DNA electrotransfer into the skin using a combination of one high- and one low-voltage pulse

doi:10.1016/j.jconrel.2005.05.003

Journal of Controlled Release

Volume 106, Issue 3, 2 September 2005, Pages 407-415

https://doi.org/10.1016/j.jconrel.2005.05.003 Get rights and content

Abstract

Electroporation is an effective alternative to viral methods to significantly improve DNA transfection after intradermal and topical delivery. The aim of the study was to check whether a combination of a short high-voltage pulse (HV) to permeabilize the skin cells and a long low-voltage pulse (LV) to transfer DNA by electrophoresis was more efficient to enhance DNA expression than conventional repeated HV or LV pulses alone after intradermal injection of DNA plasmid. GFP and luciferase expressions in the skin were enhanced by HV + LV protocol as compared to HV or LV pulses alone. The expression lasted for up to 10 days. Consistently, HV + LV protocol induced a higher Th2 immune response against ovalbumin than HV or LV pulses. Standard methods were used to assess the effect of electric pulses on skin: the application of a combination of HV and LV pulses on rat skin fold delivered by plate electrodes was well tolerated. These data demonstrate that a combination of one HV (700 to 1000 V/cm; 100 μs) followed by one LV (140 to 200 V/cm; 400 ms) is an efficient electroporation protocol to enhance DNA expression in the skin.

Introduction

When an electric field is applied to a cell or cell system, a non-uniform transmembrane potential is induced in the exposed cells. If the induced transmembrane potential is above the threshold value, cell membrane becomes permeabilized and thus more conductive. That increases the uptake of some molecules into the cells, such as drugs or DNA. Reversible increase of the cell membrane permeability caused by the electric field is called electropermeabilization or electroporation [1]. Electroporation has been used for different applications, such as electrochemotherapy, transdermal drug delivery and gene transfection. Electrochemotherapy is a treatment of solid tumors which combines a cytotoxic non-permeant drug with locally delivered permeabilizing electric pulses. It is very successful in eliminating local tumors, e.g. subcutaneous tumors and is more efficient than the chemotherapy alone [2], [3]. Transdermal drug delivery has many advantages over conventional routes of drug administration. However, the barrier properties of the skin limit transdermal drug transport. One of the methods to enhance it is electroporation which causes reversible permeabilization of the outer layer of the skin — the stratum corneum [4], [5]. Since the first report of Neumann [6], electroporation has been widely used to introduce small molecules and macromolecules, including DNA, into prokaryotic and eukaryotic cells in vitro. Electroporation is currently one of the most efficient and simple non-viral method of gene transfer in vivo [7].

Skin is an attractive target tissue for gene therapy for a variety of reasons. Its accessibility facilitates in vivo gene delivery. Skin is also a very good target organ for DNA vaccination because of the large number of potent antigen presenting cells, critical to an effective immune response. If necessary, large areas of skin can be treated and can easily be monitored [8]. Beside viral methods that are controversial because of their safety issues, chemical and physical methods have been developed to enhance gene expression in the skin [9]. Electroporation seems particularly effective to improve DNA transfection after intradermal [9], [10], [11], [12], [13], [14] and topical [15] delivery without any significant alteration of skin structure.

However, the effect of electrical parameters and electrode design on the efficacy of transfection in the skin and the mechanism of enhancement have not been studied systematically so far. It has been shown for muscle tissue that efficient cell electrotransfection can be achieved using combinations of high-voltage (HV) and low-voltage (LV) pulses. Luciferase-encoding DNA was injected in skeletal muscle and luciferase expression was studied after various pulse combinations. HV pulses alone resulted in a high level of muscle permeabilization (permeabilizing pulse), but very low DNA transfer. However, in combination with one or more LV pulses (electrophoretic pulse), a large increase in DNA transfer occurred [16], [17], [18], [19].

We hypothesized that DNA electrotransfer into the skin is also a two-step process consisting in membrane permeabilization and DNA electrophoresis and that a combination of a high-voltage pulse to permeabilize the target cells, followed by a low-voltage pulse to electrophoretically transport the DNA would improve gene transfection in the skin too. Hence, the efficacy of the delivery of DNA in the skin was investigated using a combination of HV + LV pulses in comparison to protocols reported in literature [10], [11], [12], [13], [14], [15], [16], [17], [18], [19], [20]. The qualitative and quantitative measure of the expression of two reporter genes in the skin, the kinetics of this expression, intradermal DNA vaccination and skin tolerance using HV + LV pulses were investigated.

