Nanostraw–Electroporation System for Highly Efficient Intracellular Delivery and Transfection

Abstract

Nondestructive introduction of genes, proteins, and small molecules into mammalian cells with high efficiency is a challenging, yet critical, process. Here we demonstrate a simple nanoelectroporation platform to achieve highly efficient molecular delivery and high transfection yields with excellent uniformity and cell viability. The system is built on alumina nanostraws extending from a track-etched membrane, forming an array of hollow nanowires connected to an underlying microfluidic channel. Cellular engulfment of the nanostraws provides an intimate contact, significantly reducing the necessary electroporation voltage and increasing homogeneity over a large area. Biomolecule delivery is achieved by diffusion through the nanostraws and enhanced by electrophoresis during pulsing. The system was demonstrated to offer excellent spatial, temporal, and dose control for delivery, as well as providing high-yield cotransfection and sequential transfection.

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.…”

“…Intracellular samples are collected by applying an electrical voltage through the NS, locally opening small holes in the cell membrane near the NS tip. During the subsequent 2-to 5-min interval when these pores are open (29,31,32), approximately 5-10% of the proteins, mRNA, and small molecules diffuse from cells, through the NS, and into an extraction solution below the culture well ( Fig. 1 A and B).…”

“…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.…”

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.…”