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.
- Award ID(s):
- 2125383
- NSF-PAR ID:
- 10376715
- Editor(s):
- Waldor, Matthew K.
- Date Published:
- Journal Name:
- PLOS Biology
- Volume:
- 20
- Issue:
- 9
- ISSN:
- 1545-7885
- Page Range / eLocation ID:
- e3001727
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Abstract -
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Abstract Electroporation is an electro-physical, non-viral approach to perform DNA, RNA, and protein transfections of cells. Upon application of an electric field, the cell membrane is compromised, allowing the delivery of exogenous materials into cells. Cell viability and electro-transfection efficiency (eTE) are dependent on various experimental factors, including pulse waveform, vector concentration, cell type/density, and electroporation buffer properties. In this work, the effects of buffer composition on cell viability and eTE were systematically explored for plasmid DNA encoding green fluorescent protein following electroporation of 3T3 fibroblasts. A HEPES-based buffer was used in conjunction with various salts and sugars to modulate conductivity and osmolality, respectively. Pulse applications were chosen to maintain constant applied electrical energy (J) or total charge flux (C/m2). The energy of the pulse application primarily dictated cell viability, with Mg2+-based buffers expanding the reversible electroporation range. The enhancement of viability with Mg2+-based buffers led to the hypothesis that this enhancement is due to ATPase activation via re-establishing ionic homeostasis. We show preliminary evidence for this mechanism by demonstrating that the enhanced viability is eliminated by introducing lidocaine, an ATPase inhibitor. However, Mg2+also hinders eTE compared to K+-based buffers. Collectively, the results demonstrate that the rational selection of pulsing conditions and buffer compositions are critical for the design of electroporation protocols to maximize viability and eTE.
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1371/journal.pbio.3001727