Section snippets

Reporter genes and plasmid injection

The electrotransfer of gene into the skin was evaluated and optimized with two reporter genes, pCMVluc and pCMVGFP. We prepared the plasmids using a Qiagen kit for plasmid purification. The plasmids were injected intradermally (50 μg/25 μl PBS) 30 s before the application of the electric pulses, using a Hamilton syringe with a 27-gauge needle.

Animals

The animals used in all studies except the vaccination study, were male Wistar rats from Laboratoires Janvier, France, 8–10 weeks old. They were

GFP expression in the skin

To localize the expression of a gene in the skin after intradermal injection of a plasmid followed by electroporation and to compare the efficacy of different pulsing protocols to enhance this gene expression, a plasmid coding for GFP was used as a reporter gene [15].

The control epidermis showed some autofluorescence of the hair follicles (see Fig. 1a, d, f) but no fluorescence in the dermis (data not shown). After intradermal injection of the plasmid without electroporation, a very slight and

Discussion

The efficiency of combination of HV + LV pulse on DNA electrotransfer in skin was investigated. As already proposed for gene transfection in skeletal muscle [16], [17], [18], [19], the rationale for this electroporation protocol was that the first, high-voltage pulse results in a high level of cell permeabilization (permeabilizing pulse), while the second, low-voltage pulse provides a driving force for transport of DNA into cells (electrophoretic pulse). So far the protocols reported in

Acknowledgments

This research was supported by the European Commission under the 5th framework under the grant Cliniporator QLK-1999-00484.

