In vivo assessment of skin electroporation using square wave pulses

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Abstract

The application of short-duration high-voltage pulses to the skin has been shown to enhance transdermal drug delivery by several orders of magnitude and to transiently permeabilize cells in tissue. Both exponentially decaying (ED) pulses and square wave (SW) pulses have been applied. The latter have also been used for electrochemotherapy. To date, their effect on skin integrity has not been analyzed. The scope of this work was (i) to investigate the effect induced by SW pulses on the stratum corneum and the skin, (ii) to evaluate the safety issue associated with electroporation, (iii) to contribute to the understanding of drug transport. Biophysical techniques (transepidermal water loss, chromametry, impedance and laser Doppler velocimetry or imaging measurement) and histological methods were combined to provide a global picture of the effects. Ten SW pulses applied to the skin induced a mild impairment of the skin barrier function and a dramatic decrease in skin resistance. These changes were reversible. A transient decrease (<5 min) in blood flow was observed. Neither inflammation, nor necroses were observed. These studies confirm the tolerance of the skin to square wave pulses in vivo.

Introduction

Transdermal drug delivery has the potential to be a non-invasive, user-friendly method of delivering drugs. However, because transdermal molecular passage is impeded by the barrier properties of the skin, transport of most drugs across the skin is very slow [1]. Different physical and chemical methods have been developed to overcome this barrier and enhance transdermal drug delivery [2].

The application of short-duration high-voltage pulses, i.e., electroporation (i) enhances transdermal drug delivery by several orders of magnitude [3], [4], [5], [6], [7] and (ii) transiently permeabilizes cells in tissue [8], [9], [10], [11], [12]. The resistance of the stratum corneum, which is the most important barrier of the skin, decreases dramatically within less than 1 μs upon application of high-voltage pulse. This phenomenon is attributed to electroporation that involves the creation of transient aqueous pathways by the applied electrical pulses [13], [14].

Several studies have investigated the morphological changes or integrity of the skin after exponentially decaying pulses in vitro or in vivo [15], [16]. An extended study [Freeze-Fracture electron Microscopy (FFEM), impedance, Fourier transform infrared spectroscopy (FT-IR), X-ray, Differential Thermal Analysis (DTA)] was performed to provide a complete picture of the stratum corneum structure after high voltage pulse application in vitro. Electroporation was reported to induce (i) a disorganisation of the stratum corneum lipid bilayers; (ii) an increase in skin hydration; (iii) a decrease in skin resistance induced by electroporation. These changes were partly reversible [16], [17]. Multilamellar vesicles were observed in the intercellular lipid bilayers of the stratum corneum [18]. These studies were supplemented by non-invasive bioengineering methods in vivo. Transient increase in TEWL (transepidermal water loss) was associated with an enhancement in skin hydration and alteration in stratum corneum barrier function. Reversible increase in cutaneous blood flow measured by laser Doppler velocimetry (LDV) and an erythema evaluated by chromametry was also observed [19]. Dramatic decrease in skin resistance or impedance has been reported by many authors (for a review, see Ref. [13]) but to date, there have been no measurements of skin impedance in vivo either during or after pulsing.

Besides transdermal drug delivery, another in vivo application of electroporation is electrochemotherapy, a new therapeutic approach providing delivery into the cell interiors of non-permeant drugs, which have intracellular targets. After intravenous (i.v.) or local injection of a non-permeant drug, e.g., bleomycin, which has a high intrinsic cytotoxicity, local application of short and intense high voltage pulses permeabilize exposed cells. Preclinical trials have shown the efficacy of this new therapeutic modality in various tumor models. Clinical trials have demonstrated its efficacy for the local treatment of cancers. The pulses seem to be well tolerated by patients [20], [21].

Both exponentially decaying (ED) pulses and square wave (SW) pulses have been used. In contrast to ED pulses, SW pulses can be set at constant predetermined values of voltage and pulse length. They have been used in clinical trials of electrochemotherapy and are being investigated for transdermal delivery of macromolecules. To date, their effect on skin integrity has not been analysed.

The scope of this work was: (i) to investigate the effect induced by high-voltage pulse application used for transdermal drug delivery and for electrochemotherapy (SW pulses) on the stratum corneum and the skin, (ii) to evaluate the safety associated with electroporation and (iii) to contribute to the understanding of transdermal drug transport. Non-invasive biophysical methods to assess barrier function (TEWL, impedance) and cutaneous blood flow [LDV, Laser Doppler Imaging (LDI), dye diffusion], were combined with histology to provide a global picture of the effects.

Section snippets

Animals and chemicals

Hairless male rats’ 8 weeks old were housed in standard cages at room temperature (IOPS mutant from Iffa Credo, France). Standard laboratory food (A04, UAR-France) and water were given ad libitum. Salts for buffer preparation were analytical grade (Merck Eurolab, Belgium).

Rat treatment

Experiments were carried out in an assigned laboratory room with controlled temperature (20 °C) and relative humidity (30%). Animals were anesthetized before experiments and if necessary, before a measurement session with

Results

The aim of this paper was to investigate the effect of square wave pulses on the stratum corneum and the skin. Hence, a number of different methods were used in order to provide a global understanding of the effect of electroporation on the skin. The impedance and TEWL evaluated the barrier function of the skin, LDV, LDI and patent blue staining investigated the cutaneous blood flow. The histology assessed skin integrity.

Discussion

The scope of this work was: (i) to investigate the effect induced by square wave high-voltage pulse application used for transdermal drug delivery and for electrochemotherapy on the stratum corneum and the skin, (ii) to evaluate their safety and (iii) to contribute to the understanding of transdermal drug transport. Typical electroporation conditions used for transdermal drug delivery or electrochemotherapy were used in this study (for reviews, see Refs. [11], [15]).

The effects induced by SW

Acknowledgements

The authors thank Cytopulse for the electroporation device, Dr. D. Boggetts from Moor Instruments Ltd., for the laser Doppler imaging and FRSM (Belgium) for the financial support.

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