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Nanosphere Lithography for Intracellular Delivery

  • Author(s): Wattanatorn, Natcha
  • Advisor(s): Weiss, Paul S.
  • et al.
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Abstract

Nanosphere lithography (NSL) is a simple, high-throughput technique that can be used to form large-area, close-packed monolayer arrays of nanospheres. These arrays can be directly used as an etching or as a deposition mask, to generate silicon-based nanostructures. Typically, the nanostructures produced are created by single etches of the nanosphere array resulting in limitations in fabrication of novel patterns/nanostructures. Here, we report multiple patterning nanosphere lithography for fabrication of three-dimensional periodic silicon-based nanostructures, exploiting their degradable nature during selected and repeated etching of the polymer nanospheres. As a result, the masks can be shaped in parallel for each processing step enabling the fabrication of wafer-scale three-dimensional (3D) periodic silicon nanostructures. These nanotubes and hierarchical nanostructures can be tuned precisely with independent control in three dimensions including outer/inner diameters, heights/hole-depths, and pitches. We have demonstrated our technique to construct solid/hollow nanotubes, multilevel solid/hollow nanotowers, and 3D concentric plasmonic nanodisk/nanorings with tunable optical properties on a variety of substrates.

In the second part of my dissertation, I used NSL to fabricate periodic arrays of conical nanoneedles for non-viral gene delivery for chimeric antigen receptor (CAR) T cell production. Gene delivery using non-viral methods has significant advantages in terms of safe delivery of cargo and cost. Especially, physical membrane disruption via nanoneedles has the capability to inject and deliver molecules of interest directly as well as the capability to create transient pores in the cell membrane, enabling biomolecule diffusion into the cells. However, the challenges for these systems include inconsistency of membrane penetration and slow processing throughputs. Here, we use a nanoneedle-integrated microfluidic system and gene-encapsulated supramolecular nanoparticle for production of CAR-T cells, in both model and primary T cells. Using NSL, we can achieve conical shaped silicon nanoneedles, where the height, base width, tip sharpness, and pitches are individually tunable, resulting in sturdy structures that can penetrate the cells. With this platform, we can efficiently load CAR plasmids inside nanoparticles, which are tethered to the substrate for direct injection, as well as co-flow an excess of CAR encapsulated nanoparticles, for diffusion through transient pores that are created by the nanoneedles. This platform enables continuous and sequential intracellular delivery, which provides a path for sustainable CAR-T cell production.

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This item is under embargo until June 13, 2021.