Electroporation

CONTENTS 9.1 Background...................................................................................................................... 293 9.2 Pore Models of Planar Lipid Bilayer Electromechanical Behavior ........................ 296 9.3 Cell Membrane Electromechanical Phenomena ........................................................ 298 9.4 Nonpore Theories of Electromechanical Phenomena............................................... 299 9.5 Pore Theories of Electromechanical Phenomena....................................................... 300 9.6 Molecular and Ionic Transport..................................................................................... 305 9.7 Electrotransfection .......................................................................................................... 310 9.8 Membrane Recovery....................................................................................................... 312 9.9 Cell Stress and Survival................................................................................................. 314 9.10 Tissue Electroporation and In Vivo Delivery ............................................................. 315 9.11 Electroporation of Organelles ....................................................................................... 319 Acknowledgment....................................................................................................................... 320 References ................................................................................................................................... 321

Cells and tissues contain multiple, spatially distributed barriers that compartmentalize charged and large molecules. These barriers are largely constructed out of lipids, usually phospholipids. For this reason, only very small molecules with effective high lipid solubility spontaneously penetrate the single or double phospholipid bilayerbased membranes of cells and and their organelles [1]. Of course, these membranes have a large variety of channels and transporters that facilitate transport of particular ions and molecules. Other significant barriers consist of one or more layers of cells connected by tight junctions around bladders and ducts that help retain specialized fluids, and the tough, flexible stratum corneum of mammalian skin that prevents water loss, entry of toxic molecules and infectious agents. Electroporation results in an essentially universal physical reduction of such barriers by creating membrane-spanning aqueous pathways. Aqueous pathways (large dielectric constant «w 80) across lipidcontaining (small dielectric constant «1 2) barriers greatly favor transport of even small, monovalent ions [2,3]. Applied electric field pulses with dominant frequency content below 300 MHz cause

concentration of voltage across membranes of isolated cells [4,6], and groups of cells spaced close together in a tissue [7]. If the time-dependent transmembrane voltage, Um(t),

becomes sufficiently large, then stochastic rearrangements of membrane phospholipid molecules are hypothesized to occur at a high rate, such that water-containing defects (‘‘pores’’) measurably alter the membrane’s transport properties. This is electroporation. The simplest statement is that electroporation ‘‘creates new aqueous pathways’’

through lipid-based barriers. Almost all electroporation studies to date have focused on bilayer membranes (BLMs), both artificial planar BLMs and cell membranes. In BLMs electroporation occurs under biochemically mild conditions, usually with a small temperature rise. For most easily observable phenomena such as cell transfection and molecular uptake, electroporation-related phenomena are believed to depend nonlinearly on the transmembrane voltage, Um(t). An exposure time-dependent onset within the range 0.2 < Um < 1V is usually found for single pulses with tpulse > tm, the membrane charging time. For conventional mammalian cell electroporation this is readily satisfied for widely used pulses that have tpulse in the range 1 ms to 50 ms. Both dramatic electrical behavior (‘‘reversible electrical breakdown’’ ¼ REB for cells)

and significant molecular transport occur. Most interest to date has focused on the electropermeabilization that coincides with REB conditions, which involves a large increase in membrane permeability for ions and molecules, particularly for longer pulses. Other consequences of electroporation are electrofusion of cells to other cells or tissue and electro-insertion of membrane proteins into cell membranes. The ability of a lipid-containing sheet to exclude ions and charged molecules is

fundamental and is a result of the change in ‘‘Born energy’’ associated with moving a charge from a medium with a large permittivity, e.g., water, to a region with a low permittivity, e.g., the interior of a phospholipid BLM. Here the Born energy is the electrostatic energy of an ionic charge embedded in a medium with permittivity «, written here in terms of the electric field, E, and with « ¼ K«0 where K is the dielectric constant and «0 ¼ 8.85 1012 F/m [8]:

WBorn ¼ ð

2 «E2dV (9:1)

The essential barrier function of cell membranes can be represented by a thin sheet of lipid. This allows the magnitude of the Born energy barrier, DWBorn, to be computed by the following process: a charged sphere, representing the ion or molecule, is initially located in water far from the lipid sheet (WBorn,i) and then moved to the center of the sheet (WBorn,f) which requires the expenditure of energy. The corresponding barrier height, DWBorn ¼ WBorn,f WBorn,i, is large even for small monovalent ions, e.g., Naþ and Cl, is still larger for multivalent charged molecules, and also depends slightly on the membrane thickness, sphere radius, and the amount and distribution of charge within the sphere. For a single, isolated charge such as a small ion, e.g., Naþ, the largest contribution to

