(5e) Transfection and DNA Delivery Mediated by Electroporation and Ultrasound

Electroporation and ultrasound have been shown to facilitate the loading of drugs and genes into viable cells. Optimization of gene therapy and drug delivery applications in similar systems was systematically studied before. We previously identified physical parameters influencing transfection and viability of cells, and a range of optimal conditions was found. The transfection of cells is a complex multi-step phenomenon, which includes DNA delivery across the cell membrane, trafficking within the cell to the nucleus, and synthesis of mRNA and finally protein. This study addresses the question of correlation between DNA transfection and delivery.

The study was performed on the DU145 prostate cancer cells. For transfection experiments we used gWiz-GFP plasmid. DNA plasmids were stained with YOYO-3 dye. For electroporation, electric pulses produced by a high-voltage, exponential decay pulser were applied to cell suspensions. Treatment with 500 kHz ultrasound was performed using a focused transducer in a water bath. After treatment, cells were washed and analyzed for DNA uptake and GFP expression by flow cytometery 1 and 24 h after treatment. Cells with YOYO-3?DNA complexes produce a distinct signal in the APC channel, while cells expressing GFP produced the signal in the FITC channel of flow cytometer.

Our data showed that DNA delivery and transfection after either electroporation or ultrasound treatment correlated with DNA uptake. However, only a small fraction of cells with DNA uptake expressed GFP. This might be explained by microscopy observations that intracellular DNA was mostly excluded from the nucleus. In addition, comparison of cell fluorescence over time showed that at 24 h cells lose ~75 % of delivered DNA initially present at 1 h after treatment.

As a difference between the two transfection methods, electroporation was able to deliver more than an order of magnitude greater intracellular concentrations of DNA molecules inside cells than ultrasound, but DNA transfection by these methods were comparable. Two possible explanations for this phenomenon may be greater DNA damage by electroporation or better facilitation of intracellular trafficking by ultrasound.

Conclusions from this study suggest that the bottleneck for transfection by electroporation or ultrasound is DNA delivery to the nucleus after efficient delivery into the cytosol. This is also consistent with observations from the gene therapy literature that transport across the nuclear membrane also limits DNA transfection by other methods. This hypothesis suggests that further optimizing transfection by electroporation or ultrasound may require facilitating intracellular plasmid DNA trafficking.