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Electroporation effects and applications
Electroporation (EP) of living cells and tissues is a widely used technique to enhance the permeabilization of these structures through their exposure to exogenous pulsed electric fields [22-25]. Indeed, numerous biological and medical applications to be efficacious re-quire a local uptake of low-permeant molecules [26-29] (e.g. dyes, disaccharides, vitamins, anticancer drugs, pharmaceutical compounds, proteins, and genes).
Interestingly, the effects of usPEFs on tissues in general and on living cells in partic-ular strongly depend on the pulse characteristics, as duration, amplitude and repetition frequency (see Fig. 2.2). Based on their effects, and therefore on their characteristics, the utilization of usPEFs on biological targets results in various biological and medical appli-cations.
In the past three decades traditional EP, which implies rather long pulses (duration from hundreds of microseconds to milliseconds) of low intensity (0.1-10 kV/cm) known as s-msPEFs, has been efficiently used in biotechnologies and in various biomedical applica-tions that range from calcium EP [30], electrochemotherapy (ECT) [31-33], DNA vaccina-tion [34,35], gene therapy [36,37], and irreversible EP (IRE) [38]. More recently, develop-ments in devices able to deliver shorter and more intense voltage signals allow the use of electric pulses with durations of less than 1 s (down to few ns) of magnitude in the order of several hundred kV/cm, termed nsPEFs.
The principal difference between classic and nano-EP resides in the effects induced at the cellular and intracellular level: long, low magnitude pulses affect primarily the plasma membrane (Fig. 2.3), whereas can nsPEFs pass through the outer of the cell resulting in the permeabilization of organelles. However, due to their concomitant effects on the plasma membrane these nanosecond, high intense electric pulses can also affect cell functions and are employed to promote phosphatidylserine externalization, apoptosis and cancer cell killing [39-41].
s-msPEFs: Traditional electroporation
The research on non-surgical and minimally invasive treatments of tumors or cardiac diseases has led to the development of new local ablation techniques based on electro-magnetic fields. The aim of these non-invasive treatments is to limit surgery and reduce the pain, scarring and mortality of the patients while remaining cost-effective and safe. Initially developed for gene transfer, EP is now in use for delivery of a large variety of molecules: from ions to drugs, dyes, tracers, antibodies, and oligonucleotides to RNA and DNA [29]. Traditional EP has proven to be useful both in vitro, in vivo and in patients, where drug delivery to malignant tumors has been performed [26,21,32,35,42]. Whereas initial EP pro-cedures caused considerable cell damage, developments over the past decades have led to sophistication of equipment and optimization of protocols.
Among the applications of EP in medicine we will briefly describe: i) ECT [31-33], ii) gene therapy [36,37], iii) IRE [38].
The chemical injected in ECT protocols are non-permeant or low permeant chemicals with a very high intracellular cytotoxicity. ECT uses electrical pulses to permeabilize the cells and enhance the activity of the anticancerous drug. Currently, the most commonly used drugs in ECT are bleomycin (non-permeant) and cisplatin (low permeant). One of the most interesting features of ECT is its ability to selectively kill dividing cells, and therefore tumor cells, without harming normal quiescent surrounding tissue [32,33].
ECT can only be efficient if all tumor cells are permeabilized, so the lesion needs to be entirely submitted to an electric field of sufficient amplitude. However, the electric field has to be as low as possible to ensure the safety of the procedure. Therefore, the design of the electrical parameters, of the electrodes and of their positioning must be accurately chosen such that recently patient specific treatments planning are available [43].
In 2005 and 2006, four of the leading centers in EP conducted a European clinical study on ECT called ESOPE (European Standard Operating Procedures of Electrochemotherapy) [44]. This study was necessary because of the need for a non-randomized, multi-institutional study. These clinical trials proved the efficiency of ECT with bleomycin or cisplatin for the treatment of cutaneous and subcutaneous tumors. According to standard operating pro-cedures validated in the ESOPE project monopolar, direct current electric pulses are used. As shown in Fig. 2.4 the protocol consists in an injection of the antitumor drugs followed by the application of 8 monopolar 100 s pulses with a repetition frequency ranging from 1 Hz to 5 kHz. Pulses are delivered through needle or plate electrodes with a fixed geometry (Fig. 2.5), the electric field is applied to each electrode couple in order to obtain an electric field in the tumor roughly equals to 400 V/cm [33]. The application of the pulses determines the membrane electropermeabilization with the insertion of the drugs into the cytosol of the cell. After the treatment, the cell recovers its previous state with the drugs trapped inside, expressing locally their cytotoxicity.
