Internalization of Carbon Nano-onions by Hippocampal Cells

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Biological and Medical Applications of Materials and Interfaces

Internalization of Carbon Nano-onions by hippocampal cells preserves neuronal circuit function and recognition memory Massimo Trusel, Michele Baldrighi, Roberto Marotta, Francesca Gatto, Mattia Pesce, Marco Frasconi, Tiziano Catelani, Francesco Papaleo, Pier Paolo Pompa, Raffaella Tonini, and Silvia Giordani ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b17827 • Publication Date (Web): 18 Apr 2018 Downloaded from http://pubs.acs.org on April 20, 2018

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ACS Applied Materials & Interfaces

Internalization of Carbon Nano-onions by Hippocampal Cells Preserves Neuronal Circuit Function and Recognition Memory Massimo Trusel,a, ‡ Michele Baldrighi,b, ‡ Roberto Marotta,c Francesca Gatto, d,e Mattia Pesce, a Marco Frasconi,b,† Tiziano Catelani,c Francesco Papaleo,a Pier Paolo Pompa, d Raffaella Tonini a,* and Silvia Giordani b,f* a) Neuroscience and Brain Technology, Istituto Italiano di Tecnologia, via Morego 30, Genova, Italy b) Nano Carbon Materials, Istituto Italiano di Tecnologia, via Morego 30, Genova, Italy c) Electron Microscopy Laboratory, Istituto Italiano di Tecnologia, via Morego 30, Genova, Italy d) Nanobiointeractions & Nanodiagnostics, Istituto Italiano di Tecnologia, via Morego 30, Genova, Italy e) Department of Engineering for Innovation, University of Salento, Via per Monteroni, Lecce, Italy f) Department of Chemistry, University of Turin, Via Giuria 7, Turin, Italy

KEYWORDS: Carbon nanomaterials, central nervous system, nanomedicine, fluorescence, confocal imaging, electron microscopy

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ABSTRACT: One area where nanomedicine may offer superior performances and efficacy compared to current strategies is in the diagnosis and treatment of Central Nervous System (CNS) diseases. However, the application of nanomaterials in such complex arenas is still in its infancy and an optimal vector for the therapy of CNS diseases has not been identified. Graphitic Carbon Nano Onions (CNOs) represent a class of carbon nanomaterials that shows promising potential for biomedical purposes. To probe the possible applications of graphitic CNOs as a platform for therapeutic and diagnostic interventions on CNS diseases, fluorescently labeled CNOs were stereotaxically injected in vivo in mice hippocampus. Their diffusion within brain tissues and their cellular localization were analyzed ex vivo by confocal microscopy, electron microscopy and correlative light-electron microscopy techniques. The subsequent fluorescent staining of hippocampal cells populations indicates they efficiently internalize the nanomaterial. Furthermore, the inflammatory potential of the CNOs injection was found comparable to sterile vehicle infusion, and it did not result in manifest neurophysiological and behavioral alterations of hippocampal-mediated functions. These results clearly demonstrate that CNOs can interface effectively with several cell types, which encourages further their development as possible brain diseases-targeted diagnostics and/or therapeutics nanocarriers.

INTRODUCTION The diagnosis and treatment of many CNS diseases, in particular cerebral tumors, is one of the most challenging tasks for nanotechnology to demonstrate a superior efficacy with respect to the standard chemotherapy. To date, several nanomaterials such as magnetic nanoparticles,1 gold nanoparticles,2 silica nanoparticles,3 polymeric4,5 and protein nanoparticles,6 and also carbon nanostructures,7–9 have


