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Shock waves associated with electric pulses affect cell electro-permeabilization
Bioelectrochemistry 100 (2014) 36–43
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Shock waves associated with electric pulses affect cell electro-permeabilization Luc Wasungu, Flavien Pillet, Elizabeth Bellard, Marie-Pierre Rols, Justin Teissié ⁎ CNRS, IPBS (Institut de Pharmacologie et de Biologie Structurale);205 Route de Narbonne BP64182, F-31077 Toulouse, France Université de Toulouse, UPS, IPBS, F-31077 Toulouse, France
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Article history: Received 16 October 2013 Received in revised form 6 June 2014 Accepted 20 June 2014 Available online 1 July 2014 Keywords: Electropulsation Electrotransfer High field Pulsing chamber Shock wave
a b s t r a c t New features of cell electro-permeabilization are obtained by using high field (several tens of kV/cm) with short (sub-microsecond, nanosecond) pulse duration. Arcing appears as a main safety problem when air gaps are present between electrodes. A new applicator design was chosen to obtain a closed chamber where high field pulses could be delivered in a safe way with very short pulse duration. The safety issue of the system was validated under millisecond, microsecond and nanosecond pulses. The closed chamber applicator was then checked for its use under classical electro-mediated permeabilization and electro-gene transfer (EGT). A 20 times decrease in gene expression was observed compared with classical open chambers. It was experimentally observed that shock waves were present under the closed chamber configuration of the applicator. This was not the case with an open chamber design. Electropulsation chamber design plays a role on pulsing conditions and in the efficiency of gene electro transfer. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Electric field pulses have been known for several decades to induce cell membrane permeabilization [1]. The basic mechanisms remain poorly understood [2]. Nevertheless as small hydrophilic molecules as well as large plasmids [3] can be delivered into the cell cytoplasm, this approach is routinely used in Cell Biology and Pharmacology [4]. The effect is present not only at the single cell level but can be triggered on organs. Developments in clinical applications are present using the electrochemotherapy treatment [5–7]. The method is efficient but is developed on an empirical approach due to our poor knowledge of the basic mechanisms supporting the membrane reorganization and associated transport [8,9]. The “classical” electropermeabilization approach was using pulse duration in the microsecond–millisecond time range with field intensities of the kV/cm order. More recently, technological advances on pulse generators have allowed for shorter pulses with higher field values (several tens of kV/cm “nanosecond electric pulses” or nsEP) [10–17]. Molecular processes appear to be different with the two modes of pulse delivery [18,19].
⁎ Corresponding author at: CNRS;IPBS (Institut de Pharmacologie et de Biologie Structurale);205 route de Narbonne BP64182, F-31077 Toulouse, France. Tel.: +33 561175812. E-mail address: [email protected] (J. Teissié).
http://dx.doi.org/10.1016/j.bioelechem.2014.06.011 1567-5394/© 2014 Elsevier B.V. All rights reserved.
Gene electrotransfer was supposed therefore to be enhanced by delivering nsPEF after a classical EGT sequence, that was shown to mediate only the transfer into the cytoplasm [20,21]. But one of the critical limits of high field delivery was that sparks could be ignited between the two electrodes. The design of the applicators (electrodes, pulsing chamber) was a key safety issue. In most cases, the high field was obtained by designing a narrow gap between the electrodes [22–25]. To obtain a uniform treatment on the cell population, one needs to apply a uniform field on the sample [26]. Flat parallel electrodes appeared as the most convenient designs to treat large volumes in a homogeneous way [10, 27–29]. Commercial cuvettes were available, but to avoid the breakdown through air at the edges of the electrodes due to the enhanced electric field, cuvettes were over-filled to covering the upper edges of the electrodes. As a consequence, the field was not uniform all over the sample [10]. In the present investigation, we describe a pulsing chamber with parallel flat electrodes with no air gap. The volume was 100 μL, i.e. larger than most of the other published systems. The design was chosen to obtain a closed chamber where uniform high fields can be delivered in a safe way. The system was electrically validated under millisecond, microsecond and nanosecond pulses. It was firstly validated to behave properly from the electric point of view over all the range of pulse durations (ohmic behavior over a large range of applied voltage, electrical safety, limited effect on the subnanosecond pulse rise time). It was secondly checked for its use on electropulsation of cells, i.e. classical electromediated permeabilization and electro-gene transfer (EGT), the first
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step in our strategy for gene transfer and expression. Experiments were performed on mammalian cells in suspension. An unexpected by-effect was observed in this new chamber when compared with classical cuvettes: a drastic decrease in gene expression. This appears to be resulting from the generation of shock waves during the pulse that appear to strongly affect the cell responses. 2. Material and methods 2.1. High voltage (HV) pulses applications Two different pulse generators were used for the HV experiments. Microsecond HV pulse delivery of very high voltage (up to 10 kV) was obtained by using a Cirtem MHT10 pulse generator (Labège, France) driven by a HM 8035 Hameg TTL generator in the single pulse manual mode. Nanosecond HV pulses were delivered from a Kentech PBG 2 pulse generator (up to 9 kV) (Wallingford, UK). The voltage present on the electrodes was monitored by a Barth Attenuator (Boulder city, Nevada) (6 GHz), stored on a Tektronix TDS 5104B oscilloscope (bandwidth 1 GHz) (Beaverton, Oregon). The electronic network was built in order to make sure that the load was about 50 Ω. The voltage was limited to 3 kV (field of 15 kV/cm with a 2 mm electrode gap) due to the specifications of the attenuator.
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presented here but follow the same design philosophy as the 2-mm electrodes. To prevent electric arc formation it is important in the design of the chamber that no air gap should be present between the two flat parallel stainless steel electrodes. Aluminum cannot be used due to its fast electro-oxidation (alumina being an insulator) [10]. Electric breakdown in a humid air gap occurs at about 10 kV/cm applied during less than 1 μs. We chose to use electrodes with a cylindrical symmetry (8 mm diameter), that were screwed on the two opposite sides of a nylon block milled to let a 2 mm gap between the two flat extremities. O-rings were used to prevent any leakage. A smooth surfacing of the stainless steel electrodes was obtained as their surfaces were rectified in the milling machine. The edges of the electrodes were covered by the nylon block to prevent the sharp edge exposure to the liquid. Exposed sharp edges must be avoided to prevent arcing. The volume between the two electrodes was set at 100 μl. This optimum working volume for this chamber was further checked by measuring the current intensity as a function of the filled volume. Different volumes of pulsation buffer (10 mM K2HPO4/KH2PO4 buffer, 1 mM MgCl2, 250 mM sucrose, pH 7.4, Λ = 1.6 mS/cm) ranging from 100 to 150 μL were poured into the pulsation chamber. Using a Cliniporator generator (IGEA, Carpi, Italy) one square-wave pulse of 420 V and 5 ms duration was applied. The intensity of the current during the pulse was recorded on the Cliniporator. 2.3. Cell culture
2.2. Design of a chamber system electrodes Fig. 1 describes the design of the chamber used in this study (Fig. 1A, B and C). As a comparison, the 2-mm open design electrodes are also presented in this figure (Fig. 1D and E). The 4-mm electrodes are not
Chinese Hamster Ovary (CHO WTT) cells were grown in Eagle's minimum essential medium (EMEM; Gibco-Invitrogen, Carlsbad, CA, USA), supplemented with 8% fetal calf serum (Gibco), 100 units/ml penicillin (Gibco-Invitrogen), 100 mg/ml streptomycin (Gibco-Invitrogen),
Fig. 1. Electropulsation systems, closed chamber and open electrodes. In A the different elements of the closed chamber electrodes are displayed. The two electrodes are on the left and the O-rings are on the right, the open chamber is in the middle. In B, a magnified view of the upper electrode is presented in this image. Please note the presence of a channel in this upper electrode drilled in the middle of the flat surface. In C, the experimental set up for electropulsation with this closed chamber is described. Two crocodile clips are used for pulse delivery. According to the experiment, different pulse generators are used (see methods for details). The pulse delivery is monitored either via an onboard probe on an oscilloscope or via a HV probe here represented connected to the system. In D, a general view of the 2-mm open electrodes (2-mm electrodes) is presented. This view shows the distance between electrodes of 2 mm. In E, the experimental set up for electropulsation with open electrodes is described. The material to be pulsed is placed on a Petri dish between the two electrodes and a train of pulses is applied to the system.
