Intracellular ROS mediates gas plasma-facilitated cellular transfection in 2D and 3D cultures

Abstract

This study reports the potential of cold atmospheric plasma (CAP) as a versatile tool for delivering oligonucleotides into mammalian cells. Compared to lipofection and electroporation methods, plasma transfection showed a better uptake efficiency and less cell death in the transfection of oligonucleotides. We demonstrated that the level of extracellular aqueous reactive oxygen species (ROS) produced by gas plasma is correlated with the uptake efficiency and that this is achieved through an increase of intracellular ROS levels and the resulting increase in cell membrane permeability. This finding was supported by the use of ROS scavengers, which reduced CAP-based uptake efficiency. In addition, we found that cold atmospheric plasma could transfer oligonucleotides such as siRNA and miRNA into cells even in 3D cultures, thus suggesting the potential for unique applications of CAP beyond those provided by standard transfection techniques. Together, our results suggest that cold plasma might provide an efficient technique for the delivery of siRNA and miRNA in 2D and 3D culture models.

Direct transfection of clonal organoids in Matrigel microbeads: a promising approach toward organoid-based genetic screens

“…In contrast with a previous study demonstrating low plasmid transfection efficiency (about 6%) in 3D cultures ( 30 ), we managed to reach plasmid extinction efficiency of more than 40%. Moreover, in a recent study ( 36 ) demonstrating the benefits of a novel gas plasma-facilitated cellular transfection method, siRNA extinction efficiency of about 20% (as compared to more than 80% in our system) and plasmid transfection efficiency of less than 5% (as compared to more than 40% in our system) were reached in a 3D culture system. Strikingly, we managed for the first time to reach siRNA transfection efficiencies that are comparable between 2D cultures and 3D organoids.…”

Basic Research in Plasma Medicine - A Throughput Approach from Liquids to Cells

“…This can be done by omics techniques 30 31 32 and/or up or downregulation of target genes using siRNA (redox) signaling inhibitors, antioxidants and antioxidant enzymes, as well as modulation of the plasma's reactive species output. 33 34 35 Hence, streamlined assay protocols are needed to test larger sample sets with large numbers of iterations.…”

Direct plasma stimuli including electrostimulation and OH radical induce transient increase in intracellular Ca2+ and uptake of a middle‐size membrane‐impermeable molecule

“…Among APP medical applications, transfection technology of middle‐size (500–30 000 Da) and large‐size (30 000‐ Da) molecules has attracted attention. Some research groups have reported cell membrane permeabilization of middle‐size fluorescent dye and large‐size plasmid DNA using various types of APP . Although a few reports have suggested that the multiple effects of electric and chemical stimuli are key factors in APP‐induced membrane permeabilization, most of the underlying mechanisms remain unknown.…”