direct interference of MVs similar result was observed also in the case of mvG exposure even if with synaptic activity, presumably due to MVs fusion with the the effect exerted on neuronal stiffness is less pronounced than that target neuron plasma membrane. Such an approach holds the po- induced by mvA (medianmvG ¼ 0.28 kPa, mediancontrol ¼ 0.59 kPa; tential to open new opportunities in engineering MVs for synaptic PmvG < 0.01). The reported effect of mvA and mvG on neuronal cell targeting. In this framework, it is tempting to speculate that s-GO mechanical properties is presumably a consequence of vesicular interactions with the cell membrane mimic extracellular mechan- fusion with the cellular plasma membrane, which may affect its ical signaling at the nanoscale sufficient to enable the release of lipid composition. Since the mechanical properties of a cell are MVs, thus representing unconventional tools to exploit the physics mostly defined by plasma membrane features and the ones of the governing vesicle release. We feel pertinent here to consider the underlying cytoskeleton, a change in neuronal plasma membrane fact, that the enormous potentiality of graphene-based materials in lipid composition can partially justify the observed reduction in nanomedicine has already promoted the development of new mvA and mvG-treated neurons stiffness [52]. In particular, glia- generation-nanocarriers for either gene or drug delivery [17,63]. In derived MVs are able to transport the enzyme Acid sphingomyeli- this framework, we may speculate on future developments where nase (A-SMase) involved in the metabolism of sphingomyelin (SM), engineered cells are mechanically induced to release MVs, carrying a precursor of the phospholipid sphingosine (sph). Sph and its GO nanoflakes properly functionalized to deliver genes or drugs of metabolites have been already reported to play a fundamental role interest and thus representing either the trigger and the cargo. 467 M. Musto, P. Parisse, M. Pachetti et al. Carbon 176 (2021) 458e469 Author contributions Cell. Neurosci. 13 (2019) 41, https://doi.org/10.3389/fncel.2019.00041. [7] E. Cocucci, J. Meldolesi, Ectosomes and exosomes: shedding the confusion between extracellular vesicles, Trends Cell Biol. 25 (2015) 364e372, https:// M.M. performed cell biology, electrophysiology, and immuno- doi.org/10.1016/j.tcb.2015.01.004. fluorescence experiments and analysis; M.M. and P.P. designed and [8] G. Raposo, W. Stoorvogel, Extracellular vesicles: exosomes, microvesicles, and performed AFM experiments; M.P. performed IR and UVRR exper- friends, J. Cell Biol. 200 (2013) 373e383, https://doi.org/10.1083/ jcb.201211138. iments and analysis. CM and GDM performed biology and WB ex- [9] S.L.N. Maas, X.O. Breakefield, A.M. Weaver, Extracellular vesicles: unique periments; N.L. and K.K. contributed to the synthesis and intercellular delivery vehicles, Trends Cell Biol. 27 (2017) 172e188, https:// characterization of thin graphene oxide of biological grade. BB doi.org/10.1016/j.tcb.2016.11.003. [10] C. Frühbeis, D. Fro €hlich, E.-M. Kr€amer-Albers, Emerging roles of exosomes in performed SEM micrographs of GO. L.B. and L.C. conceived the neuroneglia communication, Front. Physiol. 3 (2012) 119, https://doi.org/ study; L.B. conceived the experimental design and contributed to 10.3389/fphys.2012.00119. the analysis of data; L.B. wrote the manuscript. [11] G. Lachenal, K. Pernet-Gallay, M. Chivet, F.J. Hemming, A. Belly, G. Bodon, B. Blot, G. Haase, Y. Goldberg, R. Sadoul, Release of exosomes from differen- tiated neurons and its regulation by synaptic glutamatergic activity, Mol. Cell. CRediT authorship contribution statement Neurosci. 