Nanostraws for Direct Fluidic Intracellular Access

Abstract

Nanomaterials are promising candidates to improve the delivery efficiency and control of active agents such as DNA or drugs directly into cells. Here we demonstrate cell-culture platforms of nanotemplated "nanostraws" that pierce the cell membrane, providing a permanent fluidic pipeline into the cell for direct cytosolic access. Conventional polymeric track-etch cell culture membranes are alumina coated and etched to produce fields of nanostraws with controllable diameter, thickness, and height. Small molecules and ions were successfully transported into the cytosol with 40 and 70% efficiency, respectively, while GFP plasmids were successfully delivered and expressed. These platforms open the way for active, reproducible delivery of a wide variety of species into cells without endocytosis.

Nondestructive nanostraw intracellular sampling for longitudinal cell monitoring

“…Cells of interest are cultured on a polymer membrane with a defined region of hollow NS extending through the polymer and protruding from the surface (Fig. 1) (23,29,30) Cells grow normally over the entire polymer membrane, so that cells within the sampling region interact with surrounding cells, avoiding cell isolation. Intracellular samples are collected by applying an electrical voltage through the NS, locally opening small holes in the cell membrane near the NS tip.…”

“…1 E and F). Cells grown on the NS demonstrate normal cell behavior and mRNA expression (23,29,33). Previously, we found that NS with a diameter of 100 nm or smaller spontaneously penetrate the cell membrane, allowing the delivery of small molecules into cells.…”

“…Several groups have demonstrated the insertion of high aspectratio nanostructures for molecular delivery with minimal cell perturbation, suggesting that nanomaterials may pass through the cell membrane without dire consequences (19)(20)(21)(22)(23)(24). Extending this approach, Na et al (25) showed a nanowire "sandwich assay" in which nanowires approximately 100 nm in diameter functionalized with antibodies penetrate the cell with limited toxicity and can bind specific enzymes for extraction.…”

In vitro and ex vivo strategies for intracellular delivery

“…Prominent Nanoneedles are another key development over the last decade, involving the generation of nanometre-scaled features capable of penetrating the cell membrane and providing access to the cytosol 82 . In the reported modalities thus far, the target material is delivered through one of three modes: (1) dissociation from the penetrating structure upon cytosolic entry 82,83 ; (2) direct injection through a hollow nanoneedle or "nanostraw" 84,85 or (3) the diffusion from the extracellular medium through holes after withdrawal of the needles 44 . For the first mode, the delivery of siRNA, peptides, DNA, proteins, and impermeable inhibitors to challenging cell types such as neurons and immune cells has been demonstrated 83,86 .…”

“…In the second mode, molecules have been successfully pumped into cells through nanostraws. A key benefit of this configuration is temporal control over delivery dynamics, volume, and dosage concentration, as well as possible gating with electric fields 84,85 . In the third mode, ref.…”