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theses

Electroporation-mediated molecular delivery is of interest for many drug-delivery and gene-therapy applications. Recent studies have shown that a two-pulse protocol consisting of a short-duration high-voltage first pulse followed by a longer, low-voltage second pulse can increase the efficiency of molecular delivery and preserve more cells alive with suitably chosen pulsing parameters. In this work we investigate the effects of the first and second pulses’ field strength and the inter-pulse delay time on the delivery of two different-sized Fluorescein-Dextran conjugates (10 kDa and 70 kDa). A series of two-pulse electroporation experiments were performed on 3T3 mouse fibroblast cells, with an alternating-current first pulse to permeabilize the cell, followed by a direct-current second pulse to electrophoretically deliver the target molecule into the cell. Our results showed that the delivery amounts of Fluorescein-Dextran varies strongly with the first pulse's field strength and target molecule size. By varying the delay times between two pulses, it is shown that the delivered intracellular concentration of Fluorescein-Dextran decreased linearly with the logarithm of delay time. The data also indicate that membrane resealing after electropermeabilization occurs very rapidly, but that a non-negligible fraction of the pores can be reopened by the second pulse for times on the order of 100 s. The role of the second pulses is seen to be more than just electrophoresis, with a minimum threshold field strength required to reopen nano-sized pores or defects remaining from the first pulse. These results suggest that membrane electroporation, sealing, and reporation is a complex process that has both short-term and long-term components, which may in part explain the wide variation in membrane-resealing times reported in the literature.

ETD_5983

M.S.

Includes bibliographical references

by Yasir Demiryurek

doi:10.7282/T3M043VN

ETD graduate

The author owns the copyright to this work.