46 (2011) 409e418, https://doi.org/10.1016/j.mcn.2010.11.004. [12] K.E. van der Vos, L. Balaj, J. Skog, X.O. Breakefield, Brain tumor microvesicles: insights into intercellular communication in the nervous system, Cell. Mol. Mattia Musto: Formal analysis, performed cell biology, elec- Neurobiol. 31 (2011) 949e959, https://doi.org/10.1007/s10571-011-9697-y. trophysiology, and immunofluorescence experiments and analysis, [13] D. Ha, N. Yang, V. Nadithe, Exosomes as therapeutic drug carriers and delivery designed and performed AFM experiments. Pietro Parisse: vehicles across biological membranes: current perspectives and future chal- lenges, Acta Pharm. Sin. B. 6 (2016) 287e296, https://doi.org/10.1016/ designed and performed AFM experiments. Maria Pachetti: Formal J.APSB.2016.02.001. analysis, performed IR and UVRR experiments and analysis. [14] C. Verderio, L. Muzio, E. Turola, A. Bergami, L. Novellino, F. Ruffini, L. Riganti, Christian Memo: performed biology and WB experiments. Giu- I. Corradini, M. Francolini, L. Garzetti, C. Maiorino, F. Servida, A. Vercelli, M. Rocca, D.D. Libera, V. Martinelli, G. Comi, G. Martino, M. Matteoli, R. Furlan, seppe Di Mauro: performed biology and WB experiments. Belen Myeloid microvesicles are a marker and therapeutic target for neuro- Ballesteros: performed SEM micrographs of GO. Neus Lozano: inflammation, Ann. Neurol. 72 (2012) 610e624, https://doi.org/10.1002/ contributed to the synthesis and characterization of thin graphene ana.23627. [15] S. EL Andaloussi, I. Ma€ger, X.O. Breakefield, M.J.A. Wood, Extracellular vesicles: oxide of biological grade. Kostas Kostarelos: contributed to the biology and emerging therapeutic opportunities, Nat. Rev. Drug Discov. 12 synthesis and characterization of thin graphene oxide of biological (2013) 347e357, https://doi.org/10.1038/nrd3978. grade. Loredana Casalis: conceived the study. Laura Ballerini: [16] K.P. Loh, Q. Bao, G. Eda, M. Chhowalla, Graphene oxide as a chemically tunable Writing - original draft, conceived the experimental design and platform for optical applications, Nat. Chem. 2 (2010) 1015e1024, https:// doi.org/10.1038/nchem.907. contributed to the analysis of data, wrote the manuscript. [17] G. Reina, J.M. Gonza lez-Domínguez, A. Criado, E. Va zquez, A. Bianco, M. Prato, Promises, facts and challenges for graphene in biomedical applications, Chem. Declaration of competing interest Soc. Rev. 46 (2017) 4400e4416, https://doi.org/10.1039/c7cs00363c. [18] M. Baldrighi, M. Trusel, R. Tonini, S. Giordani, Carbon nanomaterials inter- facing with neurons: an in vivo perspective, Front. Neurosci. 10 (2016) 250, The authors declare no competing financial interest. https://doi.org/10.3389/fnins.2016.00250. [19] R. Rauti, N. Lozano, V. Leo n, D. Scaini, M. Musto, I. Rago, F.P. Ulloa Severino, A. Fabbro, L. Casalis, E. Vazquez, K. Kostarelos, M. Prato, L. Ballerini, Graphene Acknowledgment oxide nanosheets reshape synaptic function in cultured brain networks, ACS Nano 10 (2016) 4459e4471, https://doi.org/10.1021/acsnano.6b00130. We acknowledge the financial support from the European [20] R. Rauti, M. Medelin, L. Newman, S. Vranic, G. Reina, A. Bianco, M. Prato, K. Kostarelos, L. Ballerini, Graphene oxide flakes tune excitatory neurotrans- Union’s Horizon 2020 Research and Innovation Programme under mission in vivo by targeting hippocampal synapses, Nano Lett. 19 (2019) grant agreement no. 785219 and no. 881603 Graphene Flagship. 