Pulse Agile® ElectroporationIntroduction
Pulse Agile® electroporation differs from standard electroporation in that it allows control of pulse parameters from pulse to pulse within a single delivery sequence. This means that values for pulse voltage, pulse duration, interval between pulses, and number of identical pulses in a subset can be programmed to change during a series of pulses. In practice, Pulse Agile® is usually used to deliver high electric field pulses of short duration followed by lower electric field pulses of longer duration. This method increases electroporation efficiency in numerous applications using sound physical principles and is covered by Cyto Pulse U.S. patents 6,010,613 and 6,078,490.
Standard and Pulse Agile® waveform comparison
The physical principles of importance to electroporation are:
1. Applied pulsed electric fields of optimal magnitude and duration induce a transient state of permeability in cells immersed in that electric field [1]
2. An applied electric field can move charged particles in an ionic medium by electrophoresis [2]
3. An electrical current is required to maintain an electric field in an ionic medium (Ohm's Law) [3]
4. Heat production by an electrical current in an ionic medium is proportional to the square of the current (Joule's First Law) [4]These principles can be used to optimize electroporation. All four of these physical processes are active during the entire time that electric fields are applied to the area around cells. However, they vary in importance at different times during electroporation.
Induction of a state of permeability
Applying an electric field across a cell causes a redistribution of internal ions because cell membranes act as insulators. Ions move according to their charge within the electric field, but remain trapped within the cell thus accumulating at the poles in line with the electric field. This polar accumulation of ions within the cell creates an electrical potential across the cell membrane. When the transmembrane voltage exceeds a threshold of approximately one volt, conformational changes within the membrane induce a state of permeability.
The potential across a cell membrane is not equal along the entire surface of the cell. It is highest when the surface of the cell membrane is perpendicular to the electric field (in line with the electric field) and lowest when the surface of the cell membrane is parallel or tangential to the electric field (out of line with the electric field). The variation of transmembrane potential in an electric field is described by the following equation: Vtm = -1.5 r * | cosϑ | * E. The formula and the theory behind the formula are described in detail elsewhere [5].
Electroporation electric field diagram
Increasing the electric field from one value above threshold to a higher value above threshold will induce permeability in a larger surface area of the cell. This is because of the dependence upon the cosine shown in the formula above. Thus, high electric fields are used in first pulses to permeabilize a large area of the cell membrane.
The permeabilized area on each end of a cell will be asymmetrical. It will be slightly larger on the anode (+) side than the cathode (-) side. This is because the cell has a slight natural negative charge across its membrane [6].
Movement of DNA or RNA across cell membranes
The principle force influencing movement of polynucleotides across a cell membrane during electroporation is electrophoresis [7]. This has been shown both in vitro [8] and in vivo [9]. Electrophoresis is the movement of charged particles in a solution in response to an electric field. Movement of particles by electrophoresis is proportional to the total applied charge, which is in turn proportional to the magnitude of the electric field times the duration of the electric field. Assuming other factors such as solution viscosity are constant, the movement is linearly related to the total charge. This means that total distance traveled is approximately the same when half of a given electric field is applied for twice a given time.
Heat production during electroporation
Joule's first law states that ohmic or Joule heating is proportional to electrical resistance times the square of the current. Joule heating is a common cause of cell death during electroporation, so excessive increases in temperature due to this effect should be avoided. Following enough poration of the cell membrane to physically allow transfectant entry, a subsequent reduction in electric field will continue to move the transfectant by way of electrophoresis while generating significantly less heat. For example, a half decrease in electric field will reduce heat four fold.
Putting the concepts together
A summary of the proportional relationships of physical factors during electroporation are:
1. The area of a cell permeabilized by electroporation is positively correlated to the magnitude of an applied electric field (less than linearly because of the cosine in the formula)
2. The movement of a charged molecule (such as DNA) in a solution in an electric field is linearly related to the total applied charge
3. Heat produced during electroporation is proportional to the square of the applied current (or of the applied voltage if the electrical resistance is constant).Given these relationships, maximum cell permeability and maximum DNA delivery can be achieved by delivering a series of brief high electric field pulses at the beginning of electroporation to induce permeability in a large area of the cells followed by delivery of a series of lower electric field pulses to induce maximum movement of DNA with minimal heating. This is Pulse Agile® electroporation.
[1] Neumann, E, Rosenheck, K. Permeability changes induced by electric impulses in vesicular membranes, 1972, J Membr. Biol. 10:279-290
[2] Moyer, LS, A suggested standard method for the investigation of electrophoresis, 1936, J Bacteriology, 31(5):531-546
[3] Hayt, Engineering Electro-Magnetics, 1974, McGraw Hill
[4] Dictionary of Science and Technology, 1992, Academic Press
[5] Weaver, JC, Chizmadzhev, Y, Theory of electroporation, a review. 1996 Bioelectrochem. Bioenerg. 41:141-152
[6] Golzio M, Teissie J, Rols MP. Direct visualization at the single-cell level of electrically mediated gene delivery. 2002, Proc Natl Acad Sci U S A. Feb 5;99(3):1292-7
[7] Klenchin, VA, Sukharev, SI, Serov, SM, Chernomordik, LV, Chizmadzhev, YA, Electrically induced DNA uptake by cells is a fast process involving DNA electrophoresis, 1991, Biophys J, 60:804-811
[8] Andreason, GL, Evans, GA, Optimization of electroporation for transfection of mammalian cells 1989, Anal. Biochem. 180:269-275
[9] Satkauskas S, Bureau MF, Puc M, Mahfoudi A, Scherman D, Miklavcic D, Mir LM. Mechanisms of in vivo DNA electrotransfer: respective contributions of cell electropermeabilization and DNA electrophoresis. 2002, Mol Ther. Feb;5(2):133-40
