Coatings of different carbon nanotubes on platinum electrodes for neuronal devices: Preparation, cytocompatibility and interaction with spiral ganglion cells

Cochlear and deep brain implants are prominent examples for neuronal prostheses with clinical relevance. Current research focuses on the improvement of the long-term functionality and the size reduction of neural interface electrodes. A promising approach is the application of carbon nanotubes (CNTs), either as pure electrodes but especially as coating material for electrodes. The interaction of CNTs with neuronal cells has shown promising results in various studies, but these appear to depend on the specific type of neurons as well as on the kind of nanotubes. To evaluate a potential application of carbon nanotube coatings for cochlear electrodes, it is necessary to investigate the cytocompatibility of carbon nanotube coatings on platinum for the specific type of neuron in the inner ear, namely spiral ganglion neurons. In this study we have combined the chemical processing of as-delivered CNTs, the fabrication of coatings on platinum, and the characterization of the electrical properties of the coatings as well as a general cytocompatibility testing and the first cell culture investigations of CNTs with spiral ganglion neurons. By applying a modification process to three different as-received CNTs via a reflux treatment with nitric acid, long-term stable aqueous CNT dispersions free of dispersing agents were obtained. These were used to coat platinum substrates by an automated spray-coating process. These coatings enhance the electrical properties of platinum electrodes, decreasing the impedance values and raising the capacitances. Cell culture investigations of the different CNT coatings on platinum with NIH3T3 fibroblasts attest an overall good cytocompatibility of these coatings. For spiral ganglion neurons, this can also be observed but a desired positive effect of the CNTs on the neurons is absent. Furthermore, we found that the well-established DAPI staining assay does not function on the coatings prepared from single-wall nanotubes. © 2016 Burblies et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.DFG/EXC 1077/1 “Hearing4all

Chemische Strategien zur Verbesserung der Biokompatibilität und Funktion neuronaler Elektroden