2858e2870, https://doi.org/10.1021/acs.nanolett.8b04903. MM, PP and LC acknowledge CERIC-ERIC proposal grant n. [21] Z. Song, Y. Wang, Z. Xu, Mechanical responses of the bio-nano interface: a molecular dynamics study of graphene-coated lipid membrane, Theor. Appl. 20167063 for the IR measurements, performed at the SISSI-Bio Mech. Lett. 5 (2015) 231e235, https://doi.org/10.1016/j.taml.2015.11.003. beamline of Elettra Sincrotrone Trieste. PP and LC acknowledge [22] R.J. Rodrigues, A.R. Tome , R.A. Cunha, ATP as a multi-target danger signal in also the European Regional Development Fund and Interreg V-A the brain, Front. Neurosci. 9 (2015) 148, https://doi.org/10.3389/ fnins.2015.00148. Italia-Austria 2014e2020 project EXOTHERA (ITAT1036). [23] F. Calegari, S. Coco, E. Taverna, M. Bassetti, C. Verderio, N. Corradi, M. Matteoli, P. Rosa, A regulated secretory pathway in cultured hippocampal astrocytes, Appendix A. Supplementary data J. Biol. Chem. 274 (1999) 22539e22547. [24] P. Hermanowicz, M. Sarna, K. Burda, H. Gabrys, AtomicJ: an open source software for analysis of force curves, Rev. Sci. Instrum. 85 (2014), 063703, Supplementary data to this article can be found online at https://doi.org/10.1063/1.4881683. https://doi.org/10.1016/j.carbon.2021.01.142. [25] F. D’Amico, M. Saito, F. Bencivenga, M. Marsi, A. Gessini, G. Camisasca, E. Principi, R. Cucini, S. Di Fonzo, A. Battistoni, E. Giangrisostomi, C. Masciovecchio, UV resonant Raman scattering facility at Elettra, Nucl. In- References struments Methods Phys. Res. Sect. A Accel. Spectrometers, Detect. Assoc. Equip. 703 (2013) 33e37, https://doi.org/10.1016/J.NIMA.2012.11.037. [1] D.A. Shifrin, M.D. Beckler, R.J. Coffey, M.J. Tyska, Extracellular vesicles: [26] A. Bignami, L.F. Eng, D. Dahl, C.T. Uyeda, Localization of the glial fibrillary communication, coercion, and conditioning, Mol. Biol. Cell 24 (2013) acidic protein in astrocytes by immunofluorescence, Brain Res. 43 (1972) 1253e1259, https://doi.org/10.1091/mbc.e12-08-0572. 429e435, https://doi.org/10.1016/0006-8993(72)90398-8. [2] Y.J. Yoon, O.Y. Kim, Y.S. Gho, Extracellular vesicles as emerging intercellular [27] M. Musto, R. Rauti, A.F. Rodrigues, E. Bonechi, C. Ballerini, K. Kostarelos, communicasomes, BMB Rep 47 (2014) 531e539, https://doi.org/10.5483/ L. Ballerini, 3D organotypic spinal cultures: exploring neuron and neuroglia BMBREP.2014.47.10.164. responses upon prolonged exposure to graphene oxide, Front. Syst. Neurosci. [3] V. Zappulli, K.P. Friis, Z. Fitzpatrick, C.A. Maguire, X.O. Breakefield, Extracel- 13 (2019) 1, https://doi.org/10.3389/fnsys.2019.00001. lular vesicles and intercellular communication within the nervous system, [28] M. Bramini, G. Alberini, E. Colombo, M. Chiacchiaretta, M.L. DiFrancesco, J. Clin. Invest. 126 (2016) 1198e1207, https://doi.org/10.1172/JCI81134. J.F. Maya-Vetencourt, L. Maragliano, F. Benfenati, F. Cesca, Interfacing [4] C.P.-K. Lai, X.O. Breakefield, Role of exosomes/microvesicles in the nervous graphene-based materials with neural cells, Front. Syst. Neurosci. 12 (2018) system and use in emerging therapies, Front. Physiol. 3 (2012) 228, https:// 12, https://doi.org/10.3389/fnsys.2018.00012. doi.org/10.3389/fphys.2012.00228. [29] A.B. Seabra, A.J. Paula, R. De Lima, O.L. Alves, N. Dura n, Nanotoxicity of gra- [5] V. Budnik, C. Ruiz-Can ~ ada, F. Wendler, Extracellular vesicles round off phene and graphene oxide, Chem. Res. Toxicol. 27 (2014) 159e168, https:// communication in the nervous system, Nat. Rev. Neurosci. 