Theoretical analysis of localized heating in human skin subjected to high voltage pulses

Electroporation, the increase in the permeability of bilayer lipid membranes by the application of high voltage pulses, has the potential to serve as a mechanism for transdermal drug delivery. However, the associated current flow through the skin will increase the skin temperature and might affect nearby epidermal cells, lipid structure or even transported therapeutic molecules. Here, thermal conduction and thermal convection models are used to provide upper and lower bounds on the local temperature rise, as well as the thermal damage, during electroporation from exponential voltage pulses (70 V maximum) with a 1 ms and a 10 ms pulse time constant. The peak temperature rise predicted by the conduction model ranges from 19 degrees C for a 1 ms time constant pulse to 70 degrees C for the 10 ms time constant pulse. The convection (mass transport) model predicts a 18 degrees C peak rise for 1 ms time constant pulses and a 51 degrees C peak rise for a 10 ms time constant pulse. The convection model compares more favorably with previous experimental studies and companion observations of the local temperature rise during electroporation. Therefore, it is expected that skin electroporation can be employed at a level which is able to transport molecules transdermally without causing significant thermal damage to the tissue.     

Effect of high-voltage electric pulses on yeast cells: factors influencing the killing efficiency

The decisive factors determining the killing efficiency of single rectangular electric pulses of 4–28 kV cm⁻¹ amplitude and 1–300 μs duration in Saccharomyces cerevisiae S6/1 are pulse amplitude and duration, cell size and growth phase, post-pulse temperature and medium conductivity. In S. cerevisiae, the minimum pulse duration ensuring substantial killing is about 10 μs, the minimum amplitude being about 2 kV cm⁻¹. The critical pulse-induced transmembrane breakthrough voltage is 0.75 V. A pulse-induced increase in membrane permeability for small species such as inorganic ions suffices to cause cell death. A preset killing rate can be achieved by varying pulse amplitude inversely to pulse duration. Comparison of killing data on S. cerevisiae S6/1 with those on the smaller-celled Kluyveramyces lactis showed the killing pulse amplitude to be roughly proportional to cell size except for low pulse amplitudes, at which smaller cells are much more killing-prone. In exponential S. cerevisiae cells increased pulse amplitude caused a sharp increase in killing while in stationary cells this effect was much lower and occurred only at pulse amplitude above 15–20 kV cm⁻¹. Elevated post-pulse temperature lowered the killing rate whereas lowered temperature promoted it, probably by affecting the pore resealing. Lowering medium conductivity from 66 to 46 μS m⁻¹ by suspension washing reduced the killing rate by 6–20%. Reproducible killing or electroporation therefore requires standardized cell concentration, and number of cell washings.     

Perturbation of human skin due to application of high voltage

Electroporation is believed to be the effect that greatly enhances the transport of water-soluble molecules across the stratum corneum (SC) by application of short high voltage pulses. However, electroporation was originally a phenomenon investigated at the level of cell and model membranes, which is only partially comparable to the complicated structure of the stratum corneum. Here, we show, that electroporation is accompanied by other effects, which may be primarily involved in creation of new pathways and altering existing pathways, respectively. Experimental evidence shows that the dramatic increase in skin permeability is due to synergistic effect of electric field and heating by high local current density. Heating starts at small spots, not related to a visible skin structure and results in a propagating heat front. The phase transition of the SC lipids plays a major role in skin permeability during the pulse. The permeability after a high voltage pulse correlates well with the surface area showing a permanent low electrical resistance after pulsing. The main transport of water-soluble molecules is facilitated by the electric field due to the electrophoretic driving force in conjunction with the high permeability due to the breakdown of the multilamellar system of the SC lipids.     

Bipolar pulse conductometric monitoring of ion-selective electrodes: Part 1. Method development with a calcium-selective electrode and elucidation of the basic principles involved