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0.58 mg/ml L-glutamine (Eurobio, France) and vitamins. The cells were maintained in a 5% CO2 humidified incubator at 37 °C (Jouan, St Herblain, France).
electropermeabilization on a BD FACSCAN flow cytometer. The percentage of PI positive cells (i.e. highly fluorescent) in the population (permeabilization) and the average fluorescence of that sub-population were obtained.
2.4. Plasmid extraction and purification 2.8. Cell viability A 4.7 kb plasmid (pEGFP-C1) containing the gene coding for the Green Fluorescent Protein under control of the cytomegalovirus (CMV) constitutive promoter was obtained from Clonetech (Palo Alto, CA). It was purified from plasmid transfected Escherichia coli (BL 21) by using Maxiprep DNA purification system according to Qiagen instructions (Courtaboeuf, France). 2.5. Electro-mediated gene transfer Classical Electrotransfection was achieved by using a CNRS cell electropulsator (Jouan GHT 1247, St Herblain, France or Beta Tech S20, St Orens, France) that delivered millisecond square-wave electric pulses. A storage oscilloscope monitored pulse shape on line. Three types of electrodes were used: 1- a closed chamber with electrodes separated by 2 mm and adjusting a volume of 100 μl, as we just described in details (Fig. 1); 2- stainless steel flat parallel electrodes 1 cm in length and with a distance between electrodes of 2 mm (“2-mm electrodes”; from IGEA, Carpi, Italy); 3- stainless steel flat parallel electrodes 2 cm in length and with a distance of 4 mm between electrodes (“4-mm electrodes”; from our institute workshop). “2-mm electrodes” or “4-mm electrodes”were brought into contact with the bottom of a Petri dish to build an open chamber. A 100 μL drop of cell suspension was poured between the two electrodes in contact with the dish. A small meniscus was present along the electrodes on the top of the sample along the two stainless steel flat parallel electrodes. Electro pulsation was applied to 500 000 cells suspended in 100 μl of the previously described pulsation buffer. Electrotransfection was obtained in the presence of 1 μg of plasmid DNA pEGFP and 10 pulses of 700 V/cm (Electrode distance d(el) = 0.2 cm; applied voltage U(app) = 140 V; Electrode distance d(el) = 0.4 cm; applied voltage U(app) = 280 V) with duration of 5 ms, were applied at a frequency of 1 Hz. After pulsation, 1.5 ml growth medium was added and cells were kept at 37 °C in a 5% CO2 atmosphere. Transfection efficiency (being the percentage of fluorescent cells expressing the GFP in the population) and the average fluorescent intensity of positive cells were determined with a BD FACSCAN flow cytometer (BD Biosciences) 24 h after transfection. 2.6. Electro-mediated DNA/membrane interaction Electropulsation was operated under the same conditions as electrotransfection. Just after pulse delivery, DNA bound to the membrane was detected by labeling through the addition of the fluorescent intercalating probe TOTO (Invitrogen, USA) at a final concentration of 10 μM [9]. Cells were then diluted in 3 mL of PBS in a 2-chamber LabTek II culture dish (NUNC, Denmark) and observed directly with an inverted fluorescent microscope Leica DMIRB (Germany). Images were acquired with a Quantem 512 SC EMCCD camera (Roper, USA) driven by Metavue (Universal software, USA). 2.7. Electropermeabilization Electropermeabilization was obtained in a similar way than described above but the pulsation buffer contained no plasmid DNA. After electropulsation cells were transferred to the same buffer containing propidium iodide (PI) (Sigma Chemical Co. St. Louis, MO) at a 100 μM concentration. Cytometer acquisitions were run directly after
Cell viability was determined by the ability of cells to grow and divide over a 24 h period as previously described. Treated cells were allowed to grow for 24 h at 37 °C in a 5% CO2 incubator in a 35 mm Petri dish after the addition of 1.5 ml of fresh culture medium. Viability was measured by monitoring cell growth through coloration with crystal violet (CV) [30]. Briefly, cells were stained with crystal violet and after cell lysis the absorbance of the supernatant at 590 nm was measured with a spectrophotometer (Pharmacia Biotech). The viability was expressed as the ratio (in percentage) of absorbance obtained after the pulse train as compared to the extract of cells treated in the same way but without application of an electric field. 2.9. Preparation of lipid vesicles Multilamellar vesicles (MLVs) of L-α-Phosphatidylcholine (Egg PC, Avanti, USA) were prepared at 2 mg/ml. 20 mg of lipids was dissolved in 5 ml chloroform that was evaporated under nitrogen flow and vacuum until complete dryness. MLVs were obtained by adding 10 ml of NaCl solution at 500 μS/cm and hand shaking twice vigorously for 15 s. 2.10. Statistical analysis of MLV diameter The diameter distribution of MLVs was obtained as follows. A drop of the MLV suspension was observed under the stage of a microscope. The diameter measurement was performed on 300 MLVs from random images obtained with an inverted microscope DMIRB (Leica) coupled with a camera CoolSnapFx HQ2 (Photometrics, USA) and a × 40 Phaco objective (Leica). Only the MLVs in focal plan are analyzed with the Metamorph 7.1.7.0 software (Molecular Devices, USA). The diameter distribution statistical analysis was obtained with Prism5 software (GraphPad, USA). For each condition, statistical analysis was performed on two independent experiments. 3. Results 3.1. Test of the closed chamber electrodes Using the HV voltage Cirtem microsecond pulse generator and a very low conductance buffer (0.2 mS/cm), it was possible to simultaneously record the applied voltage and the delivered current on a large range of voltages. We obtained an ohmic behavior when applying 5 μs pulses (i.e. low frequency conditions) in a range of applied voltages of 0 to 9 kV, i.e. fields between 0 and 45 kV/cm. We selected short microsecond pulse duration and low conductance buffer to limit the Joule heating and its effect on the sample conductivity and due to the current safety limit of the generator (5 A). The volume of the chamber was designed to accommodate 100 μl of pulsation buffer. The overflow of liquid could then be evacuated by the hole in the upper electrode. In the following experiments we have tested whether this volume was sufficient for the current to be conducted between the electrodes and that no air bubble preventing the good flow of the current between the electrodes was present in the chamber. Applying a known voltage between the electrodes we have recorded the intensity of this current during the pulse with a Cliniporator pulser. The voltage was set at 420 V. With a volume of 100 μl and above, the intensity in the chamber remained at a stationary level of 2.6 A confirming that 100 μl was the optimum volume for the chamber. The extra volume leaks out by the upper channel and was out of the conductive pathway. This confirmed that the volume of the pulsed sample was 100 μl.