17 (2016) doi.org/10.1021/tx400385x. 160e172, https://doi.org/10.1038/nrn.2015.29. [30] F. Bianco, C. Perrotta, L. Novellino, M. Francolini, L. Riganti, E. Menna, [6] A. Grimaldi, C. Serpe, G. Chece, V. Nigro, A. Sarra, B. Ruzicka, M. Relucenti, L. Saglietti, E.H. Schuchman, R. Furlan, E. Clementi, M. Matteoli, C. Verderio, G. Familiari, G. Ruocco, G.R. Pascucci, F. Guerrieri, C. Limatola, M. Catalano, Acid sphingomyelinase activity triggers microparticle release from glial cells, Microglia-derived microvesicles affect microglia phenotype in glioma, Front. EMBO J. 28 (2009) 1043e1054, https://doi.org/10.1038/emboj.2009.45. 468 M. Musto, P. Parisse, M. Pachetti et al. Carbon 176 (2021) 458e469 [31] E. Amaral, S. Guatimosim, C. Guatimosim, Using the fluorescent styryl dye [48] F. Antonucci, E. Turola, L. Riganti, M. Caleo, M. Gabrielli, C. Perrotta, FM1-43 to visualize synaptic vesicles exocytosis and endocytosis in motor L. Novellino, E. Clementi, P. Giussani, P. Viani, M. Matteoli, C. Verderio, nerve terminals, Methods Mol. Biol. (2011) 137e148, https://doi.org/10.1007/ Microvesicles released from microglia stimulate synaptic activity via 978-1-60761-950-5_8. enhanced sphingolipid metabolism, EMBO J. 31 (2012) 1231e1240, https:// [32] W.J. Betz, F. Mao, C.B. Smith, Imaging exocytosis and endocytosis, Curr. Opin. doi.org/10.1038/emboj.2011.489. Neurobiol. 6 (1996) 365e371, https://doi.org/10.1016/S0959-4388(96)80121- [49] M.J. Carson, J.C. Thrash, B. Walter, The cellular response in neuro- 8. inflammation: the role of leukocytes, microglia and astrocytes in neuronal [33] A. Brumback, J.L. Lieber, J.K. Angleson, W.J. Betz, Using FM1-43 to study death and survival, Clin. Neurosci. Res. 6 (2006) 237e245, https://doi.org/ neuropeptide granule dynamics and exocytosis, Methods 33 (2004) 287e294, 10.1016/j.cnr.2006.09.004. https://doi.org/10.1016/j.ymeth.2004.01.002. [50] M.M. Holm, J. Kaiser, M.E. Schwab, Extracellular vesicles: multimodal envoys [34] Y. Yoshioka, Y. Konishi, N. Kosaka, T. Katsuda, T. Kato, T. Ochiya, Comparative in neural maintenance and repair, Trends Neurosci. 41 (2018) 360e372, marker analysis of extracellular vesicles in different human cancer types, https://doi.org/10.1016/j.tins.2018.03.006. J. Extracell. Vesicles 2 (2013), https://doi.org/10.3402/jev.v2i0.20424. [51] T. Gerecsei, I. Erdo}di, B. Peter, C. Ho } s, S. Kurunczi, I. Dere nyi, B. Szabo , [35] R.A. Dragovic, C. Gardiner, A.S. Brooks, D.S. Tannetta, D.J.P. Ferguson, P. Hole, R. Horvath, Adhesion force measurements on functionalized microbeads: an B. Carr, C.W.G. Redman, A.L. Harris, P.J. Dobson, P. Harrison, I.L. Sargent, Sizing in-depth comparison of computer controlled micropipette and fluidic force and phenotyping of cellular vesicles using Nanoparticle Tracking Analysis, microscopy, J. Colloid Interface Sci. 555 (2019) 245e253, https://doi.org/ Nanomedicine 7 (2011) 780e788, https://doi.org/10.1016/j.nano.2011.04.003. 10.1016/j.jcis.2019.07.102. [36] T. Visnovitz, X. Osteikoetxea, B.W. So dar, J. Mihaly, P. Lo }rincz, K.V. Vukman, [52] S. Kasas, X. Wang, H. Hirling, R. Marsault, B. Huni, A. Yersin, R. Regazzi,  To E.A. th, A. Koncz, I. Szek  acs, R. Horv ath, Z. Varga, E.I. Buza s, An improved 96 G. Grenningloh, B. Riederer, L. Forro , G. Dietler, S. Catsicas, Superficial and well plate format lipid quantification assay for standardisation of experiments deep changes of cellular mechanical properties following cytoskeleton with extracellular vesicles, J. Extracell. Vesicles 8 (2019), 1565263, https:// disassembly, Cell Motil Cytoskeleton 62 (2005) 124e132, https://doi.org/ doi.org/10.1080/20013078.2019.1565263. 10.1002/cm.20086. [37] J. Mih k, I.C. Szigya aly, R. Dea rto , A. Bo ta, T. Beke-Somfai, Z. Varga, Character- [53] E. Norman, R.G. Cutler, R. Flannery, Y. Wang, M.P. Mattson, Plasma membrane ization of extracellular vesicles by IR spectroscopy: fast and simple classifi- sphingomyelin hydrolysis increases hippocampal neuron excitability by cation based on amide and C H stretching vibrations, Biochim. Biophys. Acta sphingosine-1-phosphate mediated mechanisms, J. Neurochem. 