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Gene therapies are revolutionizing medicine by providing a way to cure hitherto incurable diseases. The scientific and technological advances have enabled the first gene therapies to become clinically approved. In addition, with the ongoing COVID-19 pandemic, we are witnessing record speeds in the development and distribution of gene-based vaccines. For gene therapy to take effect, the therapeutic nucleic acids (RNA or DNA) need to overcome several barriers before they can execute their function of producing a protein or silencing a defective or overexpressing gene. This includes the barriers of the interstitium, the cell membrane, the cytoplasmic barriers and (in case of DNA) the nuclear envelope. Gene electrotransfer (GET), i.e., transfection by means of pulsed electric fields, is a non-viral technique that can overcome these barriers in a safe and effective manner. GET has reached the clinical stage of investigations where it is currently being evaluated for its therapeutic benefits across a wide variety of indications. In this review, we formalize our current understanding of GET from a biophysical perspective and critically discuss the mechanisms by which electric field can aid in overcoming the barriers. We also identify the gaps in knowledge that are hindering optimization of GET in vivo.
Intradermal active full-length DNA Aβ42 immunization via electroporation leads to high anti-Aβ antibody levels in wild-type mice
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To make this approach more attractive for clinical use, we have here explored the use of electroporation (EP) for vaccination with DNA. A number of clinical trials which used EP for DNA delivery had shown a good safety profile and only limited and temporary side effects at the injection site (Heller and Heller, 2015; Sardesai and Weiner, 2011; Bodles-Brakhop et al., 2009; Pavselj and Préat, 2005; Roos et al., 2006; Roos et al., 2009). This study in a wild-type mouse model shows that the delivery of the DNA vaccine with a low voltage parameter setting (125 V/cm), low duration (5 ms), and high frequency (16–20 pulses) led indeed to high antibody levels with no accompanying cellular immune response (no IFNγ, no IL-17A).
Aβ immunotherapies with anti-Aβ antibody responses have high potential as possible prevention treatment for Alzheimer's disease. We have previously shown that active DNA Aβ1–42 immunization via gene gun delivery led to a non-inflammatory immune response resulting in decreased Aβ levels in brains of an immunized AD mouse model. To make DNA vaccination more applicable for clinical use, we used here intradermal electroporation. With fine tuning of the electropulse parameters, high antibody levels and low levels of inflammatory cytokines in the cellular immunoassays were observed. Full-length DNA Aβ1–42 immunization delivered via electroporation has potential to be used in the clinical setting.
Gene electrotransfer into skin using noninvasive multi-electrode array for vaccination and wound healing
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Skin is an attractive target for gene electrotransfer due to its easy accessibility and its interesting immune properties. Since electrodes are often invasive and frequently induce discomfort during pulse application, there is a fundamental need for non-invasive electrodes for skin delivery. We developed circular pin non-invasive multi-electrode array (MEA), suitable for different clinical applications. MEA was first employed to deliver a luciferase reporter gene. Then, it was used to deliver a DNA vaccine coding for ovalbumin or a plasmid encoding hCAP-18/LL-37 for promoting wound healing. The results demonstrated a strong gene expression and an efficient delivery of both, DNA vaccine and wound healing agent, dependent on the pulses applied. The use of MEA to deliver the ovalbumin plasmid demonstrated a strong immune response, as evidenced by the presence of antibodies in sera, the IFN-gamma response and the delayed tumor growth when the mice were subsequently challenged with B16-OVA cells. The delivery of a plasmid encoding hCAP-18/LL-37 significantly accelerated wound closure. The easy applicability and non-invasiveness of MEA make it suitable for various clinical applications that require gene electrotransfer to skin. Specifically, by adapting electric pulses to the expected action of a transgene, non-invasive MEA can be employed either for vaccination or for wound healing.
Electrotransfer parameters as a tool for controlled and targeted gene expression in skin
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Skin is an attractive target for gene electrotransfer. It consists of different cell types that can be transfected, leading to various responses to gene electrotransfer. We demonstrate that these responses could be controlled by selecting the appropriate electrotransfer parameters. Specifically, the application of low or high electric pulses, applied by multi-electrode array, provided the possibility to control the depth of the transfection in the skin, the duration and the level of gene expression, as well as the local or systemic distribution of the transgene. The influence of electric pulse type was first studied using a plasmid encoding a reporter gene (DsRed). Then, plasmids encoding therapeutic genes (IL-12, shRNA against endoglin, shRNA against melanoma cell adhesion molecule) were used, and their effects on wound healing and cutaneous B16F10 melanoma tumors were investigated. The high-voltage pulses resulted in gene expression that was restricted to superficial skin layers and induced a local response. In contrast, the low-voltage electric pulses promoted transfection into the deeper skin layers, resulting in prolonged gene expression and higher transgene production, possibly with systemic distribution. Therefore, in the translation into the clinics, it will be of the utmost importance to adjust the electrotransfer parameters for different therapeutic approaches and specific mode of action of the therapeutic gene.
Transport, resealing, and re-poration dynamics of two-pulse electroporation-mediated molecular delivery
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These electrical parameters, including field strength [37,43–49], pulse duration [35,44,47,50], number of pulses [34,51], and pulse shape [52–56], have been studied extensively over the past three decades. Prior studies have shown that the combination of a short-duration, high-field-strength first pulse, together with a longer-duration, lower-field-strength second pulse (denoted by ‘first pulse’ and ‘second pulse’, respectively) can increase electroporation efficiency and preserve cell viability, especially when delivering larger molecules (MW > 4 kDa [47]) or DNA [33,34,37,39,42,57–66]. Pulsing parameters have been well studied, including the strength of the first pulse [33,37,58], the strength and duration of the second pulse [34,39,57–59], and the delay between pulses [34,42,57,58,66,67].
Electroporation is of interest for many drug-delivery and gene-therapy applications. Prior studies have shown that a two-pulse-electroporation protocol consisting of a short-duration, high-voltage first pulse followed by a longer, low-voltage second pulse can increase delivery efficiency and preserve viability. In this work the effects of the field strength of the first and second pulses and the inter-pulse delay time on the delivery of two different-sized Fluorescein–Dextran (FD) conjugates are investigated. A series of two-pulse-electroporation experiments were performed on 3T3-mouse fibroblast cells, with an alternating-current first pulse to permeabilize the cell, followed by a direct-current second pulse. The protocols were rationally designed to best separate the mechanisms of permeabilization and electrophoretic transport. The results showed that the delivery of FD varied strongly with the strength of the first pulse and the size of the target molecule. The delivered FD concentration also decreased linearly with the logarithm of the inter-pulse delay. The data indicate that membrane resealing after electropermeabilization occurs rapidly, but that a non-negligible fraction of the pores can be reopened by the second pulse for delay times on the order of hundreds of seconds. The role of the second pulse is hypothesized to be more than just electrophoresis, with a minimum threshold field strength required to reopen nano-sized pores or defects remaining from the first pulse. These results suggest that membrane electroporation, sealing, and re-poration is a complex process that has both short-term and long-term components, which may in part explain the wide variation in membrane-resealing times reported in the literature.

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¹: Present address: Faculty of Electrical Engineering, University of Ljubljana, Trzaska 25, SI-1000 Ljubljana, Slovenia. Tel.: +38 6 1 476 82 69; fax: +38 6 1 426 46 58.

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DNA electrotransfer into the skin using a combination of one high- and one low-voltage pulse

Abstract

Introduction

Section snippets

Reporter genes and plasmid injection

Animals

GFP expression in the skin

Discussion

Acknowledgments

Bioelectrochem. Bioenerg.

Adv. Drug Deliv. Rev.

Adv. Drug Deliv. Rev.

Adv. Drug Deliv. Rev.

Adv. Drug Deliv. Rev.

Mol. Ther.

Adv. Drug Deliv. Rev.

Mol. Ther.

Mol. Ther.

Biochim. Biophys. Acta

Bioelectrochemistry

Med. Sci.