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Transf (GFP)/(%)
Fluorescence / AU
A
Fluorescence / AU
B
PI (cells)/(%)
The nanosecond HV pulse as observed on the electrodes is displayed in Fig. 2. The rise time of the pulse was observed to be less than 600 ps, compared to the 200 ps output value predicted from the operating manual and observed on the 10 V monitoring pulse from the auxiliary output. No major capacitive effect was induced by the chamber on the pulse delivery. From the electrical point of view, the design was therefore suitable for the delivery of nanosecond HV pulses. Pulsing induced a Joule heating and a thermal expansion of the solution. The volume thermal expansion coefficient of water is 2.6 · 10−4/K while it is only 3 10−5/K for nylon and 1.2 · 10−5/K for steel. In an attempt to prevent dramatic pressure effects due to the fast thermal expansion, a small channel was bored in the upper electrode. To avoid heterogeneity in the field distribution, the diameter of this channel must be very small and we selected a diameter of 1 mm. The temperature increase under the electro-gene transfer (EGT) conditions (ms pulses) was obtained by converting the delivered electrical energy in Joule heating. A temperature increase of 12 °C is obtained after 10 pulses (700 V/cm with a duration of 5 ms and at a frequency of 1 Hz) when assuming a poor thermal dissipation during the 1 s inter-pulse delay. This means that this is an upper bound limit. As a final conclusion, the design of this closed chamber was checked to comply with the electrical specifications (ohmic behavior, controlled volume i.e. gap, no capacitive interference on the pulse delivery on the electrodes).
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C
The original purpose of the designed chamber was to prevent the formation of electric arcs when used for electric field of high voltage and short duration pulses (nanosecond pulses or nanopulses [22,31]). The aim was to apply these nanopulses after a classical electro-gene transfer to induce a better transfection via the destabilization of the nuclear membrane. Conflicting results were reported [20,21] but the recent conclusions were that nsPEF were affecting the cell but remained non-effective for gene transfer and expression [13,21,32]. The first step towards this utilization was therefore to check the use of this chamber for classical electro-transfection. This electrode chamber efficiency was compared with other electrodes with a known classical design (see the description of the electrodes (2-mm electrodes and 4mm electrodes) in the Materials and methods section and Fig. 1). For each electrode set, the conditions for electro-transfection were the same and applied as follows: cells in suspension were subjected to a train of ten pulses of 700 V/cm with a duration of 5 ms and at a frequency of 1 Hz. In Fig. 3 results are summarized for the 3 different applicators.
Fig. 2. Record of a nanosecond HV pulse. A 3 kV nanosecond pulse was delivered on the closed pulsing chamber. The pulse was monitored by a 1 GHz storage oscilloscope and reported on the figure.
Viab (CV)/(%)
3.2. Transfection efficiency of the different systems of electrodes with associated permeabilization and viability
Chamber 2 mm
Elect . 2 mm
Elect . 4 mm
Fig. 3. Comparison of different electrodes for transfection, permeabilization, and viability. The transfection, permeabilization to PI, and viability were studied for the different electrodes systems (Closed chamber; 2-mm electrodes or 4-mm electrodes). In a), transfection (Transf (GFP) / (%) = 102. N(GFP) / N(Total), gray bars, left axis) and the average level of fluorescence is represented (thrombus, right axis). In b), permeabilization (PI (cells) / (%) = 102. F(PI) / F(Total), gray bars, left axis) and the average fluorescence of the cells (diamond; right axis) are represented. In c), the viability (Viab (CV) / (%) = 102. A(CV) / A(Basal) after 24 h associated with the transfection experiments is represented. Error bars are standard deviation of a triplicate experiment. For each electrode set, the conditions for electro-transfection were the same and applied as follows: cells in suspension were subjected to a train of ten pulses of 700 V/cm with a duration of 5 ms and at a frequency of 1 Hz.
Fig. 3a represents the transfection results. The 4-mm electrodes achieved very good transfection with 40% of transfected cells with an average fluorescence of 3493 a.u. (arbitrary unit from the BD Bioscience software analysis) in the fluorescent population. On the contrary, the closed chamber achieved very low transfection efficiency with only 2% of transfected cells. In this case the average fluorescence level of the fluorescent population of cells was about 1811 a.u. but with very few fluorescent cells, i.e. statistically not very representative of the level of transfection. The 2-mm electrodes led to an intermediate transfection level with 8% of positive cells and an average fluorescence level at 803 a.u. To understand whether this very low level of transfection was due to a poor permeabilization induced by the closed chamber, the permeabilization to propidium iodide (PI) was evaluated under the same electrical conditions. Indeed, this permeabilization to PI reflects the membrane destabilization of cells subjected to electric field pulses. As expected and shown in Fig. 3b the permeabilization induced with 4-mm electrodes was very high leading to around 84% of PI positive
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cells. The level of fluorescence in these positive cells was around 800 a.u. The closed chamber, although leading to poor transfection, induced no statistically significant differences in the level of permeabilization as 4-mm electrodes with around 66% of positive cells and a level of fluorescence of around 1000 a.u. The 2-mm electrodes induced very poor permeabilization with only 25% of PI positive cells and around 500 a.u. for the average fluorescence level. This level in permeabilization with these 2-mm electrodes may explain their low associated transfection efficiency. An important parameter when studying electrotransfection is the viability at 24 h. This experiment measures the amount of cell growth after 24 h as compared to control untreated cells (no electropulsation). In Fig. 3c this viability for the different electrodes is represented. This viability is respectively 30%, 89% and 13% for the closed chamber, 2-mm electrodes and 4-mm electrodes. 3.3. DNA/membrane interaction It was shown that DNA interaction with the membrane was a crucial step towards the transfection of cells [7]. Therefore, it appeared interesting to study this interaction for the closed chamber and to compare it with the two other electrodes. Fig. 4 shows the images of the interaction of a TOTO-labeled DNA with CHO cells using the different electrodes. In subsets 1a to 3b (Fig. 4), TOTO is added right after the pulses and in subsets 4a to 6b (Fig. 4), it is added 30 min after the pulses. Each subset of fluorescent pictures (1b, 2b, 3b, 4b, 5b and 6b) is shown with the corresponding phase contrast image (respectively 1a, 2a, 3a, 4a, 5a and 6a). In the images shot just after the pulses (1a to 3b), we can see that the DNA/ membrane interaction is present as expected for the 4-mm electrodes (Fig. 4, 3b) but also and more surprisingly for the 2-mm electrodes (Fig. 4, 2b) and the closed chamber (Fig. 4, 1b). These results are unexpected as both last set-ups led to little or no transfection (see Fig. 4, 4a).
Some DNA can also be observed at the membrane 30 min after the pulses for every type of electrodes (Fig. 4, 4b, Chamber; 5b, 2-mm electrodes; 6b, 4-mm electrodes). It is important to note that the interaction of the DNA with the membrane is present with electrodes leading to low transfection. These results suggest that a step beyond the interaction of DNA at the membrane is affected in the case of 2-mm electrodes and the closed chamber.
