Support of Neuronal Growth Over Glial Growth and Guidance of Optic Nerve Axons by Vertical Nanowire Arrays

Neural cultures are very useful in neuroscience, providing simpler and better controlled systems than the <i>in vivo</i> situation. Neural tissue contains two main cell types, neurons and glia, and interactions between these are essential for appropriate neuronal development. In neural cultures, glial cells tend to overgrow neurons, limiting the access to neuronal interrogation. There is therefore a pressing need for improved systems that enable a good separation when coculturing neurons and glial cells simultaneously, allowing one to address the neurons unequivocally. Here, we used substrates consisting of dense arrays of vertical nanowires intercalated by flat regions to separate retinal neurons and glial cells in distinct, but neighboring, compartments. We also generated a nanowire patterning capable of guiding optic nerve axons. The results will facilitate the design of surfaces aimed at studying and controlling neuronal networks

Electrically evoked intracortical field potentials recorded in the rat primary somatosensory cortex (acute measurements).

<p>(A) Simultaneous recordings using a nanowire-based electrode and a microwire electrode glued together and implanted 400 µm below the cortex surface (averaged over 32 sweeps). (B) Depth profile of evoked Aβ-fiber potential (filled boxes) recorded by the nanowire-based electrode (plotted for each depth as the peak-valley amplitude, with an onset latency between 10 ms and 20 ms after the stimulation) and correlation coefficients (filled circles) calculated for measurements performed simultaneously with the nanowire-based electrode and the microwire (calculated for the measured data sets of time interval up to 0.43 ms after the stimulation). The measurements show that the neuronal signal is primarily recorded with the nanowire-based sensing part and that the nanowire-based electrode provides acute <i>in vivo</i> recordings that are comparable to conventional microelectrodes.</p

Spontaneous neuron activity recorded with a nanowire-based electrode in the rat primary somatosensory cortex (acute measurements).

<p>(A) Raw data with 1811 spikes detected; (B) zoomed region with a neuronal burst; (C) isolated single unit from the recordings in (A), the grid size in x-direction is 0.2 ms; (D) spike cluster view in the principle component space. The cluster corresponds to an isolated single unit as presented in (C). The dashed yellow ellipse in (D) represents the standard deviation for the cluster along the principal component axes and the outer yellow border includes all 213 neural spikes in the cluster. The neuronal unit sorting is based on cluster recognition in principle component space. Here PC1 and PC2 stand for the first and second principle components. (E) The autocorrelation histogram for spike events within the unit, the bin size is 3 ms. The inter spike interval (ISI) for the spike sorting was set to 1.5 ms and resulted in 0.0% spike interference ratio.</p

SEM images of the nanowires modified sensing site.

<p>The site image presented after a single implantation (A) and after multiple implantations (B) into rat cortex. The same nanowires-based electrode before any implantation can be seen in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056673#pone-0056673-g001" target="_blank">Figure 1B</a>. Some tissue deposition on the probe after multiple implantations can be seen in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056673#pone-0056673-g005" target="_blank">Figure 5B</a>.</p

Fluid and Highly Curved Model Membranes on Vertical Nanowire Arrays

Sensing and manipulating living cells using vertical nanowire devices requires a complete understanding of cell behavior on these substrates. Changes in cell function and phenotype are often triggered by events taking place at the plasma membrane, the properties of which are influenced by local curvature. The nanowire topography can therefore be expected to greatly affect the cell membrane, emphasizing the importance of studying membranes on vertical nanowire arrays. Here, we used supported phospholipid bilayers as a model for biomembranes. We demonstrate the formation of fluid supported bilayers on vertical nanowire forests using self-assembly from vesicles in solution. The bilayers were found to follow the contours of the nanowires to form continuous and locally highly curved model membranes. Distinct from standard flat supported lipid bilayers, the high aspect ratio of the nanowires results in a large bilayer surface available for the immobilization and study of biomolecules. We used these bilayers to bind a membrane-anchored protein as well as tethered vesicles on the nanowire substrate. The nanowire-bilayer platform shown here can be expanded from fundamental studies of lipid membranes on controlled curvature substrates to the development of innovative membrane-based nanosensors

Fluorescent Nanowire Heterostructures as a Versatile Tool for Biology Applications

Nanowires are increasingly used in biology, as sensors, as injection devices, and as model systems for toxicity studies. Currently, in situ visualization of nanowires in biological media is done using organic dyes, which are prone to photobleaching, or using microscopy methods which either yield poor resolution or require a sophisticated setup. Here we show that inherently fluorescent nanowire axial heterostructures can be used to localize and identify nanowires in cells and tissue. By synthesizing GaP–GaInP nanowire heterostructures, with nonfluorescent GaP segments and fluorescent GaInP segments, we created a barcode labeling system enabling the distinction of the nanowire morphological and chemical properties using fluorescence microscopy. The GaInP photoluminescence stability, combined with the fact that the nanowires can be coated with different materials while retaining their fluorescence, make these nanowires promising tools for biological and nanotoxicological studies

Additional file 1 of Pulmonary toxicity and translocation of gallium phosphide nanowires to secondary organs following pulmonary exposure in mice

Supplementary Material 1: Experimental details and results of the pilot study and 3-months study. GaP NW synthesis and characterization in pilot study. Table S1: Pilot study design. Figure S1: Characterization of GaP NWs in the pilot study. Table S2: (Pilot study) Cellular composition of bronchoalveolar lavage 1 and 3 days after exposure to GaP NWs. Figure S2: (Pilot study) Histopathology of mouse lung 1 and 28 days after pulmonary exposure to GaP NWs. Table S3: 3-month study design. Figure S3: Additional darkfield of GaP NWs in tissues. Figure S4: Chemical identification by EDS of GaP NWs in lung tissue 1 day after exposure. Figure S5: SEM images of GaP NWs in lung tissue day 1 and 28 and 3 months post-exposure. Table S4: Diameter of gold nanoparticles and nanowires in vivo and in vitro. Figure S6, Table S5 and S6: Cellular composition of bronchoalveolar lavage 1, 3, 28 days and 3 months after exposure to GaP NWs, carbon black or MWCNT Mitsui-7. Figure S7 and Table S7: Genotoxicity in BAL cells, lung and liver tissue in 3-month study. Table S8. Mouse lung histopathology 1, 28 days and 3 months after intratracheal instillation of GaP NWs, incidence table. Table S9: Composition of phagolysosomal simulant fluid (PSF). Table S10: Composition of low-calcium Gamble’s solution