114 (2010) Biomembr. 1859 (2017) 459e466, https://doi.org/10.1016/ 430e439, https://doi.org/10.1111/j.1471-4159.2010.06779.x. j.bbamem.2016.12.005. [54] V. Amrhein, S. Greenland, B. McShane, Scientists rise up against statistical [38] D. Ami, F. Lavatelli, P. Rognoni, G. Palladini, S. Raimondi, S. Giorgetti, L. Monti, significance, Nature 567 (2019) 305e307, https://doi.org/10.1038/d41586- S.M. Doglia, A. Natalello, G. Merlini, In situ characterization of protein ag- 019-00857-9. gregates in human tissues affected by light chain amyloidosis: a FTIR micro- [55] It’s time to talk about ditching statistical significance, Nature 567 (2019), spectroscopy study, Sci. Rep. 6 (2016) 29096, https://doi.org/10.1038/ https://doi.org/10.1038/d41586-019-00874-8, 283e283. srep29096. [56] R. Frost, S. Svedhem, C. Langhammer, B. Kasemo, Graphene oxide and lipid [39] A. Nemeth, N. Orgovan, B.W. So  dar, X. Osteikoetxea, K. P czi, K. Szabo alo - membranes: size-dependent interactions, Langmuir 32 (2016) 2708e2717, Taylor, K.V. Vukman, A.  Kittel, L. Turiak, Z. Wiener, S. To  th, L. Drahos, K. Ve key, https://doi.org/10.1021/acs.langmuir.5b03239. R. Horvath, E.I. Buzas, Antibiotic-induced release of small extracellular vesicles [57] N. Li, Q. Zhang, S. Gao, Q. Song, R. Huang, L. Wang, L. Liu, J. Dai, M. Tang, (exosomes) with surface-associated DNA, Sci. Rep. 7 (2017) 1e16, https:// G. Cheng, Three-dimensional graphene foam as a biocompatible and doi.org/10.1038/s41598-017-08392-1. conductive scaffold for neural stem cells, Sci. Rep. 3 (2013) 1604, https:// [40] M.Y. Sherman, A.L. Goldberg, Cellular defenses against unfolded proteins: a doi.org/10.1038/srep01604. cell biologist thinks about neurodegenerative diseases, Neuron 29 (2001) [58] M. Basso, S. Pozzi, M. Tortarolo, F. Fiordaliso, C. Bisighini, L. Pasetto, G. Spaltro, 15e32, https://doi.org/10.1016/s0896-6273(01)00177-5. D. Lidonnici, F. Gensano, E. Battaglia, C. Bendotti, V. Bonetto, Mutant copper- [41] R. V Rao, D.E. Bredesen, Misfolded proteins, endoplasmic reticulum stress and zinc superoxide dismutase (SOD1) induces protein secretion pathway alter- neurodegeneration, Curr. Opin. Cell Biol. 16 (2004) 653e662, https://doi.org/ ations and exosome release in astrocytes: implications for disease spreading 10.1016/j.ceb.2004.09.012. and motor neuron pathology in amyotrophic lateral sclerosis, J. Biol. Chem. [42] D.J. Selkoe, Folding proteins in fatal ways, Nature 426 (2003) 900e904, 288 (2013) 15699e15711, https://doi.org/10.1074/jbc.M112.425066. https://doi.org/10.1038/nature02264. [59] G. Wang, M. Dinkins, Q. He, G. Zhu, C. Poirier, A. Campbell, M. Mayer-Proschel, [43] A. Natalello, D. Ami, S.M. Doglia, Fourier transform infrared spectroscopy of E. Bieberich, Astrocytes secrete exosomes enriched with proapoptotic cer- intrinsically disordered proteins: measurement procedures and data analyses, amide and prostate apoptosis response 4 (PAR-4), J. Biol. Chem. 287 (2012) Methods Mol. Biol. (2012) 229e244, https://doi.org/10.1007/978-1-61779- 21384e21395, https://doi.org/10.1074/jbc.M112.340513. 927-3_16. [60] R.-D. Gosselin, P. Meylan, I. Decosterd, Extracellular microvesicles from as- [44] C.J. Oldfield, A.K. Dunker, Intrinsically disordered proteins and intrinsically trocytes contain functional glutamate transporters: regulation by protein ki- disordered protein regions, Annu. Rev. Biochem. 83 (2014) 553e584, https:// nase C and cell activation, Front. Cell. Neurosci. 