3.4. Experimental evidence of apparition of shock waves One other option was that with chambers with a narrow gap between the electrodes a pressure shock was present due to the Joule heating expansion of the liquid. Therefore we investigated the occurrence of a physical stress when pulses were delivered in the closed chamber. Shock waves were a candidate. To validate the occurrence of induced shock waves, the evolution of a suspension of multilamellar liposomes was followed by measuring the diameter of the liposomes (Fig. 5). MLVs were pulsed under the same conditions as cells (700 V/cm, 5 ms, 1Hz). Pulses were delivered either with the closed chamber or with the 2mm electrodes. These conditions were shown in a previous study not to induce the electropermeabilization of the lecithin lipid bilayer [26]. Liposomes are soft lipid auto-assemblies that are destabilized by shock waves (due to their transient high pressure). They are fragmented but spontaneous reformed as smaller entities. This process is routinely used under drastic conditions for the formation of sonicated unilamellar vesicles (SUVs). A change in the diameter relative statistical frequency of the liposomes was only present when pulsing in the closed chamber (Fig. 6). Liposomes having a large diameter disappeared while we observed a sharp increase in the population centered at 5 μm. MLVs pulsed in 2-mm electrodes were unaffected. Their diameter relative statistical frequency was the same as the control samples.
Fig. 4. DNA/membrane interaction with the different electrodes. Cells were subjected to electropulsing, as described in the Material and methods with the different systems of electrodes: closed chamber 1a, 1b and 4a, 4b; 2-mm electrodes 2a and 2b and 5a and 5b; 4-mm electrodes 3a and 3b and 6a and 6b. DNA present at the membrane directly after the pulses (images 1a to 3b) or 30 min after the pulses (images 4a to 6b) was labeled with the fluorescent probe TOTO. Phase contrast images (1a, 2a, 3a, 4a, 5a and 6a) are presented with their corresponding fluorescent images (1b, 2b, 3b, 4b, 5b and 6b).
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Fig. 5. Principle of diameter determination of MLVs. This picture was an example of phase contrast image obtained in control condition. The diameter measurements were performed exclusively with the MLVs in focal plan.
4. Discussion The distance between the electrodes is a critical parameter, as shown by the results with the two parallel plate electrode designs. A
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narrow distance clearly brought a less efficient expression. This may be due to the electrical reactions present at the electrodes level [33–35] and to the electrophoretic mediated contact of DNA with the electrodes [36–39]. These electrode supported electrochemical reactions are both known to alter the nucleic acids and are larger with the 2-mm electrodes where a larger contact between the electrodes and the solution is present as we kept the volume constant. The induced degradation is a rather simple explanation of the decrease in the efficiency of gene expression. Another difference between the two stainless steel flat parallel electrodes may be the contribution of the meniscus on the field distribution, which is larger with the larger gap set-up. This suggests a positive contribution of the electrical heterogeneity in the gene transfer on a cell population. The difference between the 4-mm plate electrodes and the “closed” pulsing chamber cannot be explained that way. It was shown on cell populations that there was a direct association between the fluorescence level related with the number of plasmids associated with the pulsed cell and the level of expression [9,40]. The Joule heating occurs during the 5 ms pulse discharge and brings a volume expansion of the sample. The narrow channel is supposed to avoid a pressure increase but the volume expansion is rather fast and may result in a transient increase in pressure on the sample. By using data on hydrostatic pressure, we may suggest a pressure increase of 20 MPa. This was experimentally validated by observing a restructuration of multilamellar lipid vesicles (Fig. 6). High pressures are known to affect the membrane organization
Fig. 6. Effect of pulsation on diameter relative statistical frequency of MLV. These diagrams showed the diameter relative statistical frequency (P(N, φi) = N(φI) / ∑JN(φJ) observed under each tested condition. The abscissa axis is labeled with the center bin of the diameter interval, which width is 5 μm. For each condition, error bars are standard deviation calculated from diameter interval of 300 MLVs in two independent experiments. In A, the MLVs were in control conditions. 10 (B) or 1000 (C) pulses were applied respectively with a 2-mm open electrodes (2-mm electrodes) under an applied field of 700 V/cm, 5 ms of pulse duration, a frequency of 1Hz and a conductivity of 500μS/cm. The same parameters were used with the closed chamber with 10 pulses in D and 1000 pulses in E.
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and its function [41,42]. A decrease in the fluidity of lipid bilayer and a reversible conformational change in transmembrane proteins are induced, leading to the functional disorder of membrane-associated activity. Interdigitation of the fatty acid chains is present and gives a change in the local lateral pressure [43]. The cross talk between the membrane components results in a change in the interfacial packing [44,45]. Pressure effect may lead to a stretching of the membrane, which is known to affect the endocytic properties of membranes [46]. The interfacial changes appear to be associated with a decrease in the plasmid membrane insertion during electropulsation. However, we still observed DNA/membrane interaction with the closed chamber electrodes, possibly because the pressure increase is progressive during the pulse and insertion may be hindered only above a critical value, i.e. only after a part of the pulse duration. Nevertheless, the nature of the DNA/membrane complexes and the new membrane organization formed under higher pressure may not be as efficient for future cytoplasm translocation and efficient transfection. Stretching affects the coupling between the plasma membrane and the actin skeleton, which appears to be involved in the DNA electrotransfer to the cytoplasm [47,48]. Actin cytoskeleton appears to be actively involved in the success of the first transfer step(s). It seems that it hinders DNA motion at later stages. Therefore stretching induced alteration of its coupling to the plasma membrane may affect gene electrotransfer. The role of the interfacial organization in electrotransfection was already suggested by our previous study on the effect of membrane order affecting chemicals [49]. Osmotic pressure was inducing a new membrane interface conformation, which was not suited to give plasmid insertion when electropermeabilized. Again this was associated with a change in the structure of the interfacial water. A more practical conclusion is the need of a careful design of pulsing chamber to prevent any associated pressure effect. 5. Conclusions In this comparative study between different pulsing electrodes, we observed that, while electropermeabilization appears to be induced under the same extend for the closed chamber and the 4-mm open electrodes, only the last ones led to efficient electrotransfection. The occurrence of shock waves in the closed chamber is a new parameter that must be taken into account when applying high field pulses (N 10 kV/ cm). But coupling shock wave with electropermeabilization opens the way to a new field of applications when alterations of the cellular organization are requested. Acknowledgment This work was supported by grants from the Region Midi-Pyrénées (grant 11052700), the DGA (grant 0634024) and the ANR (ANR-06BLAN-0260-03) “Cemirbio”. Research was conducted in the scope of the EBAM European Associated Laboratory (LEA) and of the COST Action TD1104 European network for the development of electroporationbased technologies and treatments (EP4Bio2Med). Thanks are due to Ms. Natalie Lemarechal for her careful editing of the manuscript. References [1] E. Neumann, K. Rosenheck, Permeability changes induced by electric impulses in vesicular membranes, J. Membr. Biol. 10 (1972) 279–290, http://dx.doi.org/10.1007/ BF01867861. [2] J. Teissie, M. Golzio, M.P. Rols, Mechanisms of cell membrane electropermeabilization: a minireview of our present (lack of?) knowledge, Biochim. Biophys. Acta 1724 (2005) 270–280, http://dx.doi.org/10.1016/j.bbagen.2005.05.006. [3] E. Neumann, M. Schaefer-Ridder, Y. Wang, P.H. Hofschneider, Gene transfer into mouse lyoma cells by electroporation in high electric fields, EMBO J. 1 (1982) 841–845. [4] M.P. Rols, M. Golzio, B. Gabriel, J. Teissié, Factors controlling electropermeabilisation of cell membranes, Technol. Cancer Res. Treat. 1 (2002) 319–328. [5] A. Gothelf, L.M. Mir, J. Gehl, Electrochemotherapy: results of cancer treatment using enhanced delivery of bleomycin by electroporation, Cancer Treat. Rev. 29 (2003) 371–387.