The potential generated by a plastic-membrane calcium ion-selective electrode (i.s.e.) is shown to be indirectly measurable by a non-zero current method based on bipolar pulse conductance. Linear current—voltage curves are obtained using 0–5-V pulses; the current axis intercept is related to the i.s.e. potential. A simple electrical contact (e.g., platinum or stainless steel) can be used instead of a poised reference electrode as the counter electrode in this two-electrode system. Long-term exposure of the i.s.e. to calcium solutions causes an upward drift in the measured current. This drift is minimized by avoiding long exposure times to solution, rinsing the electrode between measurements, and constructing current—voltage curves for determination of the current axis intercepts. Voltage pulses lasting 100 μs are optimum for this method. Shorter pulses are subject to error from capacitive charging currents, and longer pulses yield poorer precision, and degrade the electrode through faradaic reactions. The measured signal is dependent upon Ca²⁺ concentration (rather than activity), making ionic strength adjustment unnecessary. The concentration dependence is induced by application of voltage pulses greater than ~ 15 mV in amplitude. Selectivities of the potentiometric and conductometric methods are shown to be comparable for a variety of interfering monovalent and divalent cations. The conductometric method yields a fast i.s.e. response because of induced migration of Ca²⁺ into the membrane. Response time decreases as the pulse height increases. Pulses greater than 2 V in magnitude yield response times limited by the solution mixing time rather than by the electrode.     

Determination of the lipid bilayer breakdown voltage by means of linear rising signal

Electroporation is characterized by formation of structural changes within the cell membrane, which are caused by the presence of electrical field. It is believed that "pores" are mostly formed in lipid bilayer structure; if so, planar lipid bilayer represents a suitable model for experimental and theoretical studies of cell membrane electroporation. The breakdown voltage of the lipid bilayer is usually determined by repeatedly applying a rectangular voltage pulse. The amplitude of the voltage pulse is incremented in small steps until the breakdown of the bilayer is obtained. Using such a protocol each bilayer is exposed to a voltage pulse many times and the number of applied voltage pulses is not known in advance. Such a pre-treatment of the lipid bilayer affects its stability and consequently the breakdown voltage of the lipid bilayer. The aim of this study is to examine an alternative approach for determination of the lipid bilayer breakdown voltage by linear rising voltage signal. Different slopes of linear rising signal have been used in our experiments (POPC lipids; folding method for forming in the salt solution of 100 mM KCl). The breakdown voltage depends on the slope of the linear rising signal. Results show that gently sloping voltage signal electroporates the lipid bilayer at a lower voltage then steep voltage signal. Linear rising signal with gentle slope can be considered as having longer pre-treatment of the lipid bilayer; thus, the corresponding breakdown voltage is lower. With decreasing the slope of linear rising signal, minimal breakdown voltage for specific lipid bilayer can be determined. Based on our results, we suggest determination of lipid bilayer breakdown voltage by linear rising signal. Better reproducibility and lower scattering are obtained due to the fact that each bilayer is exposed to electroporation treatment only once. Moreover, minimal breakdown voltage for specific lipid bilayer can be determined.     

Theoretical analysis of the thermal effects during in vivo tissue electroporation

Tissue electroporation is a technique that facilitates the introduction of molecules into cells by applying a series of short electric pulses to specific areas of the body. These pulses temporarily increase the permeability of the cell membrane to small drugs and macromolecules. The goal of this paper is to provide information on the thermal effects of these electric pulses for consideration when designing electroporation protocols. The parameters investigated include electrode geometry, blood flow, metabolic heat generation, pulse frequency, and heat dissipation through the electrodes. Basic finite-element models were created in order to gain insight and weigh the importance of each parameter. The results suggest that for plate electrodes, the energy from the pulse may be used to adequately estimate the heating in the tissue. However, for needle electrodes, the geometry, i.e. spacing and diameter, and pulse frequency are critical when determining the thermal distribution in the tissue.     

Influence of the physiological state on the electric field mediated transformation efficiency of intact mycobacterial cells

The sensitivity of Mycobacterium fortuitum at various growth stages to electric field pulses has been investigated in order to transform the cells by electroporation. In contrast to older cells, early- and mid-exponential growing cells are very field sensitive and, simultaneously, transformable to a relatively high degree. At an electric field strength E = 7 kV/cm and a pulse duration τ ≈ 13 ms (one pulse) an optimum transformation rate of about 2 × 10⁴ transformants/μg DNA was observed. On the basis of the different fast change of transformation rate and field sensitivity during cell growth we suppose a remarkable influence of biochemical changes in the cell wall composition on the efficiency of the electric field mediated transformation.     

