Iontophoretic Transport of High and Low Molecular Weight Plasmids through Durapore as a Model Membrane

Abstract

The aim of this study was to investigate the impact of plasmid molecular weight, plasmid aggregate size, plasmid concentration and DC current application on the transport of plasmids across Durapore, as a model membrane. Methods: Two plasmids (pUC19 and pCMVβ) with different molecular weights were amplified in Escherichia coli and purified on ion exchange columns (Qiagen). Plasmid integrity was confirmed by agarose gel electrophoresis. The concentrations of DNA were determined by the absorbance at 260 nm. Passive and Iontophoretic (cathodal) permeation experiments were performed through the sclera and the amount of plasmid was assessed using Quant-iT™ PicoGreen probe with fluorimeter. The aggregate size of plasmids in HEPES buffer (pH=7.4) were determined using particle size apparatus. Results: The passive study results revealed no trans-membrane flux of plasmids. Application of cathodal Iontophoresis at current intensities of 0.5, 1, 2, 3 and 4 mA.cm-2 resulted in a higher transport of pUC19 across the membrane in comparison with pCMVβ. The permeation increased as the current or plasmid concentration increased. Electrophoresis studies revealed that use of platinum electrodes can degrade the plasmids. The aggregate size of pUC19 and pCMVβ were 62 ± 4 and 76 ± 7 nm respectively. Conclusion: Use of platinum electrodes in gene delivery can damage the DNA molecules. Application of Iontophoresis technique could be helpful in gene delivery especially for low molecular weight plasmids. Plasmid aggregates size is a characteristic property of plasmids in gene delivery across membranes.

Keywords: pUC19, pCMVβ, Plasmid, Iontophoresis, Electrophoresis