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Shock waves associated with electric pulses affect cell electro-permeabilization Luc Wasungu, Flavien Pillet, Elizabeth Bellard, Marie-Pierre Rols, Justin Teissié ⁎ CNRS, IPBS (Institut de Pharmacologie et de Biologie Structurale);205 Route de Narbonne BP64182, F-31077 Toulouse, France Université de Toulouse, UPS, IPBS, F-31077 Toulouse, France
a r t i c l e
i n f o
Article history: Received 16 October 2013 Received in revised form 6 June 2014 Accepted 20 June 2014 Available online 1 July 2014 Keywords: Electropulsation Electrotransfer High field Pulsing chamber Shock wave
a b s t r a c t New features of cell electro-permeabilization are obtained by using high field (several tens of kV/cm) with short (sub-microsecond, nanosecond) pulse duration. Arcing appears as a main safety problem when air gaps are present between electrodes. A new applicator design was chosen to obtain a closed chamber where high field pulses could be delivered in a safe way with very short pulse duration. The safety issue of the system was validated under millisecond, microsecond and nanosecond pulses. The closed chamber applicator was then checked for its use under classical electro-mediated permeabilization and electro-gene transfer (EGT). A 20 times decrease in gene expression was observed compared with classical open chambers. It was experimentally observed that shock waves were present under the closed chamber configuration of the applicator. This was not the case with an open chamber design. Electropulsation chamber design plays a role on pulsing conditions and in the efficiency of gene electro transfer. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Electric field pulses have been known for several decades to induce cell membrane permeabilization [1]. The basic mechanisms remain poorly understood [2]. Nevertheless as small hydrophilic molecules as well as large plasmids [3] can be delivered into the cell cytoplasm, this approach is routinely used in Cell Biology and Pharmacology [4]. The effect is present not only at the single cell level but can be triggered on organs. Developments in clinical applications are present using the electrochemotherapy treatment [5–7]. The method is efficient but is developed on an empirical approach due to our poor knowledge of the basic mechanisms supporting the membrane reorganization and associated transport [8,9]. The “classical” electropermeabilization approach was using pulse duration in the microsecond–millisecond time range with field intensities of the kV/cm order. More recently, technological advances on pulse generators have allowed for shorter pulses with higher field values (several tens of kV/cm “nanosecond electric pulses” or nsEP) [10–17]. Molecular processes appear to be different with the two modes of pulse delivery [18,19].
⁎ Corresponding author at: CNRS;IPBS (Institut de Pharmacologie et de Biologie Structurale);205 route de Narbonne BP64182, F-31077 Toulouse, France. Tel.: +33 561175812. E-mail address: [email protected] (J. Teissié).
http://dx.doi.org/10.1016/j.bioelechem.2014.06.011 1567-5394/© 2014 Elsevier B.V. All rights reserved.
Gene electrotransfer was supposed therefore to be enhanced by delivering nsPEF after a classical EGT sequence, that was shown to mediate only the transfer into the cytoplasm [20,21]. But one of the critical limits of high field delivery was that sparks could be ignited between the two electrodes. The design of the applicators (electrodes, pulsing chamber) was a key safety issue. In most cases, the high field was obtained by designing a narrow gap between the electrodes [22–25]. To obtain a uniform treatment on the cell population, one needs to apply a uniform field on the sample [26]. Flat parallel electrodes appeared as the most convenient designs to treat large volumes in a homogeneous way [10, 27–29]. Commercial cuvettes were available, but to avoid the breakdown through air at the edges of the electrodes due to the enhanced electric field, cuvettes were over-filled to covering the upper edges of the electrodes. As a consequence, the field was not uniform all over the sample [10]. In the present investigation, we describe a pulsing chamber with parallel flat electrodes with no air gap. The volume was 100 μL, i.e. larger than most of the other published systems. The design was chosen to obtain a closed chamber where uniform high fields can be delivered in a safe way. The system was electrically validated under millisecond, microsecond and nanosecond pulses. It was firstly validated to behave properly from the electric point of view over all the range of pulse durations (ohmic behavior over a large range of applied voltage, electrical safety, limited effect on the subnanosecond pulse rise time). It was secondly checked for its use on electropulsation of cells, i.e. classical electromediated permeabilization and electro-gene transfer (EGT), the first
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step in our strategy for gene transfer and expression. Experiments were performed on mammalian cells in suspension. An unexpected by-effect was observed in this new chamber when compared with classical cuvettes: a drastic decrease in gene expression. This appears to be resulting from the generation of shock waves during the pulse that appear to strongly affect the cell responses. 2. Material and methods 2.1. High voltage (HV) pulses applications Two different pulse generators were used for the HV experiments. Microsecond HV pulse delivery of very high voltage (up to 10 kV) was obtained by using a Cirtem MHT10 pulse generator (Labège, France) driven by a HM 8035 Hameg TTL generator in the single pulse manual mode. Nanosecond HV pulses were delivered from a Kentech PBG 2 pulse generator (up to 9 kV) (Wallingford, UK). The voltage present on the electrodes was monitored by a Barth Attenuator (Boulder city, Nevada) (6 GHz), stored on a Tektronix TDS 5104B oscilloscope (bandwidth 1 GHz) (Beaverton, Oregon). The electronic network was built in order to make sure that the load was about 50 Ω. The voltage was limited to 3 kV (field of 15 kV/cm with a 2 mm electrode gap) due to the specifications of the attenuator.
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presented here but follow the same design philosophy as the 2-mm electrodes. To prevent electric arc formation it is important in the design of the chamber that no air gap should be present between the two flat parallel stainless steel electrodes. Aluminum cannot be used due to its fast electro-oxidation (alumina being an insulator) [10]. Electric breakdown in a humid air gap occurs at about 10 kV/cm applied during less than 1 μs. We chose to use electrodes with a cylindrical symmetry (8 mm diameter), that were screwed on the two opposite sides of a nylon block milled to let a 2 mm gap between the two flat extremities. O-rings were used to prevent any leakage. A smooth surfacing of the stainless steel electrodes was obtained as their surfaces were rectified in the milling machine. The edges of the electrodes were covered by the nylon block to prevent the sharp edge exposure to the liquid. Exposed sharp edges must be avoided to prevent arcing. The volume between the two electrodes was set at 100 μl. This optimum working volume for this chamber was further checked by measuring the current intensity as a function of the filled volume. Different volumes of pulsation buffer (10 mM K2HPO4/KH2PO4 buffer, 1 mM MgCl2, 250 mM sucrose, pH 7.4, Λ = 1.6 mS/cm) ranging from 100 to 150 μL were poured into the pulsation chamber. Using a Cliniporator generator (IGEA, Carpi, Italy) one square-wave pulse of 420 V and 5 ms duration was applied. The intensity of the current during the pulse was recorded on the Cliniporator. 2.3. Cell culture
2.2. Design of a chamber system electrodes Fig. 1 describes the design of the chamber used in this study (Fig. 1A, B and C). As a comparison, the 2-mm open design electrodes are also presented in this figure (Fig. 1D and E). The 4-mm electrodes are not
Chinese Hamster Ovary (CHO WTT) cells were grown in Eagle's minimum essential medium (EMEM; Gibco-Invitrogen, Carlsbad, CA, USA), supplemented with 8% fetal calf serum (Gibco), 100 units/ml penicillin (Gibco-Invitrogen), 100 mg/ml streptomycin (Gibco-Invitrogen),
Fig. 1. Electropulsation systems, closed chamber and open electrodes. In A the different elements of the closed chamber electrodes are displayed. The two electrodes are on the left and the O-rings are on the right, the open chamber is in the middle. In B, a magnified view of the upper electrode is presented in this image. Please note the presence of a channel in this upper electrode drilled in the middle of the flat surface. In C, the experimental set up for electropulsation with this closed chamber is described. Two crocodile clips are used for pulse delivery. According to the experiment, different pulse generators are used (see methods for details). The pulse delivery is monitored either via an onboard probe on an oscilloscope or via a HV probe here represented connected to the system. In D, a general view of the 2-mm open electrodes (2-mm electrodes) is presented. This view shows the distance between electrodes of 2 mm. In E, the experimental set up for electropulsation with open electrodes is described. The material to be pulsed is placed on a Petri dish between the two electrodes and a train of pulses is applied to the system.