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been probed for their possible use as theranostic systems in the brain,10–12 some of them being currently under clinical trial.13 However, an “all purposes” nanoparticle has not been developed yet and, moreover, in vivo investigations of many nanomaterials used in drug delivery applications have shown perturbation of the normal cell signaling and may cause pathological disorders. Indeed, deficits in cognitive and sensory–motor functions, in addition to neurotoxicity effects, have been observed after intraperitoneal injection of metal nanoparticles in rats,14 and recently concerns have been raised regarding environmental exposure to magnetic iron oxide nanoparticles, their penetration in the brain tissues, and their relation with increased incidence of Alzheimer’s disease.15 Interference with the neuronal synaptic functions has been recently identified also with graphene oxide,16,17 while in general other carbon nanomaterials display good biocompatibility with the neural tissues.8,10 These findings encourage to explore other carbon nanomaterials that can possibly deliver more efficiently drugs inside the tumor site, show optimized circulation times (high retention in the tumor site, low accumulation in liver and spleen, good excretion kinetic) and display lower toxicity. In recent years graphitic Carbon Nano Onions (CNOs),18–20 nanomaterials consisting of multiconcentric graphitic shells, have attracted interest for applications in the biomedical field.21–30 Although they can be produced in several ways and have different sizes, the most studied are those deriving from thermal annealing of ≈5 nm diameter detonation nanodiamonds.31 This synthetic process yields carbon onions with an average size of 5-8 nm that consist of continuous concentric graphitic layers 3.5 Å apart. CNOs display a number of properties that are of great interest for theranostic applications, including: (i) small size, that enables their efficient internalization by cells and possibly their diffusion in the tissues; (ii) quasi-spherical shape, that allows a favorable interaction with cells membranes;32,33 (iii) easy and efficient surface


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functionalization using the well-known chemical reactions developed for carbon nanotubes,20 which allow them to be targeted against specific biological markers and to be functionalized with molecules that endow them with imaging capabilities.23,25,26,28 Furthermore, our group was able to demonstrate that CNOs can readily and efficiently enter several cell types and their internalization does not give rise to evidences of toxicity in single cells as well as in small living beings.23–30,34 In addition, they have a remarkably low inflammatory potential,23 which is particularly important when dealing with neurons that are tipically very sensitive to toxicants. Nevertheless, the behavior of these nanoparticles in the CNS in terms of preferences for specific cell types and compatibility with the neuronal tissue remains unexplored. Addressing these questions, before going further with a costly, time consuming and potentially unfruitful development of a complex nanomedical platform to be used in the brain is thus crucial. The aim of this work is to examine whether CNOs positively interface in vivo with the CNS. The specific questions we wish to answer are i) can CNOs disperse from the injection site to be internalized from neuronal and non-neuronal cell populations? ii) what is the CNOs inflammatory potential towards neuronal cells? iii) what is their effect towards the neuronal electric conduction and the CNS functions? The outcome of these investigations therefore will provide an in vivo assessment of the CNOs capabilities to perform as a biocompatible nanomaterial for applications in the CNS. We investigated the cellular and subcellular distribution of fluorescently labeled CNOs in vivo infused in murine hippocampus by combining confocal microscopy, transmission electron microscopy and correlative methodologies. We then tested whether CNOs brain infusion affected hippocampal functions by identifying and quantifying inflammation markers, and by assessing neurophysiological and behavioral parameters. The results reported herein on fluorescently


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labeled CNOs demonstrate the potential of this carbon nanostructure for applications in the diagnosis and treatment of CNS diseases.

RESULTS Synthesis of fluorescently labeled CNOs Fluoresceinamine-functionalized CNOs (fluo-CNOs) were chosen to perform the in vivo studies because they are internalized efficiently by cells, they provide bright fluorescence imaging and they are free from toxic effects towards different cell types, including non-tumoral ones.23 Covalent introduction of a fluorescent label (fluoresceinamine) was achieved by reacting pristine CNOs (p-CNOs) with p-carboxyphenyldiazonium chloride to functionalize the surface of the nanomaterial with benzoic acid moieties, obtaining benz-CNOs, and then performing a carbodiimide-mediated amide coupling between the carboxylic acid groups on the CNOs surface and the amino groups of fluoresceinamine molecules, to give fluoresceinamine-conjugated CNOs (fluo-CNOs) (Figure 1a). After the first functionalization step, the TGA of the nanomaterial indicates the occurrence of weight loss in the range 100 °C - 450 °C (11.2%) in benzoic acid functionalized CNOs (benzCNOs) which is absent in p-CNOs and can be attributed to the decomposition of the surface functional moieties, and a decrease in the decomposition temperature of the carbon core from 700 °C to 558 °C (Figure 1c). Furthermore, ζ potential decreases from -18.3 mV for p-CNOs to -38.8 mV for benz-CNOs, suggesting the effective introduction of negatively charged functional groups on the nanomaterial surface (see the Supporting Information). From TGA we can estimate that the first functionalization step provides CNOs with approx. 57 benzoic acid moieties per CNO. After the second functionalization step, which yields fluo-CNOs, TGA shows


