Investigations of electroporation physics using optically transparent polymer devices and molecular dynamics simulation

Abstract

New technologies have emerged in the medical field over the last decade to treat problems such as cancer and tumors. Electroporation is one such technique that has been extensively used in electrochemotherapy, drug delivery, tumor ablation, electrogenetherapy, transdermal drug delivery, food processing, and many more emerging applications. In this thesis, we focus on several aspects of the physics of electroporation by developing new polymer devices for experimental studies and by conducting atomistic molecular dynamics simulations. The thesis is arranged as follows. After an introduction to the phenomenon of electroporation, Chapters 2 and 3 describe the experimental studies, whose highlights are:

Design and fabrication of a transparent polymer device: We developed an optically transparent polymer device made of polydimethylsiloxane (PDMS) with transparent indium tin oxide (ITO) parallel plate electrodes in a horizontal geometry. The device was characterized and used to enhance the electroporation efficiency of C2C12 (mouse myoblast cells) and yeast cells. An Olympus IX51 fluorescence microscope was used for in situ observation of electroporation in different buffers. Electroporation efficiency was also measured by fluorescence assisted flow cytometry using a BD FACSCalibur™ instrument (BD Biosciences) under in situ conditions.

Rapid optimization of electroporation parameters: Using the polymer device, we rapidly optimized pulse strength, duration, and number using minimal sample volumes. Unlike conventional glass cuvette electroporators, the transparent horizontal geometry allowed continuous in situ monitoring of the same cell population during electroporation using fluorescence microscopy. This enabled rapid determination of optimum pulse conditions, saving both time and sample volume.

Free energy estimation for pore formation: Using our unique device geometry, we estimated the free energy of pore formation to be 45 ± 0.5 kBT at room temperature. Such measurements were possible due to the ability to observe electroporated cells continuously during electric field application.

Advantages of horizontal electrode geometry: In the horizontal arrangement, cells settle due to gravity near the bottom electrode. This interfacial region experiences a high space charge electric field, enhancing electroporation efficiency even at low applied voltages. This leads to simpler drive circuits and reduced sample heating.

In Chapters 4 and 5, molecular dynamics simulations of electroporation are presented, focusing on several new insights. The main results are:

Activation energies of pore formation: Activation energies for pore formation in POPC and DPPC lipid bilayers were estimated under different external electric fields. These simulations were carried out over a range of temperatures, unlike most MD studies which only use room temperature.

Pore opening and closing dynamics: The complete dynamics of pore opening and closing were studied in simulations-an aspect difficult to capture experimentally.

Comparison of gel and liquid crystalline phases: The wide temperature range used in simulations allowed, for the first time, comparison of pore dynamics in both gel and liquid crystalline phases of lipid bilayers.

Long time pore stabilization using low sustaining fields: Using low sustaining electric fields, stable pores were maintained for 50 ns in the simulations. This novel method enabled calculation of the electric field and potential distribution inside the pore region for the first time using MD simulations.