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0.58 mg/ml L-glutamine (Eurobio, France) and vitamins. The cells were maintained in a 5% CO2 humidified incubator at 37 °C (Jouan, St Herblain, France).
electropermeabilization on a BD FACSCAN flow cytometer. The percentage of PI positive cells (i.e. highly fluorescent) in the population (permeabilization) and the average fluorescence of that sub-population were obtained.
2.4. Plasmid extraction and purification 2.8. Cell viability A 4.7 kb plasmid (pEGFP-C1) containing the gene coding for the Green Fluorescent Protein under control of the cytomegalovirus (CMV) constitutive promoter was obtained from Clonetech (Palo Alto, CA). It was purified from plasmid transfected Escherichia coli (BL 21) by using Maxiprep DNA purification system according to Qiagen instructions (Courtaboeuf, France). 2.5. Electro-mediated gene transfer Classical Electrotransfection was achieved by using a CNRS cell electropulsator (Jouan GHT 1247, St Herblain, France or Beta Tech S20, St Orens, France) that delivered millisecond square-wave electric pulses. A storage oscilloscope monitored pulse shape on line. Three types of electrodes were used: 1- a closed chamber with electrodes separated by 2 mm and adjusting a volume of 100 μl, as we just described in details (Fig. 1); 2- stainless steel flat parallel electrodes 1 cm in length and with a distance between electrodes of 2 mm (“2-mm electrodes”; from IGEA, Carpi, Italy); 3- stainless steel flat parallel electrodes 2 cm in length and with a distance of 4 mm between electrodes (“4-mm electrodes”; from our institute workshop). “2-mm electrodes” or “4-mm electrodes”were brought into contact with the bottom of a Petri dish to build an open chamber. A 100 μL drop of cell suspension was poured between the two electrodes in contact with the dish. A small meniscus was present along the electrodes on the top of the sample along the two stainless steel flat parallel electrodes. Electro pulsation was applied to 500 000 cells suspended in 100 μl of the previously described pulsation buffer. Electrotransfection was obtained in the presence of 1 μg of plasmid DNA pEGFP and 10 pulses of 700 V/cm (Electrode distance d(el) = 0.2 cm; applied voltage U(app) = 140 V; Electrode distance d(el) = 0.4 cm; applied voltage U(app) = 280 V) with duration of 5 ms, were applied at a frequency of 1 Hz. After pulsation, 1.5 ml growth medium was added and cells were kept at 37 °C in a 5% CO2 atmosphere. Transfection efficiency (being the percentage of fluorescent cells expressing the GFP in the population) and the average fluorescent intensity of positive cells were determined with a BD FACSCAN flow cytometer (BD Biosciences) 24 h after transfection. 2.6. Electro-mediated DNA/membrane interaction Electropulsation was operated under the same conditions as electrotransfection. Just after pulse delivery, DNA bound to the membrane was detected by labeling through the addition of the fluorescent intercalating probe TOTO (Invitrogen, USA) at a final concentration of 10 μM [9]. Cells were then diluted in 3 mL of PBS in a 2-chamber LabTek II culture dish (NUNC, Denmark) and observed directly with an inverted fluorescent microscope Leica DMIRB (Germany). Images were acquired with a Quantem 512 SC EMCCD camera (Roper, USA) driven by Metavue (Universal software, USA). 2.7. Electropermeabilization Electropermeabilization was obtained in a similar way than described above but the pulsation buffer contained no plasmid DNA. After electropulsation cells were transferred to the same buffer containing propidium iodide (PI) (Sigma Chemical Co. St. Louis, MO) at a 100 μM concentration. Cytometer acquisitions were run directly after
Cell viability was determined by the ability of cells to grow and divide over a 24 h period as previously described. Treated cells were allowed to grow for 24 h at 37 °C in a 5% CO2 incubator in a 35 mm Petri dish after the addition of 1.5 ml of fresh culture medium. Viability was measured by monitoring cell growth through coloration with crystal violet (CV) [30]. Briefly, cells were stained with crystal violet and after cell lysis the absorbance of the supernatant at 590 nm was measured with a spectrophotometer (Pharmacia Biotech). The viability was expressed as the ratio (in percentage) of absorbance obtained after the pulse train as compared to the extract of cells treated in the same way but without application of an electric field. 2.9. Preparation of lipid vesicles Multilamellar vesicles (MLVs) of L-α-Phosphatidylcholine (Egg PC, Avanti, USA) were prepared at 2 mg/ml. 20 mg of lipids was dissolved in 5 ml chloroform that was evaporated under nitrogen flow and vacuum until complete dryness. MLVs were obtained by adding 10 ml of NaCl solution at 500 μS/cm and hand shaking twice vigorously for 15 s. 2.10. Statistical analysis of MLV diameter The diameter distribution of MLVs was obtained as follows. A drop of the MLV suspension was observed under the stage of a microscope. The diameter measurement was performed on 300 MLVs from random images obtained with an inverted microscope DMIRB (Leica) coupled with a camera CoolSnapFx HQ2 (Photometrics, USA) and a × 40 Phaco objective (Leica). Only the MLVs in focal plan are analyzed with the Metamorph 7.1.7.0 software (Molecular Devices, USA). The diameter distribution statistical analysis was obtained with Prism5 software (GraphPad, USA). For each condition, statistical analysis was performed on two independent experiments. 3. Results 3.1. Test of the closed chamber electrodes Using the HV voltage Cirtem microsecond pulse generator and a very low conductance buffer (0.2 mS/cm), it was possible to simultaneously record the applied voltage and the delivered current on a large range of voltages. We obtained an ohmic behavior when applying 5 μs pulses (i.e. low frequency conditions) in a range of applied voltages of 0 to 9 kV, i.e. fields between 0 and 45 kV/cm. We selected short microsecond pulse duration and low conductance buffer to limit the Joule heating and its effect on the sample conductivity and due to the current safety limit of the generator (5 A). The volume of the chamber was designed to accommodate 100 μl of pulsation buffer. The overflow of liquid could then be evacuated by the hole in the upper electrode. In the following experiments we have tested whether this volume was sufficient for the current to be conducted between the electrodes and that no air bubble preventing the good flow of the current between the electrodes was present in the chamber. Applying a known voltage between the electrodes we have recorded the intensity of this current during the pulse with a Cliniporator pulser. The voltage was set at 420 V. With a volume of 100 μl and above, the intensity in the chamber remained at a stationary level of 2.6 A confirming that 100 μl was the optimum volume for the chamber. The extra volume leaks out by the upper channel and was out of the conductive pathway. This confirmed that the volume of the pulsed sample was 100 μl.