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increased weight loss in the range 100 °C - 450 °C (18.8%) indicating the successful covalent functionalization of the nanomaterial (Figure 1c). Moreover, the increase in ζ potential value (-22.5 mV, see the Supporting Information) with respect to benz-CNOs is a good indication of the reaction success. Analysis of TGA data indicates that the reaction allowed the introduction of approx. 19 fluoresceinamine molecules per CNO. Once dispersed in PBS pH 7.3 to a final concentration of 10 µg/mL, the synthesized fluo-CNOs display a small but clear absorption peak in the visible region with maximum at λ=488 nm, corresponding to the absorption peak of the fluorescent label, as well as a classical broad absorption throughout the whole wavelength range, that is typical of CNOs. In contrast, both p-CNOs and benz-CNOs do not show any clear absorption peak in the range 300-1000 nm (see Supporting Information). Irradiation of the fluoCNOs dispersion at λ=490 nm provides the emission of bright fluorescence with maximum emission peak at λ= 512 nm (Figure 1d). The spectroscopic data indicate therefore that fluorescein molecules were successfully grafted onto the CNOs surface and that the labeled nanomaterial exhibit bright fluorescence when dispersed in physiological medium.


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Figure 1: Synthesis and characterization of fluo-CNOs. a) Synthetic scheme for the production of benz-CNOs and fluo-CNOs b) HRTEM images of p-CNOs (left), benz-CNOs (center), and fluo-CNOs (right) showing the concentric graphitic structure. Scale bars are 5 nm. c) TGA plots of p-CNOs (black), benz-CNOs (blue), and fluo-CNOs (orange); weight loss and weight loss first derivative are in solid and dashed lines respectively. d) UV/Vis absorption (red line) and emission (green line) spectra of a 10 µg/mL solution in PBS of fluo-CNOs. In vivo diffusion and internalization in the CNS


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To test the ability of Carbon Nano Onions to interface in vivo with the neuronal tissue, fluoCNOs were stereotaxically injected in mice brains (n=8). For our study we targeted the brain region denominated hippocampus, which is involved in multiple functions spanning from memory to spatial processing.35 The hippocampus is conventionally divided in various zones: the dentate gyrus (DG) and the cornus ammonis areas, namely CA4, CA3, CA2 and CA1. These zones are connected to one-another forming a loop: neuronal signal incoming from the enthorinal cortex mainly enters the hippocampal circuit through the perforant path impinging on the DG. The signal is then conveyed to the CA3 zone through the mossy fibers connection, and CA3 neurons project their axons to the CA1 portion. The latter serves as an output structure, conveying the signal to the efferent areas. With the idea of targeting the input and output zones of the hippocampus, the fluo-CNOs were suspended in sterile PBS (1.6 mg/ml). The concentration was chosen balancing the need to obtain a sufficient loading of fluorescent nanomaterial to allow its detection in the tissues by confocal microscopy, the need to avoid undesired inflammation due to an excessive dosage, and the low volume allowed for the injection (0.5 µL). Indeed, the injected fluo-CNOs dose in this study was 0.8µg, about 3 orders of magnitude lower than the one we showed to provide only little inflammation after i.p. administration.23 Fluo-CNOs were infused in two neuron-dense sub-regions, the DG and CA1 areas (see Figure 2a). Next, we perfused mice brains with a fixative 48 h after the CNOs infusion, and we obtained slices of the area of interest to evaluate whether CNOs could diffuse from the injection site and penetrate the cell’s membrane in vivo (see the Experimental Section for more details). Owing to their bright fluorescence, fluo-CNOs were easily localized within the cerebral tissues by means of epifluorescence microscopy. The nuclear staining DAPI allowed for the fluorescent visualization of the cells nuclei (Figure 2b). Fluorescence confocal imaging of the