Die vorliegende Arbeit befasst sich mit der Herstellung und Charakterisierung funktionaler Elektroden-Beschichtungen auf der Basis verschiedener Kohlenstoff-Nanomaterialien. Im Hinblick auf eine mögliche Anwendung für Elektroden neuraler Schnittstellen werden die Vorgehensweise und Methoden dementsprechend ausge-wählt. Der Fokus liegt dabei auf der Untersuchung im Hinblick auf die Eignung des Einsatzes als Bestandteil des Elektroden-Arrays des Cochlea-Implantats. Das Cochlea-Implantat ist eine Hörprothese, die es ermöglicht von Schwerhö-rigkeit und Taubheit betroffenen Menschen wieder einen Höreindruck zu vermitteln bzw. eine deutliche Verbesserung des Hörvermögens zu ermöglichen. Dabei werden die Audiosignale der Umgebung von einem am Kopf befindlichen Mikrofon aufge-nommen und an einen im Schädel befindlichen Signalprozessor weitergeleitet, der die-se Signale in elektrische Impulse umsetzt, mit denen über das im Innenohr implantierte Elektroden-Array die Nervenzellen des Innenohres direkt stimuliert werden. Als Beschichtungsmaterial kommen dabei Kohlenstoff-Nanoröhren sowie nano-poröser Kohlenstoff zum Einsatz. Im Falle der Kohlenstoff-Nanoröhren (CNTs; engl.: carbon nanotubes) werden kommerziell erhältliche CNTs unterschiedlichen Typs und Reinheit verwendet. Die CNTs werden mittels eines nasschemischen Verfahrens aufge-reinigt und mit sauerstoff-haltigen funktionellen Gruppen modifiziert. Dieses Verfah-ren ermöglicht die Herstellung stabiler wässriger CNT-Dispersionen. Der exemplarisch für die Gruppe der nanoporösen Kohlenstoffe ausgewählte CMK 3 (Carbon Mesostruc-tured by KAIST-3) wird über eine Templat-gestützte Synthese hergestellt. Der Einsatz verschiedener Polymere als Dispergierhilfsmittel macht es möglich auch vom nanopo-rösen Kohlenstoff stabile Dispersionen zu erhalten. Über die Beschichtungsverfahren der Rakel- und der automatisierten Sprühbeschichtung lassen sich mit Hilfe dieser Dis-persionen homogene Filme mit einheitlicher guter Qualität reproduzierbar in größerer Anzahl herstellen. Die Schichtdicken der Filme liegen für die sprühbeschichteten CNT-Filme im Bereich von 100 nm und für die sprüh- und rakelbeschichteten Filme des na-noporösen Kohlenstoffs im unteren µm-Bereich. Neben der detaillierten Charakterisie-rung der morphologischen und strukturellen Eigenschaften sowie der chemischen Zu-sammensetzung der Filme konnten zudem die mechanische Stabilität sowie die Stabili-tät gegenüber wässrigen Lösungen gezeigt werden. Die elektrische Leitfähigkeit der Filme und ihre elektrochemischen Eigenschaften wurden mit Hilfe der Impedanzspekt-roskopie und Cyclovoltammetrie nachgewiesen. Im Hinblick auf die mögliche Anwendung im menschlichen Körper als Implan-tat-Material wird in ausführlichen Zellkultur-Untersuchungen mit verschiedenen Fib-ro¬blasten-Zelllinien aber auch mit Spiral-Ganglion Zellen – den Nervenzellen des In-nenohres – die Zytokompatibilität untersucht, um erste Aussagen über die allgemeine Biokompatibilität und generelle Eignung der Materialien treffen zu können.This thesis examines the manufacturing and characterization of functionalized coatings for electrodes based on carbon nanomaterials. With regard to the possible ap-plication for neural interfaces electrodes, procedures and methods are chosen accord-ingly. The focus is set on the investigation of the feasibility of a usage as component of the electrode arrays of the cochlear implant. The cochlear implant is a neuroprosthetic device that enables people with severe hearing loss to improve their hearing ability or deaf people even to (re-)gain auditory impressions. Surrounding audio signals are recorded by a microphone that is located at the patient’s head. The signals are transmitted to a signal processor implanted in the skull that transduces these signals into electric pulses. These electric pulses stimulate the neurons of the inner ear by an electrode array implanted directly in the inner ear. For the electrode coatings, carbon nanotubes (CNTs) and nanoporous carbons are used. In case of the carbon nanotubes, commercially available CNTs of different type and purity are utilized. The CNTs are treated via a wet chemical process in order to become purified and modified with oxygen-containing functional groups. This treatment enables after further processing to obtain stable aqueous dispersion of the CNTs. The CMK 3 (Carbon Mesostructured by KAIST-3) is selected as an example for the group of nanoporous carbons and synthesized via a template-based procedure. Using different polymers as dispersing agent, it is possible to obtain stable dispersions of the nanoporous carbon as well. With these dispersions, homogenous films with uniformly good quality can be produced with good reproducibility via two coating methods – doc-tor blading and automated spray-coating. Spray-coated CNT films have a thickness of about 100 nm. The spray-coated and doctor-bladed films of the nanoporous carbon show film thickness in the low µm-range. In addition to the detailed characterization of the morphological and structural properties and the chemical composition of the films, the mechanical stability and the stability against aqueous solvents is also achieved and tested. The electrical conductivity of the films and their electrochemical properties are proven via impedance spectroscopy and cyclic voltammetry. For the possible application in the human body as implant material, the cyto-compatibility is investigated via detailed cell culture investigations with different cell lines of fibroblasts and spiral ganglion cells – the neurons of the inner ear. With these investigations it is possible to make first statements regarding the biocompatibility and general suitability of the used carbon nanomaterials.Cluster of Excellence "Hearing4all"/DFG Cluster of Excellence EXC 1077/1 “Hearing4all”./EXC 1077/1/E

Survival rate of spiral ganglion neurons after two days of cultivation (<i>N</i> = 1, <i>n</i> = 9).