nsPEFs: Recent applications and perspectives
The nsPEF experiments were made possible because of major improvements in the de-sign of nanopulsers. Their fabrication is highly complicated due to the high voltages (sev-eral hundred kV/cm) and ultrashort duration (ns) needed, being the main challenge the delivery of a repeatable pulse with a minimum of oscillations and reflections in the wave-form. For a detailed discussion on this technological issue see Section 2.3. These extremely short electrical pulses have attracted much attention during the last 10 years because they could represent a purely electrical cancer therapy that does not require drugs, hyperther-mia, or local pH changes. It must be noted that the shorter the pulses, the higher the field strength necessary to observe the biological effects of nsPEFs. However, the energy trans-mitted to the treated area by the electric pulses is very low and leads to a very weak heating [50]. Despite the great deal in understanding the pore dimension, its lifetime and trans-port properties through the plasma cell membranes [40,51-53] caused by nsPEFs, they have been extensively investigated to trigger other effects. Indeed, nsPEFs allow the targeting of intracellular organelles, the initiation of apoptosis in cells, and the inhibition of tumor growth [41,54].
Among the effects of nsPEFs on living cells we will briefly describe: i) effect at the plasma membrane level, ii) intracellular effects, iii) impact on cell viability [55-57].
EFFECT AT THE PLASMA MEMBRANE LEVEL
Effects of electric field pulses on membrane permeabilization are are usually investi-gated indirectly through uptake of fluorescent molecules, which are able to pass or not this cell barrier. However, the molecules used in the early studies were too large for the po-rated membrane to allow their crossing in the case of submission of a reduced numbers of nsPEFs. Since 2003 the possibility of nsPEFs to affect the plasma membrane has been supported by experimental observations [58,59], among which the phosphatidylserine (PS) external-ization from the inner leaflet of the lipid bilayer to the outer leaflet, a marker of cell apop-tosis [60].
Moreover, cell swelling (Fig. 2.6) and small molecules uptake (Fig. 2.7) were proved [40,51,53], showing that the mechanism undergoing s-msPEFs permeablization takes place also under nsPEFs exposure. Nevertheless the debate on the pore size, its duration and up-take properties under this condition is still open.
Figure 2.7: High-field, nanosecond pulses at high repetition rates permeabilize cell mem-branes, permitting entry of the small molecule YO-PRO-1 into the cell. Fluorescence mi-croscopic images of Jurkat cells in growth medium containing YO-PRO-1 (5.0 M) at 0.0, 1.0, and 5.0 s after exposure of the cells to 100, 4 ns, 8 MV/m pulses at a repetition rate of 1 kHz. Influx of YO-PRO-1 occurs primarily at the anode pole of the cells. Images were obtained from [40]
INTRACELLULAR EFFECTS
nsPEFs were first considered for their potential selective effect on the inner organelles of the cell, e.g. endoplasmic reticulum and mitochondria, nuclei, Golgi vacuoles and secre-tory or endocytic vesicles [41]. Few studies have targeted the effect of nanosecond pulses on the cytoskeleton. Actin has been shown to stabilize the plasma membrane of plant cells during the application of nsPEFs and to act on its permeabilization [41].
The release of intra-cytoplasmic calcium (Fig. 2.8) has been specifically studied since it is present in organelles, mainly in the endoplasmic reticulum (ER) and mitochondria. Several works have shown a release of calcium independently of intra-cytoplasmic mem-brane calcium channels, which was thus directly linked to the destabilization of organelle envelopes by pulses shorter than 100 ns [56].
Effects of the nuclear envelope were reported indirectly by the ability to increase the level of plasmid expression following electrotransfection only when the intensity of the pulse is high enough [62]. However, so far there are no experiments that show the desta-bilization of the nuclear envelope itself by nsPEFs. This can be explained first by the size of the nucleus, which is much larger than that of other intracellular organelles and could prevent any effect of the nsPEFs on the nuclear envelope; second by the complexity of the envelope; and finally by the technical limitations of detection of this expected destabilizalectroperturbation]Real-time imaging of [Ca2+] electroperturbation following the application of 10 pulses 30-ns, 2.5 MV/m [56].
IMPACT ON CELL VIABILITY
Because of their very short duration, nsPEFs do not transfer a large amount of energy to the sample and thus the observed effects are probably non-thermal. However, any impact on the external or internal membranes of the cell can profoundly affect its performance and therefore can cause its death. As previously described, nsPEFs could induce the desta-bilization of the internal membranes and cause the release of important intracellular me-diators of cell signaling, or second messengers such as calcium.
Weaver el at. [63] reported that the initial effect of the application of the pulse could be the release of the calcium from the ER and also a flow of the Ca2+ from the external to the internal medium due to the EP of the external membrane. This determines calcium redistribution inside the cell. The calcium could enter in the mitochondria and cause the mitochondria destruction with the release of the cytochrome c and other molecules such as SMAC/Diablo, EndoG, and AIF that are signal for cell death.
