Microfluidic Electroporation Coupling Pulses of Nanoseconds and Milliseconds to Facilitate Rapid Uptake and Enhanced Expression of DNA in Cell Therapy

An-Yi Chang,Shengnian Wang,Zhaogang Yang,Hong Tian,Xuan Liu,Liping Hua

doi:10.1038/s41598-020-63172-8

An-Yi Chang, Shengnian Wang + Show 4 more

Open Access

https://doi.org/10.1038/s41598-020-63172-8

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Abstract

Standard electroporation with pulses in milliseconds has been used as an effective tool to deliver drugs or genetic probes into cells, while irreversible electroporation with nanosecond pulses is explored to alter intracellular activities for pulse-induced apoptosis. A combination treatment, long nanosecond pulses followed by standard millisecond pulses, is adopted in this work to help facilitate DNA plasmids to cross both cell plasma membrane and nuclear membrane quickly to promote the transgene expression level and kinetics in both adherent and suspension cells. Nanosecond pulses with 400–800 ns duration are found effective on disrupting nuclear membrane to advance nuclear delivery of plasmid DNA. The additional microfluidic operation further helps suppress the negative impacts such as Joule heating and gas bubble evolution from common nanosecond pulse treatment that lead to high toxicity and/or ineffective transfection. Having appropriate order and little delay between the two types of treatment with different pulse duration is critical to guarantee the effectiveness: 2 folds or higher transfection efficiency enhancement and rapid transgene expression kinetics of GFP plasmids at no compromise of cell viability. The implementation of this new electroporation approach may benefit many biology studies and clinical practice that needs efficient delivery of exogenous probes.

Highlights

Electroporation works by applying short, high-voltage electrical pulses across the cell membrane to make it transiently permeable to exogeneous cargos[1]
Nanosecond pulses are first imposed on cells to make the nuclear membrane permeable, followed by a standard millisecond pulse treatment which breaks down the cell plasma membrane
Our comparison was made among three different cases: standard electroporation alone, TCHD treatment followed by standard electroporation, and nanosecond pulse treatment followed by standard electroporation

Summary

Introduction

Electroporation works by applying short, high-voltage electrical pulses across the cell membrane to make it transiently permeable to exogeneous cargos[1]. Owing to its balance of operation simplicity, transfection effectiveness, and broad allowance of probe or cell types, such reversible electroporation systems are often chosen as the preferred delivery approach in the past two decades to facilitate cellular internalization of plasmid DNA, oligonucleotides, and molecule drugs in many biological and clinical studies[2,3,4] Besides such reversible electroporation treatment, ultra-sharp, but ultra-short electrical pulses (10–300 kV/cm with 10–300 ns pulse duration) were recently found useful to induce cell apoptosis for cancer treatment[5,6,7,8,9,10]. Demonstrated with quick kinetics and significantly improved transgene expression without compromise of cell viability, the success of this new electroporation system on these representative cell lines as transfection host or cancer study models may help advance many biology studies and clinical practice on cell function regulation and therapeutic effectiveness verification

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