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Transf (GFP)/(%)
Fluorescence / AU
A
Fluorescence / AU
B
PI (cells)/(%)
The nanosecond HV pulse as observed on the electrodes is displayed in Fig. 2. The rise time of the pulse was observed to be less than 600 ps, compared to the 200 ps output value predicted from the operating manual and observed on the 10 V monitoring pulse from the auxiliary output. No major capacitive effect was induced by the chamber on the pulse delivery. From the electrical point of view, the design was therefore suitable for the delivery of nanosecond HV pulses. Pulsing induced a Joule heating and a thermal expansion of the solution. The volume thermal expansion coefficient of water is 2.6 · 10−4/K while it is only 3 10−5/K for nylon and 1.2 · 10−5/K for steel. In an attempt to prevent dramatic pressure effects due to the fast thermal expansion, a small channel was bored in the upper electrode. To avoid heterogeneity in the field distribution, the diameter of this channel must be very small and we selected a diameter of 1 mm. The temperature increase under the electro-gene transfer (EGT) conditions (ms pulses) was obtained by converting the delivered electrical energy in Joule heating. A temperature increase of 12 °C is obtained after 10 pulses (700 V/cm with a duration of 5 ms and at a frequency of 1 Hz) when assuming a poor thermal dissipation during the 1 s inter-pulse delay. This means that this is an upper bound limit. As a final conclusion, the design of this closed chamber was checked to comply with the electrical specifications (ohmic behavior, controlled volume i.e. gap, no capacitive interference on the pulse delivery on the electrodes).
39
C
The original purpose of the designed chamber was to prevent the formation of electric arcs when used for electric field of high voltage and short duration pulses (nanosecond pulses or nanopulses [22,31]). The aim was to apply these nanopulses after a classical electro-gene transfer to induce a better transfection via the destabilization of the nuclear membrane. Conflicting results were reported [20,21] but the recent conclusions were that nsPEF were affecting the cell but remained non-effective for gene transfer and expression [13,21,32]. The first step towards this utilization was therefore to check the use of this chamber for classical electro-transfection. This electrode chamber efficiency was compared with other electrodes with a known classical design (see the description of the electrodes (2-mm electrodes and 4mm electrodes) in the Materials and methods section and Fig. 1). For each electrode set, the conditions for electro-transfection were the same and applied as follows: cells in suspension were subjected to a train of ten pulses of 700 V/cm with a duration of 5 ms and at a frequency of 1 Hz. In Fig. 3 results are summarized for the 3 different applicators.
Fig. 2. Record of a nanosecond HV pulse. A 3 kV nanosecond pulse was delivered on the closed pulsing chamber. The pulse was monitored by a 1 GHz storage oscilloscope and reported on the figure.
Viab (CV)/(%)
3.2. Transfection efficiency of the different systems of electrodes with associated permeabilization and viability
Chamber 2 mm
Elect . 2 mm
Elect . 4 mm
Fig. 3. Comparison of different electrodes for transfection, permeabilization, and viability. The transfection, permeabilization to PI, and viability were studied for the different electrodes systems (Closed chamber; 2-mm electrodes or 4-mm electrodes). In a), transfection (Transf (GFP) / (%) = 102. N(GFP) / N(Total), gray bars, left axis) and the average level of fluorescence is represented (thrombus, right axis). In b), permeabilization (PI (cells) / (%) = 102. F(PI) / F(Total), gray bars, left axis) and the average fluorescence of the cells (diamond; right axis) are represented. In c), the viability (Viab (CV) / (%) = 102. A(CV) / A(Basal) after 24 h associated with the transfection experiments is represented. Error bars are standard deviation of a triplicate experiment. For each electrode set, the conditions for electro-transfection were the same and applied as follows: cells in suspension were subjected to a train of ten pulses of 700 V/cm with a duration of 5 ms and at a frequency of 1 Hz.
Fig. 3a represents the transfection results. The 4-mm electrodes achieved very good transfection with 40% of transfected cells with an average fluorescence of 3493 a.u. (arbitrary unit from the BD Bioscience software analysis) in the fluorescent population. On the contrary, the closed chamber achieved very low transfection efficiency with only 2% of transfected cells. In this case the average fluorescence level of the fluorescent population of cells was about 1811 a.u. but with very few fluorescent cells, i.e. statistically not very representative of the level of transfection. The 2-mm electrodes led to an intermediate transfection level with 8% of positive cells and an average fluorescence level at 803 a.u. To understand whether this very low level of transfection was due to a poor permeabilization induced by the closed chamber, the permeabilization to propidium iodide (PI) was evaluated under the same electrical conditions. Indeed, this permeabilization to PI reflects the membrane destabilization of cells subjected to electric field pulses. As expected and shown in Fig. 3b the permeabilization induced with 4-mm electrodes was very high leading to around 84% of PI positive
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cells. The level of fluorescence in these positive cells was around 800 a.u. The closed chamber, although leading to poor transfection, induced no statistically significant differences in the level of permeabilization as 4-mm electrodes with around 66% of positive cells and a level of fluorescence of around 1000 a.u. The 2-mm electrodes induced very poor permeabilization with only 25% of PI positive cells and around 500 a.u. for the average fluorescence level. This level in permeabilization with these 2-mm electrodes may explain their low associated transfection efficiency. An important parameter when studying electrotransfection is the viability at 24 h. This experiment measures the amount of cell growth after 24 h as compared to control untreated cells (no electropulsation). In Fig. 3c this viability for the different electrodes is represented. This viability is respectively 30%, 89% and 13% for the closed chamber, 2-mm electrodes and 4-mm electrodes. 3.3. DNA/membrane interaction It was shown that DNA interaction with the membrane was a crucial step towards the transfection of cells [7]. Therefore, it appeared interesting to study this interaction for the closed chamber and to compare it with the two other electrodes. Fig. 4 shows the images of the interaction of a TOTO-labeled DNA with CHO cells using the different electrodes. In subsets 1a to 3b (Fig. 4), TOTO is added right after the pulses and in subsets 4a to 6b (Fig. 4), it is added 30 min after the pulses. Each subset of fluorescent pictures (1b, 2b, 3b, 4b, 5b and 6b) is shown with the corresponding phase contrast image (respectively 1a, 2a, 3a, 4a, 5a and 6a). In the images shot just after the pulses (1a to 3b), we can see that the DNA/ membrane interaction is present as expected for the 4-mm electrodes (Fig. 4, 3b) but also and more surprisingly for the 2-mm electrodes (Fig. 4, 2b) and the closed chamber (Fig. 4, 1b). These results are unexpected as both last set-ups led to little or no transfection (see Fig. 4, 4a).
Some DNA can also be observed at the membrane 30 min after the pulses for every type of electrodes (Fig. 4, 4b, Chamber; 5b, 2-mm electrodes; 6b, 4-mm electrodes). It is important to note that the interaction of the DNA with the membrane is present with electrodes leading to low transfection. These results suggest that a step beyond the interaction of DNA at the membrane is affected in the case of 2-mm electrodes and the closed chamber.