7 (2013) 251, https://doi.org/ doi.org/10.1146/annurev-biochem-072711-164947. 10.3389/fncel.2013.00251. [45] D. V Caccamo, M.M. Herman, A. Frankfurter, C.D. Katsetos, V.P. Collins, [61] A.R. Taylor, M.B. Robinson, D.J. Gifondorwa, M. Tytell, C.E. Milligan, Regulation L.J. Rubinstein, An immunohistochemical study of neuropeptides and of heat shock protein 70 release in astrocytes: role of signaling kinases, Dev. neuronal cytoskeletal proteins in the neuroepithelial component of a spon- Neurobiol. 67 (2007) 1815e1829, https://doi.org/10.1002/dneu.20559. taneous murine ovarian teratoma. Primitive neuroepithelium displays [62] S. Wang, F. Cesca, G. Loers, M. Schweizer, F. Buck, F. Benfenati, M. Schachner, immunoreactivity for neuropeptides and neuron-associated beta-tu, Am. J. R. Kleene, Synapsin I is an oligomannose-carrying glycoprotein, acts as an Pathol. 135 (1989) 801e813. oligomannose-binding lectin, and promotes neurite outgrowth and neuronal [46] R.C. Paolicelli, G. Bergamini, L. Rajendran, Cell-to-cell communication by survival when released via glia-derived exosomes, J. Neurosci. 31 (2011) extracellular vesicles: focus on microglia, Neuroscience 405 (2019) 148e157, 7275e7290, https://doi.org/10.1523/JNEUROSCI.6476-10.2011. https://doi.org/10.1016/j.neuroscience.2018.04.003. [63] H. Zhao, R. Ding, X. Zhao, Y. Li, L. Qu, H. Pei, L. Yildirimer, Z. Wu, W. Zhang, [47] Y. Yang, A. Boza-Serrano, C.J.R. Dunning, B.H. Clausen, K.L. Lambertsen, Graphene-based nanomaterials for drug and/or gene delivery, bioimaging, T. Deierborg, Inflammation leads to distinct populations of extracellular ves- and tissue engineering, Drug Discov. Today 22 (2017) 1302e1317, https:// icles from microglia, J. Neuroinflammation 15 (2018) 168, https://doi.org/ doi.org/10.1016/j.drudis.2017.04.002. 10.1186/s12974-018-1204-7. 469 Update Carbon Volume 178, Issue , 30 June 2021, Page 824 DOI: https://doi.org/10.1016/j.carbon.2021.04.026 Carbon 178 (2021) 824 Contents lists available at ScienceDirect Carbon journal homepage: www.elsevier.com/locate/carbon Corrigendum Corrigendum to Shedding plasma membrane vesicles induced by graphene oxide nanoflakes in brain cultured astrocytes [CARBON 176 (2021) 458e469] Mattia Musto a, Pietro Parisse b, Maria Pachetti b, Christian Memo a, Giuseppe Di Mauro a, Belen Ballesteros c, Neus Lozano c, Kostas Kostarelos c, d, Loredana Casalis b, **, Laura Ballerini a, * a International School for Advanced Studies (SISSA), 34136, Trieste, Italy b ELETTRA Synchrotron Light Source, 34149, Basovizza, Italy c Catalan Institute of Nanoscience and Nanotechnology (ICN2), Campus UAB, Bellaterra, 08193, Barcelona, Spain d Nanomedicine Lab, National Graphene Institute and Faculty of Biology, Medicine & Health, The University of Manchester, Manchester, M13 9PT, United Kingdom The authors regret that the printed version of the above article contained a number of errors. The correct and final version follows. The authors would like to apologise for any inconvenience caused. The authors would like to make the following addition to the Acknowledgment section of the article: The authors are grateful to Dr. Gesmi Milcovich for contributing to the recording and analyses in the AFM biomechanics measurements and related fruitful discussions. DOI of original article: https://doi.org/10.1016/j.carbon.2021.01.142. * Corresponding author. ** Corresponding author. E-mail addresses: loredana.casalis@elettra.eu (L. Casalis), laura.ballerini@sissa.it, ballerin@sissa.it (L. Ballerini). https://doi.org/10.1016/j.carbon.2021.04.026 0008-6223/© 2021 The Author(s). Published by Elsevier Ltd. All rights reserved.