Bioelectrochemistry 103 (2015) 28–33

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Time domain dielectric spectroscopy of nanosecond pulsed electric field induced changes in dielectric properties of pig whole blood Jie Zhuang, Juergen F. Kolb ⁎ Leibniz Institute for Plasma Science and Technology, Felix-Hausdorff-Strasse 2, 17489 Greifswald, Germany

a r t i c l e

i n f o

Article history: Received 26 January 2014 Received in revised form 29 July 2014 Accepted 12 August 2014 Available online 23 August 2014 Keywords: Nanosecond pulsed electric field Time domain dielectric spectroscopy Whole blood Electroporation

a b s t r a c t The dielectric spectra of fresh pig whole blood in the β-dispersion range after exposure to 300–nanosecond pulsed electric fields (nsPEFs) with amplitude higher than the supra-electroporation threshold for erythrocytes were recorded by time domain reflectometry dielectric spectroscopy. The implications of the dielectric parameters on the dynamics of post-pulse pore development were discussed in light of the Cole–Cole relaxation model. The temporal development of the Cole–Cole parameters indicates that nsPEFs induced significant poration and swelling of erythrocytes within the first 5 min. The results also show that the majority of erythrocytes could not fully recover from supra-electroporation up to 30 min. The findings of this study suggest that time domain dielectric spectroscopy is a promising label-free and real-time physiological measuring technique for nsPEFblood related biomedical applications, capable of following the conformational and morphological changes of cells. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Recently, nanosecond pulsed electric fields (nsPEFs), i.e., electric pulses of nanosecond duration with electric field strength on the order of 10 kV/cm, have attracted rising interests for its potential in a broad spectrum of biomedical applications, e.g., it has shown promising future as a novel tumor therapy by inducing apoptosis of cancer cells [1,2]. A unique aspect of nsPEFs is the creation of the so-called nanopores, i.e., pores with a diameter on the order of 1 nm in cell membranes [3,4]. The typical size of pores created by nsPEFs is believed significantly smaller but much denser than the pores induced by conventional electroporation pulses which usually span from microsecond to millisecond range [3]. Several studies have indicated that a portion of nsPEF-induced nanopores has a lifetime longer than several minutes [5,6]. These features may be attractive to biomedical applications such as controlled drug release from cellular carrier system, e. g., carrier erythrocytes, at targeted location for a sustained period of time. Carrier erythrocytes are very suitable for controlled drug delivery, thanks to their biodegradability, biocompatibility and large carrier volumes [7]. Several methods have been proposed for the targeted drug release using carrier erythrocytes, including loading paramagnetic particles or photosensitive agents in erythrocytes, sonoporation using ultrasound together with microbubbles and attaching site-specific antibody to erythrocyte membrane [8]. Recent reports have suggested delivering electrical pulses to biological matter using focusing antennas [9,10], which make it possible to remotely release drugs from erythrocytes by nsPEFs. Potential ⁎ Corresponding author. E-mail address: [email protected] (J.F. Kolb).

http://dx.doi.org/10.1016/j.bioelechem.2014.08.009 1567-5394/© 2014 Elsevier B.V. All rights reserved.

advantages of nsPEFs over other techniques are as follows: (1) no extra agent is required to be encapsulated or attached to erythrocyte thus eliminates corresponding side effects; (2) nsPEF-treatment is nonthermal and non-ionizing therefore prevents hyperthermia or phototoxicity induced by ultrasound or laser; (3) the pores induced by nsPEFs are relatively small compared to hemoglobin molecules so hemolysis can be minimized by selecting appropriate exposure parameters, in the mean time small drug molecules can still be released into extracellular environment. There have been a number of reports investigating the effects of pulsed electric fields, mostly electroporation pulses, on erythrocytes [11–15]. However these studies were exclusively conducted in an environment which is usually different from physiological conditions, i.e., whole blood at body temperature. Erythrocytes were exposed to pulsed electric fields in saline solutions instead of blood plasma and had a volume fraction different from physiological hematocrit. To explore the feasibility of using nsPEFs as a controlled drug release technique with some degree of selectivity, it is essential to investigate nsPEF-induced conformational and morphological changes in erythrocytes under physiological conditions. To the best of our knowledge, such an examination has not been reported yet. In this work, nsPEF-induced conformational and morphological changes in pig erythrocytes under physiological conditions were tentatively investigated by means of time domain reflectometry (TDR) dielectric spectroscopy, which delivers the dielectric spectrum of pig whole blood in a wide frequency range. Dielectric and impedance spectroscopy are often employed in characterizing the dynamics of membrane poration after PEF-exposures [16–18]. TDR dielectric spectroscopy is superior to other methods for physiological measurements in the

