Elsevier

Materialia

Volume 9, March 2020, 100577
Materialia

Full Length Article
ZnO nanowire florets promote the growth of human neurons

https://doi.org/10.1016/j.mtla.2019.100577Get rights and content

Highlights

  • hNT neuron growth was promoted on short, densely packed nanowires.

  • hNT neuron growth was inhibited on long, sparsely packed nanowires.

  • Neurons clustered around individual florets of nanowires.

  • Neurons on nanowire florets demonstrated functionality in response to glutamate.

Abstract

Vertical nanowires, due to their 1-dimensional structure, chemical stability, biocompatibility and relative ease of manufacture are ideal candidates for biological interfaces in nanomedicine applications. The majority of live cell studies performed on vertical nanowires have mainly employed Si and GaP nanowires, whereas ZnO nanowires have been relatively unexplored. In this article, we demonstrate that the growth and adhesion of human NTera2.D1/hNT neurons can be modulated on ZnO nanowires. Vertical ZnO nanowires were fabricated using a time-controlled low temperature hydrothermal synthesis on pre-patterned seed regions that allows for precise control over nanowire morphology. We demonstrate that neuronal adhesion was enhanced when ZnO nanowires were below 500 nm long and at a density of 350 nanowires per µm2, increasing up to 33% compared to SiO2 surfaces. In contrast neuron adhesion was inhibited by longer and less dense nanowires. Furthermore, we demonstrate that neurons grew preferentially on individual florets of nanowires when the nanowires were fabricated into arrays of varying dimensions. Finally, we demonstrate that the neurons on individual nanowire florets were viable and displayed functional Ca2+ responses to glutamate stimulation that were comparable to neurons that were grown on SiO2.

Introduction

Nanowires are 1-dimensional cylindrical nanostructures that are typically manufactured from inorganic, conducting or semiconducting materials [1]. Nanowires typically have a diameter below 100 nm, a length in the micrometre range and have unique physical properties, such as high aspect ratios and large surface areas, which are not observed in bulk materials [2]. There is a great deal of interest in the application of ZnO nanowires to cell biology due to the ease of synthesis in varying geometries and morphologies. Their size makes them ideal for interactions with cells, which have a typical cell membrane width of approximately 10 nm and a cell diameter of approximately 10 µm. In recent years, the interaction of nanowires with living cells has been reviewed extensively for biological applications [3], [4], [5], [6], [7], [8]. Specifically, nanowires have been used for cell guidance and spatial patterning [9,10], electrical recording [11], [12], [13], [14], transfection [15], mechanical sensing [16] and biomolecule transport [17]. In particular, ZnO nanowires have demonstrated a potential to be used on microelectrode arrays for measuring electrical signals from neurons since their 3D structure can reduce the electrical impedance of the electrodes [18].

Despite previous use of ZnO nanowires for biological applications there are conflicting reports of cytotoxicity under different experimental conditions. The interaction between nanowires and cells can vary considerably depending on application and parameters such as the cell type, nanowire material, geometry, morphology and the fabrication process [3].

ZnO nanowires can easily be grown via a low temperature (below 100 °C) hydrothermal synthesis [19] with a low cost and high scalability on various substrates [20], [21], [22]. The morphology and geometry of nanowires can be controlled by varying both of the hydrothermal growth parameters (e.g. precursor concentrations, growth time) and through the area and spacing of the seed layers available, via photolithographic patterning [23], [24], [25].

To date, no study has been reported on interaction of ZnO nanowires with human neurons. In this article, we assess the biocompatibility of ZnO nanowires of various morphologies with human neurons that were derived from the human teratocarcinoma cell line (hNT). hNT neurons express a variety of characteristic neuronal markers [26], exhibit spontaneous electrical activity [27], and have been validated as an alternative to primary human neurons [28]. hNT neurons have been used in cell patterning applications [29] and have been used in clinical applications as a cell transplantation treatment to aid in recovery following stroke [30]. In addition, hNTs raise few ethical concerns as neurons are derived from an stem cell line and provide an accessible way to produce large quantities of human neurons.

Section snippets

ZnO nanowire fabrication

ZnO nanowire samples were fabricated in a pattern comprised 8 arrays nanowire florets with inter-floret gaps (G) varying from 5 µm to 100 µm. The floret diameter was 15 µm and was similar to the average size of a neuron soma. Fig. 1A and B shows brightfield images of a single ZnO nanowire sample and one of the nanowire arrays, respectively. The nanowire fabrication process is depicted in Fig. 1C and is described below. The nanowires were formed on 7 × 7 mm2 silicon substrates with a 100 nm

Results

In this section, we describe the growth of hNT neurons on ZnO nanowire samples. First, we characterised the morphology of ZnO nanowires that were hydrothermally grown under various conditions. Next, we evaluated whether neuron growth was promoted on individual nanowire florets. Then, we evaluated the influence of both the array geometry and the nanowire morphology on neuron growth. Finally, we evaluated whether the neurons within the nanowire arrays were functionally viable by examining their

Discussion

In this article, we have described how vertical ZnO nanowires either promote or inhibit the growth of human hNT neurons depending on their morphology. Using a hydrothermal growth method developed by Maddah et al. [23], combined with photolithography to generate spatially patterned nanowire florets, we were able to examine the growth of neurons over a wide range of nanowire morphologies. Unlike previous literature, we report either an increase or a decrease in neuron growth that was modulated by

Conclusions

In this article we have demonstrated both the promotion and inhibition of hNT neuron growth on ZnO nanowires. It was found that neuron growth was promoted when ZnO nanowires were less than 500 nm in length with a density of greater than 350 nanowires per µm2, however, as the nanowires grew longer, they became less dense and supported less extensive neuron growth. The promotion of neuron growth was attributed to the nanowire covered surfaces presenting a favourable surface roughness. In

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

We acknowledge funding support from The Royal Society of New Zealand Marsden fund (UOA1510/3709273).

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