Facile fabrication of ordered discontinuous nanotopography on photosensitive substrates for enhanced neuronal differentiation

Abstract

Controlling the development and morphology of neurons is important for basic neuroscience research as well as for applications in nerve regeneration and neural interfaces. Various studies have shown that nanoscale topographies can promote the development of neuronal cells and the differentiation of neural stem cells; however, the fabrication of these nanotopographical features often involves expensive and sophisticated techniques. Here, we employ nanosphere lens lithography combined with UV-LED technology to create nanopatterns on an SU-8 photoresist. We develop a facile method to create a reusable polystyrene nanosphere (PS-NS) lens array by the spontaneous formation of a hexagonal close-packed array of PS-NSs at a water–air interface and its subsequent transfer to a polydimethylsiloxane carrier film without using any special equipment. We show that this simple technique can create ordered arrays of nanodots on an SU-8 film, the dimensions of which can be controlled by the size of the PS-NSs. When used as a substrate for the neuronal differentiation of pheochromocytoma (PC12) cells, the nanopatterned SU-8 films exhibit enhanced differentiation parameters with respect to conventional tissue culture plastic as compared with their flat counterparts. The method proposed here can greatly facilitate the nanopatterning of various photosensitive substrates for the development of implants for nerve regeneration and neural interfacing.

Hierarchical Micro-/Nanotopographies Patterned by Tandem Nanosphere Lens Lithography and UV–LED Photolithography for Modulating PC12 Neuronal Differentiation

“…The preparation of the SU-8 films and the nanopatterning process were similar to the ones we reported previously . The films were formed on clean 16 mm-diameter round glass coverslips (Figure A).…”

“…We used PS spheres (also called latex beads) with two different nominal diameters, namely, 460 nm and 1.1 μm (Sigma; Merck KGaA, Germany), to create arrays of SU-8 nanodots and nanopillars, respectively. The preparation of a reusable PS-NS lens array with 460 nm PS nanospheres is described in our previous study . Briefly, a 1% solids PS-NS suspension containing 75% v/v ethanol was dispensed on a 45°-inclined silicon wafer partially immersed in ultrapure water (type I; resistivity of 18.2 MΩ·cm) containing a small amount of Triton X-100 (Sigma-Aldrich; Merck KGaA, Germany).…”

“…The PS-NS array formation shown in (A) and all the SEM images are for the 1.1 μm-diameter PS spheres. For the 460 nm-diameter PS nanospheres, please refer to ref .…”

“…A low-cost, custom-made uncollimated UV–LED exposure system consisting of a square array of 360 5 mm LEDs with a peak wavelength between 380 and 385 nm (Centenary Materials Co., Ltd., Taiwan) and a ground glass diffuser (Edmund Optics Inc., NJ, USA) was used for all the UV exposure steps. , All of the baking steps were done on a hotplate. During the photoresist processing, the room temperature was maintained between 23 and 25 °C, while the humidity was maintained between 51 and 55% RH.…”

“…Previously, we have shown that nanosphere lens lithography (NLL; also called colloidal phase-shift lithography) , can be used in conjunction with a custom-made uncollimated ultraviolet light-emitting diode (UV–LED) light source to create ordered arrays of nanodots on the negative-tone, epoxy-based SU-8 photoresist . This nanopatterned film was then used as the substrate for the neuronal differentiation of PC12 cells and was shown to improve neurite sprouting as compared to a conventional tissue culture plastic substrate.…”