Micro pulsed radio-frequency electroporation chips

Electroporation (EP) is one of the most important physical methods in biotechnology, which employs electrical pulses to transiently permeabilize cell membranes. In this study, a new micro pulsed radio-frequency electroporation cell (microPREP) chip was fabricated using a lift-off technique and SU-8 photolithography. The biological tests were carried out using three different plant protoplasts (cabbage, spinach and oil rape) on the micro EP chip and a pulsed RF electric field was applied to the microchip. The variations of fluorescent intensity and cell viability as functions of the electric pulse amplitude and duration time during the electroporation process were studied in detail at the single-cell level. Using such chip design and test method, one can easily optimize the efficiency and cell viability. Also, a large amount of statistical data can be quickly obtained. Finally, results of this parametric study were presented in the "phase diagram", from which the critical electric field for inducing single-cell electroporation under different conditions can be clearly determined.     

Kinetics of the temperature rise within human stratum corneum during electroporation and pulsed high-voltage iontophoresis

Electroporation is believed to be a nonthermal phenomenon at the membrane level. However, the effects of associated processes, such as Joule heating, should be considered. Because electroporation of skin, specifically the stratum corneum (SC), occurs at highly localized sites, the heating is expected to conform locally to the sites of electroporation. Significant localized heating was found to be strongly dependent on the voltage and duration of the high-voltage pulses. Specifically, a localized temperature rise was predicted theoretically and confirmed by experiments, with only a small rise (about 17 degrees C) for short, large pulses (1 ms, 100 V across the SC), but was increased (about 54 degrees C) for long, large pulses (300 ms, 60 V across the SC). The latter case appears to result in irreversible structural changes like vesicularization of the lipid lattice. These results support the hypothesis that electroporation occurs within the SC and that additional processes, such as localized heating, may be important.     

Surface area involved in transdermal transport of charged species due to skin electroporation

The electroporative effect on the stratum corneum (SC) is highly localized. However, the fractional area for the transport of small ions and larger ionic species differs considerably during and after high voltage (HV) application. Electroporation of SC creates new aqueous pathways, accessible for small ions, such as Cl(-) and Na(+) ions. The pores are distributed across the skin surface yielding a fractional area for current flow during electroporation of up to 0.1%. An increased permeability after high voltage application persists within a fractional area on the order of 10(-3)%. The permeabilization of SC for larger, charged molecules (M > 200 g/mol) involves Joule heating and a phase transition of the long chain sphingolipids within local transport regions (LTR). The transport area for these molecules (approximately 10(-3)%) changes only negligibly after high voltage application.     

Effects of application voltage and cathode and anode positions at electroporation on the in vitro permeation of benzoic acid through hairless rat skin

The enhancing effect of electroporation on the in vitro skin permeation of benzoate was evaluated. Needle and ring electrodes made of Ag/AgCl were connected to an electrical power source, which produced exponentially decaying pulses. The needle electrode was kept in contact with the skin surface, and the ring electrode was positioned either on or under the skin. The electrical pulse was applied to abdominal hairless rat skin at 150-600 V every minute from 4 to 6 h during the 10-h permeation experiment. Skin permeation of benzoate was promoted by electroporation and the effect was increased by application of a higher voltage. No immediate recovery to the control flux, however, was observed for high voltage groups after turning off the voltage application. When the cathode and anode were separated by the skin membrane by setting in the epidermal and dermal sides, respectively, an iontophoretic effect may also play a role in benzoate flux. These results indicated that the drug permeation by electroporation is the result of passive diffusion and an iontophoretic effect as well as the electroporation effect.     

Cell membrane electropermeabilization by symmetrical bipolar rectangular pulses. Part I. Increased efficiency of permeabilization

The paper presents a comparative study of electropermeabilization of cells in suspension by unipolar and symmetrical bipolar rectangular electric pulses. While the parameters of electropermeabilization by unipolar pulses have been investigated extensively both in cell suspensions and in tissues, studies using bipolar pulses have been rare, partly due to the lack of commercially available bipolar pulse generators with pulse parameters suitable for electropermeabilization. We have developed a high-frequency amplifier and coupled it to a function generator to deliver high-voltage pulses of programmable shapes. With symmetrical bipolar pulses, the pulse amplitude required for the permeabilization of 50% of the cells was found to be approximately 20% lower than with unipolar pulses, while no statistically significant difference was detected between the pulse amplitudes causing the death of 50% of the cells. Bipolar pulses also led to more than 20% increase in the uptake of lucifer yellow. We show that these results have a theoretical background, because bipolar pulses (i) counterbalance the asymmetry of the permeabilized areas at the poles of the cell which is introduced by the resting transmembrane voltage, and (ii) increase the odds of permeabilization of cells having a nonspherical shape or a nonhomogeneous membrane. If similar results are also obtained in tissues, bipolar pulse generators could in due course gain a wide, or even a predominant use in cell membrane electropermeabilization.     