DWBorn arises from the region close to the ion. The small diameter of an ion (solute) of type ‘‘s’’ (typically 2rs 0.4 nm) is significantly smaller than a typical membrane thickness of dm,l 4 nm) for the lipid hydrocarbon chains. The corresponding full thickness is dm 5 nm, which includes the phospholipid’s headgroups. As noted above DWBorn can be estimated by calculating the change in energy to move the ion from bulk water to bulk lipid. This allows DWBorn to be estimated by neglecting the membrane thickness and instead considering bulk lipid. This is justified because the greatest contribution to the electric field is in the volume near the ion. This estimate yields

DWBorn e 2

8pß0rs

Km 1 Kw

100 kT (9:2)

and Medical Aspects of Electromagnetic

where the relevant temperature is T ¼ 378C ¼ 310K. Numerical solutions to the electrostatic problem for a thin, low dielectric constant sheet immersed in water yields relatively small corrections. A barrier of this size is surmounted at a negligible rate by thermal fluctuations (spontaneous ion movement). Moreover, a transmembrane voltage, Um,direct, which is much larger than physiological values, would be needed to provide this energy. Uncharged molecules can partition into the membrane and then cross the membrane by diffusion; these species are not significantly affected by DWBorn. Instead, their transport is governed by a passive permeability due to the combined effect of dissolution and diffusion. Spontaneous barrier crossing is therefore negligible. An early calculation considered

not only the ‘‘intact sheet’’ case, but also the case of a fixed cylindrical pore [2,3]. Both aqueous configurations lowered DWBorn, but the greater reduction was achieved by the pore. The basic pore structure, penetration of the lipid membrane by an aqueous pathway, is of course the essence of channels based on proteins. It is also the basis of the ‘‘transient aqueous pore’’ theory of electroporation [9-19], but with the significant difference that the fluctuating and expandable electroporation aqueous pathways can be created rapidly (time scale of nanoseconds), but are metastable, with inferred pore lifetimes ranging from milliseconds to minutes. To our knowledge, the first experimental report of electroporation-related phenomena

were the irreversible [20] and reversible [21] observations of ‘‘breakdown’’ of the excitable membrane of the node of Ranvier. Almost a decade later nonthermal killing of microorganisms by electric field pulses was reported [22-24], followed a few years later by the observation of a large, field-induced molecular permeability increase in natural vesicles [25]. Increasing numbers of experimental reports involving electrical behavior of fieldpulsed cell membranes came in the next few years [26-28]. Artificial planar BLMs [29,30] exhibited dramatic electromechanical behavior, and the first pore-based theory was advanced to explain the fate of pulsed planarmembranes [9-15] and then extended in a series of further experimental [31-33] and additional theoretical studies [16,17,34]. Other reports confirmed and extended observation of cell membrane transport due to field-induced permeability increases [35-39]. This included introduction of ‘‘inactive’’ DNA into red blood cells [40], followed by the critically important demonstration of transformation of cells by electrically mediated DNA uptake [41]. An important feature of artificial and cell membranes is that they concentrate electric

field pulses with slow rise times (relative to the membrane charging time) because of the large membrane resistance relative to that of the extra-and intracellular media [4-6,42,43]. This form of field amplification can be regarded as voltage concentration due to a spatially distributed voltage divider effect within a single or multicellular system. For the case of current injection (current clamp), field amplification can also arise from current density concentration, arising frommultiple cells in close proximity or nearby insulating objects. In the case of cell and organelle membranes providing the predominant barriers and fast pulseswith rise times smaller than 3 ns (significant frequency content above 300MHz), spatially distributed dielectric voltage division emerges, electric fields tend toward approximate uniformity, and voltage concentration at membranes is much smaller [7]. However, most electroporation studies and applications have utilized pulses with

rise times that exceed a typical cell charging time. In the case of artificial planar BLMs the membrane completely blocks the current pathway, so that the entire voltage across the experimental apparatus electrodes appears, after a characteristic charging time, tm 106 s, across the membrane. In the case of cells, the situation is more complicated, but an approximate guide is obtained for the case of spherical cell for which Um,max 1.5Eapprcell, which shows that the change in transmembrane voltage is given approximately by the product of the electric field and the cell size.