Transfection is a critical step for gene editing and cell‐based therapies. Nanoscale technologies have shown great promise in providing higher transfection efficiency and lower cell perturbation than conventional viral, biochemical, and electroporation techniques due to their small size and localized effects. Although this has significant implications for using cells post‐transfection, it has not been thoroughly studied. Here, the nano‐electro‐injection (NEI) platform is developed, which makes use of localized electric fields to transiently open pores on cell membranes followed by electrophoretic delivery of DNA into cells. NEI provides twofold higher net transfection efficiency than biochemicals and electroporation in Jurkat cells. Analysis of cell doubling time, intracellular calcium levels, and messenger ribonucleic acids (mRNA) expression changes after these gene delivery methods reveals that viruses and electroporation adversely affected cell behavior. Cell doubling times increase by more than 40% using virus and electroporation methods indicative of higher levels of cell stress, unlike NEI which only minimally affects cell division. Finally, electroporation, but not NEI, greatly alters the expression of immune‐associated genes related to immune cell activation and trafficking. These results highlight that nanoscale delivery tools can have significant advantages from a cell health perspective for cell‐based research and therapeutic applications.
In vitro and ex vivo intracellular delivery methods hold the key for releasing the full potential of tissue engineering, drug development, and many other applications. In recent years, there has been significant progress in the design and implementation of intracellular delivery systems capable of delivery at the same scale as viral transfection and bulk electroporation but offering fewer adverse outcomes. This review strives to examine a variety of methods for in vitro and ex vivo intracellular delivery such as flow‐through microfluidics, engineered substrates, and automated probe‐based systems from the perspective of throughput and control. Special attention is paid to a particularly promising method of electroporation using micro/nanochannel based porous substrates, which expose small patches of cell membrane to permeabilizing electric field. Porous substrate electroporation parameters discussed include system design, cells and cargos used, transfection efficiency and cell viability, and the electric field and its effects on molecular transport. The review concludes with discussion of potential new innovations which can arise from specific aspects of porous substrate‐based electroporation platforms and high throughput, high control methods in general.
more » « less- NSF-PAR ID:
- 10454636
- Publisher / Repository:
- Wiley Blackwell (John Wiley & Sons)
- Date Published:
- Journal Name:
- Small
- Volume:
- 16
- Issue:
- 51
- ISSN:
- 1613-6810
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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