3.4. Experimental evidence of apparition of shock waves One other option was that with chambers with a narrow gap between the electrodes a pressure shock was present due to the Joule heating expansion of the liquid. Therefore we investigated the occurrence of a physical stress when pulses were delivered in the closed chamber. Shock waves were a candidate. To validate the occurrence of induced shock waves, the evolution of a suspension of multilamellar liposomes was followed by measuring the diameter of the liposomes (Fig. 5). MLVs were pulsed under the same conditions as cells (700 V/cm, 5 ms, 1Hz). Pulses were delivered either with the closed chamber or with the 2mm electrodes. These conditions were shown in a previous study not to induce the electropermeabilization of the lecithin lipid bilayer [26]. Liposomes are soft lipid auto-assemblies that are destabilized by shock waves (due to their transient high pressure). They are fragmented but spontaneous reformed as smaller entities. This process is routinely used under drastic conditions for the formation of sonicated unilamellar vesicles (SUVs). A change in the diameter relative statistical frequency of the liposomes was only present when pulsing in the closed chamber (Fig. 6). Liposomes having a large diameter disappeared while we observed a sharp increase in the population centered at 5 μm. MLVs pulsed in 2-mm electrodes were unaffected. Their diameter relative statistical frequency was the same as the control samples.
Fig. 4. DNA/membrane interaction with the different electrodes. Cells were subjected to electropulsing, as described in the Material and methods with the different systems of electrodes: closed chamber 1a, 1b and 4a, 4b; 2-mm electrodes 2a and 2b and 5a and 5b; 4-mm electrodes 3a and 3b and 6a and 6b. DNA present at the membrane directly after the pulses (images 1a to 3b) or 30 min after the pulses (images 4a to 6b) was labeled with the fluorescent probe TOTO. Phase contrast images (1a, 2a, 3a, 4a, 5a and 6a) are presented with their corresponding fluorescent images (1b, 2b, 3b, 4b, 5b and 6b).
L. Wasungu et al. / Bioelectrochemistry 100 (2014) 36–43
Fig. 5. Principle of diameter determination of MLVs. This picture was an example of phase contrast image obtained in control condition. The diameter measurements were performed exclusively with the MLVs in focal plan.
4. Discussion The distance between the electrodes is a critical parameter, as shown by the results with the two parallel plate electrode designs. A
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narrow distance clearly brought a less efficient expression. This may be due to the electrical reactions present at the electrodes level [33–35] and to the electrophoretic mediated contact of DNA with the electrodes [36–39]. These electrode supported electrochemical reactions are both known to alter the nucleic acids and are larger with the 2-mm electrodes where a larger contact between the electrodes and the solution is present as we kept the volume constant. The induced degradation is a rather simple explanation of the decrease in the efficiency of gene expression. Another difference between the two stainless steel flat parallel electrodes may be the contribution of the meniscus on the field distribution, which is larger with the larger gap set-up. This suggests a positive contribution of the electrical heterogeneity in the gene transfer on a cell population. The difference between the 4-mm plate electrodes and the “closed” pulsing chamber cannot be explained that way. It was shown on cell populations that there was a direct association between the fluorescence level related with the number of plasmids associated with the pulsed cell and the level of expression [9,40]. The Joule heating occurs during the 5 ms pulse discharge and brings a volume expansion of the sample. The narrow channel is supposed to avoid a pressure increase but the volume expansion is rather fast and may result in a transient increase in pressure on the sample. By using data on hydrostatic pressure, we may suggest a pressure increase of 20 MPa. This was experimentally validated by observing a restructuration of multilamellar lipid vesicles (Fig. 6). High pressures are known to affect the membrane organization
Fig. 6. Effect of pulsation on diameter relative statistical frequency of MLV. These diagrams showed the diameter relative statistical frequency (P(N, φi) = N(φI) / ∑JN(φJ) observed under each tested condition. The abscissa axis is labeled with the center bin of the diameter interval, which width is 5 μm. For each condition, error bars are standard deviation calculated from diameter interval of 300 MLVs in two independent experiments. In A, the MLVs were in control conditions. 10 (B) or 1000 (C) pulses were applied respectively with a 2-mm open electrodes (2-mm electrodes) under an applied field of 700 V/cm, 5 ms of pulse duration, a frequency of 1Hz and a conductivity of 500μS/cm. The same parameters were used with the closed chamber with 10 pulses in D and 1000 pulses in E.
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and its function [41,42]. A decrease in the fluidity of lipid bilayer and a reversible conformational change in transmembrane proteins are induced, leading to the functional disorder of membrane-associated activity. Interdigitation of the fatty acid chains is present and gives a change in the local lateral pressure [43]. The cross talk between the membrane components results in a change in the interfacial packing [44,45]. Pressure effect may lead to a stretching of the membrane, which is known to affect the endocytic properties of membranes [46]. The interfacial changes appear to be associated with a decrease in the plasmid membrane insertion during electropulsation. However, we still observed DNA/membrane interaction with the closed chamber electrodes, possibly because the pressure increase is progressive during the pulse and insertion may be hindered only above a critical value, i.e. only after a part of the pulse duration. Nevertheless, the nature of the DNA/membrane complexes and the new membrane organization formed under higher pressure may not be as efficient for future cytoplasm translocation and efficient transfection. Stretching affects the coupling between the plasma membrane and the actin skeleton, which appears to be involved in the DNA electrotransfer to the cytoplasm [47,48]. Actin cytoskeleton appears to be actively involved in the success of the first transfer step(s). It seems that it hinders DNA motion at later stages. Therefore stretching induced alteration of its coupling to the plasma membrane may affect gene electrotransfer. The role of the interfacial organization in electrotransfection was already suggested by our previous study on the effect of membrane order affecting chemicals [49]. Osmotic pressure was inducing a new membrane interface conformation, which was not suited to give plasmid insertion when electropermeabilized. Again this was associated with a change in the structure of the interfacial water. A more practical conclusion is the need of a careful design of pulsing chamber to prevent any associated pressure effect. 5. Conclusions In this comparative study between different pulsing electrodes, we observed that, while electropermeabilization appears to be induced under the same extend for the closed chamber and the 4-mm open electrodes, only the last ones led to efficient electrotransfection. The occurrence of shock waves in the closed chamber is a new parameter that must be taken into account when applying high field pulses (N 10 kV/ cm). But coupling shock wave with electropermeabilization opens the way to a new field of applications when alterations of the cellular organization are requested. Acknowledgment This work was supported by grants from the Region Midi-Pyrénées (grant 11052700), the DGA (grant 0634024) and the ANR (ANR-06BLAN-0260-03) “Cemirbio”. Research was conducted in the scope of the EBAM European Associated Laboratory (LEA) and of the COST Action TD1104 European network for the development of electroporationbased technologies and treatments (EP4Bio2Med). Thanks are due to Ms. Natalie Lemarechal for her careful editing of the manuscript. References [1] E. Neumann, K. Rosenheck, Permeability changes induced by electric impulses in vesicular membranes, J. Membr. Biol. 10 (1972) 279–290, http://dx.doi.org/10.1007/ BF01867861. [2] J. Teissie, M. Golzio, M.P. Rols, Mechanisms of cell membrane electropermeabilization: a minireview of our present (lack of?) knowledge, Biochim. Biophys. Acta 1724 (2005) 270–280, http://dx.doi.org/10.1016/j.bbagen.2005.05.006. [3] E. Neumann, M. Schaefer-Ridder, Y. Wang, P.H. Hofschneider, Gene transfer into mouse lyoma cells by electroporation in high electric fields, EMBO J. 1 (1982) 841–845. [4] M.P. Rols, M. Golzio, B. Gabriel, J. Teissié, Factors controlling electropermeabilisation of cell membranes, Technol. Cancer Res. Treat. 1 (2002) 319–328. [5] A. Gothelf, L.M. Mir, J. Gehl, Electrochemotherapy: results of cancer treatment using enhanced delivery of bleomycin by electroporation, Cancer Treat. Rev. 29 (2003) 371–387.
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