PROJECT TITLE: Engineering Vertically Aligned Silicon Nanowire Arrays for delivering genes to mammalian cells

Supervisor: Prof. Dr. Nicolas Voelcker, Mawson Institute, University of South Australia, Tel +61883023190

Email: nico.voelcker@unisa.edu.au

Location: Mawson Institute, University of South Australia, Adelaide, Australia

PROJECT OUTLINE: The emerging field of nanotechnology, which involves the development of nanomaterials with well-defined architectures at the nanoscale, has been pivotal in the development of powerful tools for interfacing with biological systems. In particular, vertically-aligned silicon nanowire (VAS-NW) arrays are a remarkable example of these well-defined nanoscale architectures, which are frequently being used to create functional NW-to-cell interface1. Achieving direct communication between VAS-NW arrays and mammalian cells, through the ability to engineer the architectures of VAS-NW arrays and the surface chemistry, holds great opportunities to monitor and orchestrate gene delivery into mammalian cells2,3. It is conceivable that this new VAS-NW array technology will allow us to overcome significant limitations associated with the existing strategies for delivering genes into cells that are limited either by the range of chemical and biological species that can be delivered, by low efficiency of delivery or by high toxicity4. In this project, we will use functionalised, pre-designed VAS-NW arrays, as a platform to transport genes into various types of living cells, with minimal impact on the cells’ viability and function Fig.1B. The physical design of these well-defined patterned VAS-NW arrays is achieved through a combination of techniques including colloidal assembly, nanosphere lithography (NSL), and metal-assisted chemical etching (MACE) Fig.1A. Significantly, the interplay of two parameters – the physical design of the VAS-NW arrays and their surface chemistry – will improve our ability to efficiently introduce genes into targeted mammalian cells.

Engineering patterned VAS-NW arrays: Pre-designed VAS-NW arrays with fine control over the aspect ratio, density, location and orientation of individual NW elements within the array, will establish reproducible, hybrid and smart VAS-NW array–cellular interfaces for gene delivery application.

 Engineering surface chemistry: Tuning the surface chemistry and functionalization of the VAS-NW arrays, will offer greater flexibility and specificity in controlling key parameters of the VAS-NW arrays – favourable biocompatibility, tailored biodegradability and higher chemical stability – enabling one for instance to overcome existing limitations in terms of sustained release of genes from the NWs element, across the mammalian cell membrane.

METHODS AND EQUIPMENT: Fabrication of VAS-NW array: In metal-assisted chemical etching (MACE), the fabrication of 1D NWs comprises of two steps. First, a lithographically structured noble metals thin film (e.g., Ag, and Au) is deposited on the substrate. Second, the etching in hydrofluoric acid-based solution (HF+ oxidative agent) is performed. A patterned VAS-NWs array using MACE is schematically shown in Fig.1A. Hexagonal close-packed monolayers of monodisperse polystyrene particles (1a) are transferred into hexagonal non-close-packed particle arrays via oxygen plasma etching (1b). The arrays were used as a mask for metal deposition e.g. Ag by sputter coater (1c). After removing the spheres by lift-off, an ordered array of nano-holes was produced in the Ag film (1d). Subsequently, the metal layer served as a catalyst for the wet-etching of silicon in a tuneable etching solution. Using this method, VAS-NW arrays were fabricated with different aspect ratios and porosities (1e). The silver layer was lifted away with nitric acid (1f).

Cell transfection: First, the surface of the VAS-NW arrays is functionalized with cargo molecules such as gWIZ-GFP plasmid. Second, mammalian cells are seeded onto specific architecture design (e.g., VAS-NW arrays) and surface chemistry. Finally, we monitor

the efficacy in transporting genes as well as the interactions between the array and the cells using confocal microscopy, SEM and FIB-SEM. Our predesigned functionalised VAS-NW arrays have been shown to be able to deliver plasmids into a number of mammalian cells including human dental pulp-derived mesenchymal stem cells (hDPMSCs) that are considered particularly difficult to transfect, with efficiency greater than 90%.

 Fig 1 A) Main steps in the fabrication of a patterned VAS-NW arrays using MACE. B) Schematic illustration of cells transfection by VAS-NW arrays. C) SEM image of hDPMSCs grown on VAS-NW array. D) Fluorescence confocal image of the transfected hDPMSCs via the use VAS-NW array coated with gWIZ-GFP plasmid (> 90 % transfection efficiency, nuclear staining with Hoechst 33342).

References 1. Shalek, A. K., et al., Proc Natl Acad Sci U S A (2010) 107 (5), 1870 2. Mendes, P. M., Chemical Society Reviews (2013) 3. Buriak, J. M., et al., Journal of the American Chemical Society (1999) 121 (49), 11491 4. Lechardeur, D., et al., Adv Drug Deliv Rev (2005) 57 (5), 755

About Adelaide:

Adelaide is the capital of South Australia and offers a very high standard of living (top 5 most liveable city in the world according to a survey by “The Economist”), with great climate, food, wine, beautiful unspoiled nature and beach environments, in an inexpensive setting. Adelaide was also recently voted into the Lonely Planet top 10 cities in the world.

The Mawson Institute is a leading centre of expertise in materials science and its application to novel products. Based on strong fundamental research in selected areas of materials science, we innovatively combine materials science with engineering for translation to prototype products. New technology platforms developed at the Mawson Institute are intended to underpin next generation manufacturing via integration into new products and processes. At our Institute, scientists and engineers work in parallel on concepts and commercialisation, significantly reducing the time and steps in product development. At the same time, research by PhD students and staff provides the next generation of knowledge, ideas, and technologies.

The institute is based in two new state-of-the-art buildings with outstanding research facilities (see photo of the MM building).

Prof. Nico Voelcker, Associate Director (Research) Mawson Institute Adelaide, South Australia 5000 GPO Box 2471 Adelaide South Australia 5001 Australia t: +61 8 8302 5508 f: +61 8 8302 5613 e: nico.voelcker@unisa.edu.au