Cell membrane electropermeabilization with arbitrary pulse waveforms.

xposure of a biological cell to an electric field can produce a variety of responses [1]-[6]. If the field strength exceeds a certain threshold value, this leads to a large transient increase in membrane conductivity and permeability for ions and molecules (electropermeabilization, often also named electroporation) or to fusion of adjacent cells (electrofusion) [5], [6]. Nowadays, these phenomena are widely used in applications such as gene transfection [7]-[8], preparation of monoclonal antibodies [9], and drug delivery, especially in electrochemotherapy of tumors [10]-[12]. For optimal effectiveness of these applications, one must choose the most appropriate amplitude, duration, and waveform of the applied electric pulses. With 2 mm distance between plate electrodes, which is an established setup for electropermeabilization in vitro, the threshold voltages typically range from 120 to 300 V [13], with pulse durations from several microseconds to several milliseconds [5]. Due to these demands, electropermeabilization is performed using specialized devices, often referred to as electroporators or electropulsators. Today, several such devices are commercially available [14], delivering either exponential or unipolar rectangular pulses with adjustable duration and amplitude. Often, the number of pulses and the intervals in which they are delivered can also be chosen. However, it has been reported that efficiency of electropermeabilization can be appreciably improved with other waveforms, such as bipolar rectangular pulses [15], or rectangular pulses with superimposed sine waves [16]. In addition, while unipolar pulses necessarily lead to electrolytic effects that are detrimental to the exposed cells [17], these effects can be reduced by use of symmetrical bipolar waveforms [18]. Commercially available generators of arbitrary waveforms cannot provide amplitudes in the range of several hundred volts that are necessary for electropermeabilization. Furthermore, commercially available bipolar amplifiers that can be used to amplify the generated signals are i) limited in voltage to 400 V peak-to-peak, ii) limited in current to 2 A, iii) limited in frequency to several kHz, and, last but not least, iv) very costly. Thus, research of the role of pulse waveforms in the efficiency of electropermeabilization has until now been limited to several laboratories with custom-built devices. It is only after an improved efficiency of a certain waveform has been well established that a commercial interest arises and these devices become available—usually, again at a considerable cost—to other laboratories. In this article, we present a detailed design of a system for in vitro electropermeabilization with arbitrary waveforms. A low-voltage signal is generated by a programmable function generator and amplified by a bipolar amplifier cir-