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brain slices shows the presence of a bright fluorescence signal of fluo-CNOs close to the injection trace (Figure 2). Interestingly, the fluo-CNOs signal is found not only as diffuse fluorescence but also inside the cells body, which is indicative of the nanomaterial’s ability to permeate the cell’s membrane (Figure 2b). Notably, the same fluorescence signal is detected also in cells belonging to the surrounding tissues, the number of stained cells decreasing with the distance from the injection site. This suggests that fluo-CNOs can disperse from the injection site to the neighboring regions (Figure 2a). The specificity of fluo-CNO fluorescence was qualitatively assessed in a subset of brain slices from naïve, saline- and fluo-CNO-injected mice (48 h and 14 days after CNOs infusion) (Figure S15).

Figure 2: Fluorescein-tagged CNOs infused in-vivo render hippocampal cells fluorescent. a) Schematic representation of the intra-hippocampal infusion, highlighting the injection sites in the


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DG and CA1 areas (left). Confocal image of the hippocampal CA1 and DG regions infused with fluo-CNOs (right). b) Sequential magnification of the area enclosed in Figure 2a (left). Highmagnification confocal image of CA1 area from a mouse infused with fluo-CNOs (right). Green: fluo-CNOs. Red: cell nuclei stained by DAPI. Transmission (TEM) and scanning transmission (STEM) electron microscopy analyses were performed on CA1 and DG regions from mouse hippocampal slices selected among those presenting a clearly recognizable injection site. STEM imaging was performed using a high angular annular dark field (HAADF) detector to increase overall image contrast. Both TEM imaging approaches have been employed to discriminate the fluo-CNOs from the surrounding carbon-rich cellular structures thanks to their electron-dense multi-shell graphitic structure and their size (observed fluo-CNOs average diameter = 5 nm, s.e.m. 0.05, n=976, see the Supporting Information).27 Close to the injection site fluo-CNOs are mainly observed as large aggregates of thousands of particles (up to 4-5 µm in size) inside late endosome-phagosomes of not neural microglial cells (Figure 3a-c and the Supporting Information; the cell type is determined according to Peters and Folger36). In the same region fluo-CNOs are also observed inside hippocampal neurons partially filling the neurite cytoplasm (Figure 4a,b and the Supporting Information). Far from the injection site the fluo-CNOs are instead exclusively confined in small endosomal compartments scattered in the hippocampal neuron cell body and forming small subspherical clusters (Figure 4c-f and the Supporting Information). To corroborate the hypothesis that fluo-CNOs are responsible for the fluorescent staining of neuronal cells (Figure 2) and to further investigate their subcellular localization, we integrated confocal microscopy with TEM in a correlative light-electron microscopy (CLEM) approach.37,38 Fluorescent neuronal cells were localized in hippocampal slices by confocal microscopy and ultrathin slices of this region of


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interest were prepared for electron microscopy imaging (Figure 5a). Although cell ultrastructure is not optimally preserved, TEM analysis of the same fluo-CNOs labeled cells reveals the presence of spherical carbon nanoparticles both as tight clusters in cytoplasmic confined membrane-bound vesicles (Figure 5b,c) and as looser small aggregates free-floating in the cytoplasm (Figure 5d,e) coherently with our previous EM observation. These results demonstrate that the green-fluorescent staining of neuronal cells is ascribed to the internalization of fluoCNOs, and that the integrity of the fluorescent label is preserved in the intracellular environment.