<p>The survival rates were determined by the amount of neurons in relation to the seeding control after cultivation in supernatants obtained from different CNT-coated samples. Supernatants were obtained from CNT-coated (Bayer MWNT-coating, Fraunhofer SWNT-coating, SWeNT SWNT-coating) and non-coated platinum substrates incubated in serum-containing medium (FCS control). SGC medium supplemented with BDNF (BDNF control) and serum-containing medium (FCS control) as well as SGN medium without the addition of any growth factors (medium control) were used as controls. Values are given as mean ± standard error of the mean (<i>N</i> = 1, <i>n</i> = 9). Asterisks indicate the significance of the survival rates of the different conditions compared to the negative control. Statistical assessment was performed using one-way ANOVA with Bonferroni's multiple comparison test (n.s. = not significant, *p < 0.05; **p < 0.01; ***p < 0.001).</p

Calculated crystallite sizes <i>L</i>_<i>G</i> for as-received and purified CNT powders.

<p>The listed values are the mean of three independent measurements and the corresponding standard deviation.</p

Transmission light microscopic images of spiral ganglion cells (SGCs) (<i>N</i> = 1, <i>n</i> = 9) after two days of cultivation.

<p>The samples differed in the media composition. The media contained supernatants from incubating CNT-coated (Bayer MWNT-coating, Fraunhofer SWNT-coating, SWeNT SWNT-coating) and non-coated platinum substrates in serum-containing medium. For comparison, SGCs were also cultivated in SGC medium supplemented with BDNF (BDNF control) and serum-containing medium (FCS control) as well as in serum-free SGC medium (medium control).</p

Metal contents of the different CNT dispersions determined via ICP-OES.

<p>Metal contents refer to the dispersions with CNT mass fractions of 0.1%. DL: detection limit.</p

Coatings of Different Carbon Nanotubes on Platinum Electrodes for Neuronal Devices: Preparation, Cytocompatibility and Interaction with Spiral Ganglion Cells - Fig 2

<p><b>SEM images of carbon nanotube films on platinum substrates: Bayer MWNTs, Fraunhofer SWNTs, SWeNT SWNTs (<i>left to right</i>).</b> The low magnifications show homogenously coated samples; in the higher magnifications in the insets, the randomly aligned networks of the carbon nanotubes become visible.</p

Immunofluorescence staining of spiral ganglion cell cultures cultivated for 48 h on CNT-coated platinum samples and laminin- and poly-D/L-ornithine-coated control surfaces (<i>N</i> = 4, <i>n</i> = 2).

<p>The spiral ganglion neurons (<i>green</i>) of the mixed SGC culture were neurofilament positive. Additionally, cell nuclei (<i>blue</i>) were fluorescence-stained with 4',6-diamidin-2-phenylindol (DAPI) and the glial cells (<i>red</i>) were stained with S100. These images are depicted as overlay combined with the anti-neurofilament images; ccp: cell culture plastic.</p

Fluorescence microscopic images of GFP-expressing NIH3T3 fibroblasts (<i>N</i> = 4, <i>n</i> = 3) after four days of cultivation on CNT-coated platinum substrates as well as on non-coated platinum and on cell culture plastic.

<p>Fluorescence microscopic images of GFP-expressing NIH3T3 fibroblasts (<i>N</i> = 4, <i>n</i> = 3) after four days of cultivation on CNT-coated platinum substrates as well as on non-coated platinum and on cell culture plastic.</p

Impedance spectroscopy scan of the CNT-coated platinum substrates in comparison to a non-coated platinum electrode.

<p><i>Red</i>: Bayer MWNTs, <i>blue</i>: Fraunhofer SWNTs, <i>green</i>: SWeNT SWNTs, <i>dashed black</i>: uncoated platinum substrate.</p