J. Zhuang, J.F. Kolb / Bioelectrochemistry 103 (2015) 28–33

following aspects: (1) this method is label-free and non-invasive, meaning no dye or agent is added to the sample under test, which guarantees that the intrinsic responses of erythrocytes to nsPEFs are not disturbed; (2) the dielectric properties of erythrocytes in the β-dispersion range (typically from 100 kHz to 100 MHz) are extremely sensitive to the changes in membrane conductivity and cell morphology, which are often consequences of membrane poration; (3) TDR dielectric spectroscopy can be performed in a real-time manner therefore it is more efficient than other labor-intensive methods such as microscopy examination or flow cytometry, which makes it especially convenient for recording the time course of nsPEF-cell interactions. It has been reported that the dielectric properties of preserved rabbit blood can be used to monitor the time course of blood deterioration accompanied by morphological changes of erythrocytes [19]. Measurements, under physiological conditions, remain a very challenging task for dielectric spectroscopy. The strong polarization effect at electrode/electrolyte interface due to charge accumulation often dominates the dielectric spectrum in the audio to radio frequency range, obscuring the β-dispersion of cells. We have developed a strategy to overcome the impact from electrode polarization [20]. By plating a layer of platinum black to the measuring electrodes, the equivalent surface area was significantly enlarged which leads to greater polarization capacitance. Then the magnitude of the electrode polarization impedance dropped notably in the frequency domain. The contribution from electrode polarization could be reduced by two orders of magnitude in the permittivity spectrum. The residual impedance of electrode polarization was then modeled as a constant-phase-angle (CPA) element in series with the sample. In this study the bulk dielectric spectra of control and exposed samples were parameterized by the Cole–Cole relaxation model for further analysis, which has been suggested to provide the best fitting results for the β-dispersion of animal whole blood [21]. 2. Materials and methods

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The reflected time domain signals of empty sensor and sensor filled with the blood sample were collected by the TDR oscilloscope and a Running Laplace Transform algorithm was applied to obtain the raw dielectric spectrum of the sample under test. The details of signal capture and the Running Laplace Transform can be found elsewhere [20]. 2.2. Blood sample preparation Fresh heparinized pig whole blood was obtained from Dr. Barbara Y. Hargrave (Department of Biological Sciences, Old Dominion University). Mammalian blood consists of blood plasma and blood cells including erythrocytes, leukocytes, and thrombocytes. Usually the number and volume faction of erythrocytes are 2 orders of magnitude higher than other cells so it is a practical approximation considering a whole blood sample as an erythrocyte suspension with respect to dielectric modeling. The blood samples were always subjected to nsPEF-exposure and dielectric inspection no more than 2 h after collection in order to avoid blood deterioration, such as formation of echinocytes. 2.3. Nanosecond pulsed electric field exposure Electrical pulses of 300 ns were generated by an in-house-made transmission line pulse generator with a characteristic impedances of 7 Ω [23]. The rise and fall time of the pulse is better than 5 ns. The blood samples were contained in a standard 2-mm gap electroporation cuvette during exposure to 8 pulses with 1 Hz repetition rate. The electric field strength was chosen to be 30 kV/cm which falls into the range of typical values used for nsPEF-cell treatment [24]. For exposed blood samples, TDR dielectric spectroscopy was performed consecutively from 1 min to 30 min following nsPEF-exposure. Prolonged measurements were not pursued mainly due to evaporation of exposed blood samples at longer times. At each time point, the raw data were collected from 3 experiments at different days using fresh blood samples from the same pig.

