Molecular Dynamics Simulations to Probe Effects of Ultra-Short, Very-High Voltage Pulses on Cells

The use of very high electric fields (∼ 100kV/cm or higher) with pulse durations in the nanosecond range (Ultra-short) has been a very recent development in bioelectrics. Traditionally, the electric field effects have mostly been confined to: (a) low field, long-duration pulses, and (b) focused mainly on electroporation studies. Thus, aspects such as possible field-induced DNA damage, calcium release, alterations in neuro-transmitters, or voltage-gating have generally been overlooked. Ultra-short, high-field pulses open the way to targeted and deliberate apoptotic cell killing (e.g., of tumor cells). Though experimental data is very useful, it usually yields information on macroscopic variables that is inherently an average over time and/or space. Measurements often do not provide the molecular level information or details, as might be possible through numerical simulations. Also, the relevance and relative role of underlying physical mechanisms cannot be probed. With developments in computer technology, rapid advances in numerical algorithms for parallel computing, and with increasing computational resources, computer simulations of cellular dynamics and biological phenomena is gaining increasing popularity. A range of simulation methods exist that span the macroscopic continuum approaches (e.g. the Smoluchowski equation), to those based on the semi-classical retarded Langevin and Green\u27s functions, to microscopic-kinetic analyses based on Brownian dynamics or Molecular Dynamics (MD). Here we focus on the MD technique, as it provides the most comprehensive, time-dependent, three-dimensional nanoscale analyses with inclusion of the many-body aspects. This dissertation research presents simulations and analyses of lipid membrane poration and its dynamics, predictions of transport parameters under high-field, non-equilibrium conditions, electric fields effects on DNA, micelle disintegration, protein unfolding and intra-cellular calcium release. The following results have been found as a result of the application of external electric fields on cells: (a) Poration due to the re-orientation of the lipid molecules within the lipid bilayer, (b) Externalization of charged molecules such as Phosphotidyl Serine (PS), (c) Dramatic lowering of permittivity and diffusion coefficient with spatially structured layering of the membrane nanopore, (d) DNA alignment in the direction of electric field and eventual fragmentation, (e) Calcium release from the endoplasmic reticulum (ER) leading to time-dependent oscillatory waves and (f) Membrane fragmentation upon the application of high external fields

SINGLE-CELL ELECTROPORATION USING ELECTROLYTE-FILLED CAPILLARIES -EXPERIMENTAL AND MODELING INVESTIGATIONS

Electrolyte-filled capillaries (EFCs) with fine tips provide a highly concentrated electric field for local single-cell electroporation (SCEP) with high spatial resolution. A complete circuit for SCEP experiments was built that consisted of a test circuit and an electroporation circuit, with the ability to monitor electrically the electroporation pulses. SCEP itself was monitored in real time by observing the loss of a fluorescent adduct of glutathione (Thioglo-1-GSH) from the intracellular space. SCEP can be applied for transfection of individual adherent cells. We hypothesize that transfection of single cells can be accomplished with the plasmid contained in a single capillary. During SCEP, electroosmotic flow can pump electrolyte out of the capillary enhancing plasmid transfer into cells. This was confirmed from both simulation and transfection experiments. Cells were successfully transfected with EGFP-C2 plasmid when the loss of Thioglo-1-GSH upon SCEP (ΔF) is larger than 10% and its mass transfer rate (M) through the membrane exceeds 0.03 s-1. A series of SCEP experiments has been carried out on PC-3 cells (with 2-µm tip opening) and A549 cells (with 4~5-µm tip opening) to investigate how the parameters such as cell-to-tip distance (dc), cell size (dm) and shape, temperature, current, and the cell cycle affect SCEP outcomes (M and resealing rate α) via statistical analysis. A good linear regression is achieved only at a low temperature of 15℃. The main factors affecting small molecule transport across cell membrane are dc, dm and electric current. A range of M (0.03 s-1 ~ 0.4 s-1 for PC-3 cells, or 0.03 s-1 ~ 0.5 s-1 for A549 cells) gives the best linear regressions. M is also affected by the cell cycle of A549 cells, and correlated with cell roundness only for PC-3 cells. Cells reseal faster at higher temperature; while lower temperature provides better survivability with identical ΔF. Lastly, numerical models were elaborated as a platform for better understanding of the SCEP process and prediction of the trends of SCEP under various experimental conditions. A mass transport model involving potential distribution, diffusion, convection and electrokinetic flow was extended to study mass transport at a buffer-filled pipette tip/porous medium interface