Micaela Schaefer High Voltage
Phyllis Sterlin
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Gene induction and/or inhibition provide powerful tools to understand gene functions1, control cellular signals2, and develop new therapeutic technologies3. The emerging exploration in RNA interference4,5 and cell reprogramming6,7 for cancer treatment and/or personalized medicine pushes the expectation on the effectiveness of gene delivery to a new high level. Safe delivery of healthy copies of DNA or RNA probes in majority treated cells with high efficiency and excellent survival rate becomes essential for the success of these applications. Viral transduction is highly stable and efficienct8, but has limited carrying capacity and high risk of oncogenesis and inflammation9. This largely stimulates the pursuit of nonviral delivery strategies, including both chemical and physical approaches, which however have not yet become competitive to their viral counterpart10,11,12,13,14. Compared to the chemical delivery strategies, physical approaches grew fast in recent years, benefited from their direct delivery to desired intracellular locations15,16,17,18,19. Among them, electroporation is often favorable for its balance of simplicity, transfection effectiveness, broad allowance on probe or cell types, and operation convenience20,21,22. In electroporation, short, high-voltage electric pulses are applied to surpass the cell membrane capacitance, making the subjected cells transiently permeable20. It has two active but relatively independent research directions: single cell electroporation (SCE) and bulk electroporation (BE). The former focuses on the discovery of cellular transport dynamics and mechanism (i.e., electrophysiology) while the latter targets at high transfection efficiency to cells in a large population. Both fields are important but difficult to support each other. For example, according to single cell electroporation theory, the transmembrane potential (ΔVm, in V) for reversible breakdown of the cell lipid bilayer can be estimated by:
Such transfection enhancement of MAE is attributed to the synergistic effects of the electric field focusing, localized electroporation, and size-dependent treatment. The first two effects benefit for cell membrane permeabilization at benign pulse conditions and its better recovery afterwards, while the size-dependent treatment allocates the number and area of the transient openings on individual cell membrane to ensure homogeneous treatment on cells of various sizes. Their specific contributions are addressed as following:
Physical observation of such millisecond membrane opening process in live cell electroporation is still very challenging, considering the requirement on integrating the electroporation setup within Cryo-TEM facility56. However, reasonable speculation based on current available electroporastion theory and some recent cell electroporation simulation findings57,58 could approximately reveal the pore formation dynamics occurs in MAE. According to equation (1), transmembrane potential on an individual cell also varies by locations or the local cell surface orientation to the imposed electric field. In bulk electroporation, this means the highest transmembrane potential appears in two locations (i.e., 0 and 180 degrees, facing the two large plate electrodes) of individual cells and drops continuously in between, according to their suspension status and spherical geometry57,58. As the consequence, heterogeneous permeability presents across the whole cell membrane: with some large pores close to the two poles of the spherical cell and many other small, incomplete openings elsewhere. On the contrary, as the electrical pulses are highly focused by many tiny micropillars of the same size, the local transmembrane potential in locations facing these microelectrodes on an individual cell is similar in MAE. Small pores of similar size are therefore generated in these locations and distributed evenly on the cell membrane. Although the input total energy in both systems (bulk electroporation and MAE) is same, different polarization consequence occurs on individual cells for their different electrode configurations: more small pores of uniform size are generated in MAE while a mixture of large pores and many other small, incomplete openings in bulk electroporation.
To conclude, our MAE system could enhance the electroporation-mediated DNA and RNA delivery to both adherent and suspension cells. Its well-defined micropillar array configuration ensures size specific treatment to a large number of cells regardless their random dispersion. Besides the benefits we demonstrated here, the cellular uptake dynamics of individual cells in MAE could be representative as it works like many size-dependent treatments are done in parallel. This could provide useful information to help simplify the tedious, cell-specific protocol searching process for bulk electroporation and help mutual support between two long separated electroporation fields, single cell electroporation and bulk electroporation. Its success may benefit many research communities where a safe and effective non-viral gene delivery approach is needed on a daily basis.
Y.Z. and S.W. conceived the study. Y.Z. and S.H. design the experiments. Y.Z., S.H., Y.L. and X.L. performed the experiments. Y.Z., S.H. and S.W. analyzed and interpreted the data. Y.Z. and S.W. wrote the manuscript. All authors reviewed the manuscript.
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