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Importance <strong>of</strong> electrophoretic force for successful gene electrotransfer<br />
for suboptimal plasmid concentrations<br />
Mojca Pavlin, Maša Kandušer, Damijan Miklavčič<br />
University <strong>of</strong> Ljubljana, Faculty <strong>of</strong> Electrical Engineering, Trzaška 25, SI-1000 Ljubljana, Slovenia<br />
Electroporation is a versatile biotechnology technique that among others enables gene<br />
electrotransfer, i.e. transfer <strong>of</strong> DNA into biological cells. High-voltage pulses induce<br />
structural changes in the cell membrane that thus becomes transiently permeable for ions,<br />
molecules and macromolecules. Gene electrotransfer is already an established method for<br />
gene delivery in vitro and in vivo. Currently, majority <strong>of</strong> research is focused on improving<br />
in vivo transfection efficiency and first clinical trials are in progress, while mechanisms<br />
involved in electrogene transfer have not been yet completely understood.<br />
In this paper we analyze the mechanisms <strong>of</strong> gene electrotransfer by using combinations <strong>of</strong><br />
high-voltage (HV) and low-voltage pulses (LV) in vitro. We applied different combinations<br />
<strong>of</strong> HV and LV pulses to CHO cells and determined the transfection efficiency using<br />
plasmid DNA coding for green fluorescent protein (GFP). Our results show that short<br />
HV pulses alone are sufficient to successfully deliver DNA into cells when optimal plasmid<br />
concentrations were used. For optimal plasmid concentration LV pulse did not contribute<br />
significantly to the transfection efficiency, in contrast to the reported results <strong>of</strong> several in<br />
vivo and one in vitro study, where combinations <strong>of</strong> HV and LV pulses markedly increased<br />
transfection efficiency compared to protocols where only HV pulses were applied. With this<br />
we demonstrated that short HV pulses are not only crucial for efficient permeabilization<br />
<strong>of</strong> the cell membrane, which enables transfection, but are alone sufficient to successfully<br />
deliver DNA into cells in vitro for optimal plasmid concentrations. Therefore in general it<br />
is difficult to separate the role <strong>of</strong> HV pulses as being only permeabilizing and LV pulses as<br />
being electrophoretic since both effects are usually presented in both cases.<br />
However, for suboptimal plasmid concentrations we obtained that LV pulses which follow<br />
HV pulses increase transfection rate similarly as in in vivo conditions. Our results therefore<br />
suggest that low-voltage pulses are contributing to increased transfection in in vivo<br />
conditions due to limited mobility <strong>of</strong> plasmid DNA in the extracellular matrix resulting<br />
due to low local plasmid concentration. Namely, low-voltage pulses provide additional<br />
electrophoretic force which drags DNA toward the cell membrane and contributes to<br />
better interaction <strong>of</strong> the DNA with the membrane and better transfection efficiency, while<br />
for optimal concentrations <strong>of</strong> the plasmid (usually used in vitro) electrophoresis does not<br />
have important role.<br />
p35117
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