Nontoxic nanopore electroporation for effective intracellular delivery of biological macromolecules

Abstract

We present a simple nanopore-electroporation (NanoEP) platform for delivery of nucleic acids, functional protein, and Cas9 single-guide RNA ribonucleoproteins into both adherent and suspension cells with up to 80% delivery efficiency and >95% cell viability. Low-voltage electric pulses permeabilize a small area of cell membrane as a cell comes into close contact with the nanopores. The biomolecule cargo is then electrophoretically drawn into the cells through the nanopores. In addition to high-performance delivery with low cell toxicity, the NanoEP system does not require specialized buffers, expensive materials, complicated fabrication processes, or cell manipulation; it simply consists of a generic nanopore-embedded water-filter membrane and a low-voltage square-wave generator. Ultimately, the NanoEP platform offers an effective and flexible method for universal intracellular delivery.

A versatile bulk electrotransfection protocol for mouse embryonic fibroblast and iPS cells

“…A > 95 % electrotransfection rate was achieved with the optimized pulsing condition. Even the rate of transient expression of reporter genes as well as the cell viability using the OptiMEM-GlutaMAX in the current study were as high as that of newly reported mechanical-electrical (7) and nanopore-electrical approaches (12). We used plasmids with various sizes, from 6.2 to 13.5 kb, and found a high transfection rate and cell viability irrespective of the plasmid size.…”

Nanoneedle Platforms: The Many Ways to Pierce the Cell Membrane

“…Using electric pulses, cell membranes can be transiently perforated so that a large variety of materials can be intracellularly delivered. In recent years, electroporation coupled with micro/nanostructures, [ 151–154 ] called as micro/nanoscale electroporation (MEP/NEP), has significantly improved cell viability while ensuring efficient transfection. These micro/nanostructure‐mediated local electric fields can more gently penetrate the cell membrane, and the resulting cell damage can be almost negligible.…”

Methods for protein delivery into cells: from current approaches to future perspectives

“…This allows the application of lower voltage without reducing the electric field strength. This approach minimizes heating and pH changes [55,56]; as a consequence cell viability after electroporation and transfection efficiency are both increased [57][58][59][60][61][62].…”

“…In the nanopore-electroporation (NanoEP) platform cells are deposited onto a polycarbonate membrane that it contains 100 nm diameter nanopores with density of 0.2 pores per mm 2 . The pores are connected to a liquid reservoir and cargoes can then be delivered into numerous cells via localized electroporation after establishing an electric field [58]. NanoEP has been used to deliver a variety of cargoes, including mRNAs, DNA plasmids, CRISPR/Cas9 RNA-protein complexes and functional stromal interaction molecule 1 (STIM1) proteins into the cell cytoplasm [58].…”

“…The pores are connected to a liquid reservoir and cargoes can then be delivered into numerous cells via localized electroporation after establishing an electric field [58]. NanoEP has been used to deliver a variety of cargoes, including mRNAs, DNA plasmids, CRISPR/Cas9 RNA-protein complexes and functional stromal interaction molecule 1 (STIM1) proteins into the cell cytoplasm [58]. The nanostraws electroporation system (NES) has essentially the same set-up as the nanostraws system described above, but the addition of electrodes in the liquid reservoir underneath the straws and in the cell culture medium reservoir allows localized electroporation to be performed [59,66].…”