Figure 3: TEM analysis of fluo-CNOs in mice hippocampus microglia. a) and b) low magnification images of non-neural cells with fluo-CNOs clusters confined in large late endosome-phagosome compartments (asterisks); c) higher magnification of large fluo-CNOs aggregates (asterisks) inside late endosomes-phagosomes. Inset: high magnification of the framed region in c) showing smaller fluo-CNOs clusters inside endosomal compartments


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(arrowheads). Scale bars are 2 µm in a), 1 µm in b) and c) and 0.5 µm in c) inset. Abbreviations: n, nucleus.

Figure 4. EM analysis of fluo-CNOs in mice hippocampal neurons. a) and b) TEM images of fluo-CNOs clusters (asterisks) inside dentate gyrus neurites. c) TEM low magnification of two dentate gyrus neurons; d) STEM higher magnification of the region boxed in c); e) TEM and f) STEM high magnification of the region boxed in d). Abbreviations: n, nucleus. Scale bars are 0.5 µm in a) and b), 2 µm in c), 1 µm in d) and 0.25 µm in e) and f).


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Figure 5: CLEM analysis of fluo-CNOs in mouse hippocampus (CA1). a) Inset: 2PLSM image showing fluo-CNOs labeled neurons (green) inside of a 100 µm-thick brain section. The ROI was laser branded (laser cut marked with *) to allow the matching of 2PLSM and EM images. The white and black arrowheads point to the cells of interest. a) The same ROI retraced at the TEM on the corresponding ultra-thin plastic section. Black and white arrowheads point to the same cells highlighted in the inset. b) TEM image of the cell highlighted with the white arrowhead. c) magnification of the region boxed in b) showing the presence of fluo-CNOs inside cytoplasm vesicles; inset: magnification of the vesicle inside the white box. d) TEM image of the cell highlighted with the black arrowhead. e) magnification of the region boxed in d) showing fluo-CNOs loose aggregates floating in the cell cytoplasm; inset: magnification of the aggregate inside the white box. Scale bars are 2 µm in b and d, 1 µm in e and 0.5 µm in c, c inset and d inset. Abbreviation: cyt, cytoplasm; n, nucleus. Determination of immune reaction to fluo-CNOs injection


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The insertion of exogenous material in tissues, even when sterile, often induces the activation of inflammatory responses, including the recruitment of specialized cells to cope with the intruder.39 To understand whether fluo-CNO infusion would induce neuroinflammation, we measured the levels of known inflammation markers in the hippocampus at 48, 72, and 120 hours after the injection, which are indicated in literature as timepoints where these markers are well detectable.40 We injected fluo-CNOs or their vehicle (sterile PBS), and compared these to naïve animals (not injected). We then measured the levels of cytokines that are known as biomarkers of inflammation.41,42 IL-1β and IL-6 and the chemokines MCP1 and MIP-1α were evaluated by using the Multiplex MAGPIX Immunoassay (Figure 6a), while TNFα and COX-2, strongly induced by pro-inflammatory challenges in astrocytes, were quantified by means of qPCR (Figure 6b).43 The results indicate that fluo-CNOs or vehicle infusion caused a significant increase in some of the inflammatory response biomarkers with respect to non-injected mice. However, we found no significant differences in the inflammatory response when comparing the fluo-CNOs injection and the injection of sterile PBS (vehicle) at all the timepoints considered (Figure 6 and the Supporting Information). In other words, this indicates that the infusion of fluoCNOs does not induce a specific activation of microglial or astrocytic cells compared to vehicleinjected mice.


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Figure 6: Analysis of neuroinflammatory markers in the hippocampus in naïve, vehicleinfused and fluo-CNO infused mice. a) Histograms representing IL-1β, IL-6, MCP1 and MIP1α levels measured via Multiplex MAGPIX Immunoassay. b) Histograms representing COX-2 and TNFα levels measured via Q-PCR. Experiments were performed after 48, 72 and 120 hours


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from the surgical procedure. Data obtained from 5 mice/treatment are reported as average ± SEM. * p

Internalization of Carbon Nano-onions by Hippocampal Cells

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