However, the inability to systematically connect the release of these molecules to apop-tosis prevents setting the direct involvement of nanosecond pulses in apoptosis.
Interestingly, it was suggested [41] that a precise adjustment of electrical parameters could help to destabilize cell membranes (internal and external) without causing cell death, using very short pulses (less than 10 ns) and moderate intensity (tens of kV/cm).
Theoretical descriptions of the electoporation process
Despite the large use of these techniques, the exact molecular description of the pore formation process, of its dimensions, shape, morphology, electrical and mechanical pros-perities and molecules uptake remains not fully clarified because of the limitation in time and spatial resolution of the today experimental tools. One hypothesis regarding the molec-ular mechanism of EP is that the application of electrical pulses induces rearrangements of the membrane components (water and lipids) that ultimately lead to the formation of aqueous hydrophilic pores [64-67], whose presence increases substantially the ionic and molecular transport through the otherwise impermeable membranes. The pore existence was recently confirmed by [68]. In this work the authors showed for the first time the di-rect imaging of the ionic flux through individual electropores employing total internal re-flection fluorescence. This approach yet does not allow straight visualization of the pore needed for a thorough characterization of the pore formation, of its dimensions, and of the mechanisms molecular transport.
Aside the experimental observations, atomistic and continuum models provided a dual and complementary view of the EP phenomena with different levels of spatial and temporal resolution. Theoretical continuum models (see Subsection 2.2.1) of single cells or cell clusters ne-glect the molecular structure and the membrane is simply viewed as a thin homogenous dielectric layer surrounded by an electrolyte solution [24,69-73]. Such models constitute an important part of EP research as they enable the exploration on length scales (more than hundreds of micrometers) and time scales (seconds and more) currently not achievable by molecular dynamics (MD) simulations [74] or molecular models based on the mean field theory [75]. On the other hand, MD descriptions have proven to be effective in providing atomic insights (nanometer length range, hundreds of nanosecond time scale) into both the structure and the dynamics of model lipid membrane models. This tool can be used to investigate physiological [76-89] and exposure to usPEFs conditions, hence to study the following cascade of events taking place at the membrane level and providing perhaps the most complete molecular model of the EP process of complex bilayers (see Subsection 2.2.2 for a detailed review of the related state-of-the-art).
Table of contents :
1 Introduction / Introduction
2 Electroporation: Treatments,ModelsandDevices / Électroporation: Traitements, Modèles et Dispositifs
2.1 Electroporation effects and applications
2.1.1 s-msPEFs: Traditional electroporation
2.1.2 nsPEFs: Recent applications and perspectives
2.2 Theoretical descriptions of the electoporation process
2.2.1 Continuum models
2.2.2 Atomic representations
2.3 Exposure setups for in vitro electroporation
2.3.1 s-msPEFs technology
2.3.2 nsPEFs technology
2.4 References
3 Methods: ClassicalMolecular Dynamics/Méthodes de modélisation: Dynamique Moléculaire Classique
3.1 Newtonian dynamics
3.2 Integration algorithms
3.3 Force fields and potential energy function
3.4 Periodic boundary conditions
3.5 Long-range interactions
3.6 Thermodynamic ensembles
3.6.1 Controlled temperature
3.6.2 Controlled pressure
3.7 References
4 Properties of Electropores, aMolecular Characterization /Propriétés des Électropores, une CaractérisationMoléculaire
4.1 Water-lipid Interface characterization
4.1.1 Choice of the bilayers
4.1.2 Systems andMethods
4.1.3 Results and Discussions
4.1.4 Conclusions
4.1.5 References
4.2 Electroporation of pure lipid and cholesterol rich bilayers
4.2.1 Introduction
4.2.2 Systems andMethods
4.2.3 Results
4.2.4 Discussions
4.2.5 Conclusions
4.2.6 References
4.3 Electropores characterization
4.3.1 Introduction
4.3.2 Systems and methods
4.3.3 Results and Discussions
4.3.4 Conclusions
4.3.5 References
4.4 Electro-transfer of small molecules through electropores
4.4.1 Introduction
4.4.2 Systems andMethods
4.4.3 Results and Discussions
4.4.4 Conclusions
4.4.5 References
5 Exposure Devices for nsPEFs / Dispositifs d’Exposition pour les nsPEFs
5.1 A VersatileMicrochamber for nsPEFs experiments
5.1.1 Introduction
5.1.2 Methods
5.1.3 Results
5.1.4 Discussions and Conclusions
5.1.5 References
6 Perspectives / Perspectives
6.1 Permittivity of water/lipid interfaces
6.2 Transport of fluorescent dyes
7 Summary and Conclusions / Résumé et Conclusions