Nonlinear current-voltage relationship of the plasma membrane of single CHO cells

The application of electric field pulses to Chinese Hamster Ovary (CHO) cells causes membrane electroporation (MEP). If a voltage or current ramp is applied to the cellular membrane of a single CHO cell, the membrane conductance increases nonlinearly with field strength reaching saturation. In particular, the kinetics of the induced conductance changes represents the data basis for the interpretation in terms of underlying structural changes. The current/voltage characteristic is found to be continuous, but displays occasionally a sharp increase in the conductance. The step-like increases are interpreted to reflect the formation of one (or more) larger pore(s). The analysis of current clamp data yields pores of radius (r(p)) in the range of 2.5< or =r(p)/nm< or =20; the pores of the voltage clamp data are in the range of 2.5< or =r(p)/nm< or =55. The larger pores occur predominantly during hyperpolarising and less frequently during depolarising conditions, respectively. The different kinetics of pore formation in the hyperpolarising condition, where the inward field increases, and the depolarising condition, where the inward field first decreases and then increases in the opposite direction, suggests structural asymmetry with respect to the direction of the electric membrane field. At the required higher voltage, the effect of the resting potential is negligibly small.     

High-voltage electroconduction in molten chlorides of alkaline-earth metals

Effects of high-voltage pulses on electroconduction of molten chlorides of alkaline-earth metals and their mixtures with potassium chloride are studied experimentally. It is established that the electroconductivity of the melts subjected to the pulses increases proportionally to the voltage amplitude and the number of activating pulses in a pulse series. Oscillatory relaxation of nonequilibrium melts is discovered.     

Using a micro electroporation chip to determine the optimal physical parameters in the uptake of biomolecules in HeLa cells

In this study, a new micro electroporation (EP) cell chip with three-dimensional (3D) electrodes was fabricated by means of MEMS technology, and tested on cervical cancer (HeLa) cells. Extensive statistical data of the threshold electric field and pulse duration were determined to construct an EP "phase diagram", which delineates the boundaries for 1) effective EP of five different size molecules and 2) electric cell lysis at the single-cell level. In addition, these boundary curves (i.e., electric field versus pulse duration) were fitted successfully with an exponential function with three constants. We found that, when the molecular size increases, the corresponding electroporation boundary becomes closer to the electric cell lysis boundary. Based on more than 2000 single-cell measurements on five different size molecules, the critical size of molecule was found to be approximately 40 kDa. Comparing to the traditional instrument, MEMS-based micro electroporation chip can greatly shorten the experimental time.     

Local and transient structural changes in stratum corneum at high electric fields: contribution of Joule heating

Electroporation of skin is accompanied by local heating, such that thermally induced structural changes of the stratum corneum (SC) accompany the field effect. Comparing on the time scale, the local changes in structure, temperature and conductance of the SC, during and after the pulse, it is seen that Joule heating also facilitates the subsequent molecular transport. It is found that the transport of medium-sized, ionic molecules occurs through localized transport regions (LTR). The size of a LTR increases with the pulse length, whereas the density of the LTRs increases with increasing voltage, for instance at U(SC=)80 V, the LTR cover approximately 0.02--1% of the surface area. The state of low resistance within the LTR is long-lived. During high voltage application, the center of the LTR is heated above the phase transition temperature of the SC lipids (70 degrees C) and the heat front propagates outwards. Inside the SC, the pulse causes aggregates of small-sized vesicles. At a higher temperature, the aggregate formation and their disappearance are delayed. Multiple pulses with the applied voltage of U(appl)=80 V induce the formation of long-lasting vesicle aggregates with a diameter of slashed circle=1--30 microm, covering 0.05--0.5% of the total sample area. The electric energy dissipated within the LTR during high voltage application is apparently sufficient to raise the temperature well above the phase transition temperature of the lipids of the SC, accounting for the conformational changes from the multi-lamella to the vesicular structures.     

Electrotransformation of Saccharomyces cerevisiae protoplast by a plasmid of Escherichia coli

The experiments reported here are aimed at obtaining optimum conditions for gene transformation after electric field pulses. Saccharomyces cerevisiae DBY746 is used as the recipient strain for shuttle plasmid (YRp group). From the relationship between the optimum electric field conditions and the transformation efficiency it is discovered that the maximum transformation efficiency appears at a wide pulse length of 400 μs with an electric field strength of 4 kV/cm, yielding up to 273 transformants/μg DNA. The electroporation unit used in the experiment is a home-made set featuring simplicity, readiness and practicality.     

Mechanisms involved in gene electrotransfer using high- and low-voltage pulses--an in vitro study

Gene electrotransfer is an established method for gene delivery which uses high-voltage pulses to increase permeability of cell membrane and thus enables transfer of genes. Currently, majority of research is focused on improving in vivo transfection efficiency, while mechanisms involved in gene electrotransfer are not completely understood. In this paper we analyze the mechanisms of gene electrotransfer by using combinations of high-voltage (HV) and low-voltage pulses (LV) in vitro. We applied different combinations of HV and LV pulses to CHO cells and determined the transfection efficiency. We obtained that short HV pulses alone were sufficient to deliver DNA into cells for optimal plasmid concentrations and that LV pulse did not increase transfection efficiency, in contrast to reported studies in vivo. However, for sub-optimal plasmid concentrations combining HV and LV pulses increased transfection rate. Our results suggest that low-voltage pulses increase transfection in conditions where plasmid concentration is low, typically in vivo where mobility of DNA is limited by the extracellular matrix. LV pulses provide additional electrophoretic force which drags DNA toward the cell membrane and consequently increase transfection efficiency, while for sufficiently high concentrations of the plasmid (usually used in vitro) electrophoretic LV pulses do not have an important role.     

Control of molecular fragmentation using shaped femtosecond pulses

The possibility that chemical reactions may be controlled by tailored femtosecond laser pulses has inspired recent studies that take advantage of their short pulse duration, comparable to intramolecular dynamics, and high peak intensity to fragment and ionize molecules. In this article, we present an experimental quest to control the chemical reactions that take place when isolated molecules interact with shaped near-infrared laser pulses with peak intensities ranging from 1013 to 1016 W/cm2. Through the exhaustive evaluation of hundreds of thousands of experiments, we methodically evaluated the molecular response of 16 compounds, including isomers, to the tailored light fields, as monitored by time-of-flight mass spectrometry. Analysis of the experimental data, taking into account its statistical significance, leads us to uncover important trends regarding the interaction of isolated molecules with an intense laser field. Despite the energetics involved in fragmentation and ionization, the integrated second-harmonic generation of a given laser pulse (ISHG), which was recorded as an independent diagnostic parameter, was found to be linearly proportional to the total ion yield (IMS) generated by that pulse in all of our pulse shaping measurements. Order of magnitude laser control over the relative yields of different fragment ions was observed for most of the molecules studied; the fragmentation yields were found to vary monotonically with IMS and/or ISHG. When the extensive changes in fragmentation yields as a function of IMS were compared for different phase functions, we found essentially identical results. This observation implies that fragmentation depends on a parameter that is responsible for IMS and independent from the particular time-frequency structure of the shaped laser pulse. With additional experiments, we found that individual ion yields depend only on the average pulse duration, implying that coherence does not play a role in the observed changes in yield as a function of pulse shaping. These findings were consistently observed for all molecules studied (p-, m-, o-nitrotoluene, 2,4-dinitrotoluene, benzene, toluene, naphthalene, azulene, acetone, acetyl chloride, acetophenone, p-chrolobenzonitrile, N,N-dimethylformamide, dimethyl phosphate, 2-chloroethyl ethyl sulfide, and tricarbonyl-[eta5-1-methyl-2,4-cyclopentadien-1-yl]-manganese). The exception to our conclusion is that the yield of small singly-charged fragments resulting from a multiple ionization process in a subset of molecules, were found to be highly sensitive to the phase structure of the intense pulses. This coherent process plays a minimal role in photofragmentation; therefore, we consider it an exception rather than a rule. Changes in the fragmentation process are dependent on molecular structure, as evidenced in a number of isomers, therefore femtosecond laser fragmentation could provide a practical dimension to analytical chemistry techniques.