2.1. Time domain reflectometry dielectric spectroscopy 2.4. Dielectric modeling with the Cole–Cole relaxation model The dielectric properties, i.e., relative permittivity and conductivity, of pig blood samples were measured by a TDR dielectric spectrometer consisting of an Agilent 86100C TDR oscilloscope and an Agilent 54754A differential plug-in (Fig. 1). Similar technology has been successfully employed to measure the dielectric properties of cancer cells [16,22]. The blood samples were contained in a cut-off type coaxial dielectric sensor with Pt black-plated electrodes. Detailed description of the sensor can be found in literature [20]. A thermostat (Julabo, San Diego, CA) was utilized to keep the temperature of the sensor stabilized at 38 °C which is close to a pig's body temperature. The dielectric sensor was connected to the spectrometer with an Agilent 8120-4948 semirigid coaxial cable. The measuring signal is a voltage step of 200 mV with 35-ps rise time. Non-linear changes in the dielectric properties of the blood samples during dielectric measurement is not expected because the electric field generated by the test signal in the dielectric sensor (b1 V/cm) is 4 orders lower than the electric field of applied nsPEFs.

Fig. 1. Block diagram of the TDR dielectric spectroscopy system. The fast rising voltage pulse, V0(t), is generated by a pulse generator and applied to the sample through a coaxial cable with a characteristic impedance of Z0. The reflected signal, Rx(t), is digitized by a sampling head and stored in a TDR oscilloscope [20].

Analysis with the broadband dielectric spectrum is a straightforward method but the underlying relationship between the frequencydependent values and cell structure and morphology is ambiguous. In this study further analysis was achieved by parameterization of the dielectric spectrum for control and exposed samples through the Cole–Cole relaxation model which provides the best fitting results for the beta-dispersion of whole blood [21]:



ε ðωÞ ¼ εh þ

Δε σ þ 1 þ ðjωτr Þα jωε0

ð1Þ

where j2 = −1, ω is the angular frequency, ε0 is the permittivity of free space, ε⁎(ω) is the complex permittivity of the sample, εh is the high frequency permittivity, Δε is the relaxation intensity of β-dispersion, σ is the dc conductivity, τr is the relaxation time and α is the broadening parameter (0 b α b 1). The contribution of electrode polarization, which is inevitable using high conductivity physiological medium as the supernatant, has to be considered during the fitting procedure. A constant-phase-angle (CPA) element was used to model the impedance of electrode polarization, which is always in series with the bulk impedance of the blood sample under test. The corresponding impedance, ZEP, can be modeled as: ZEP ¼ K

−1

−η

ðjωÞ

;

where K is a constant and 0 ≤ η ≤ 1 [25].

ð2Þ

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J. Zhuang, J.F. Kolb / Bioelectrochemistry 103 (2015) 28–33

3. Results The immediate measurement results of TDR spectroscopy are time domain responses of the blood samples. Typical time domain responses of several mammalian cell suspensions can be found in literature [20,22] and are not shown here. The raw dielectric spectrum of pig blood is obtained by transforming the time domain response into frequency domain using the Running Laplace Transform [26]. By fitting the raw dielectric spectrum into the Cole–Cole relaxation model together with the electrode polarization impedance modeled as a CPA element, the Cole–Cole parameters in Eq. (1) are derived for both control and exposed pig blood samples. For each parameter, the statistical significance of the difference was examined by the Student's t-test. The raw dielectric spectrum and some of the Cole–Cole parameters (Δε, τr, α and σ) are found sensitive to the application of nanosecond pulses. The details of the nsPEF-induced changes are shown in the following section. 3.1. Raw dielectric spectra of control and exposed pig whole blood Fig. 2 shows the relative permittivity and conductivity spectrum of control and exposed pig blood samples (5 min after exposure), ranging from 10 kHz to 400 MHz. The β-dispersion exists as a gradual decrease in the relative permittivity and a gradual increase in the conductivity, approximately from 100 kHz to 100 MHz for both control and exposed samples. The gradual decrease in the relative permittivity, or β-dispersion, means that the charging speed of plasma membrane cannot follow the oscillating electric field as frequency increases, which in consequence leads to decreases in the intensity of membrane interface polarization. In the meantime, the plasma membrane of erythrocytes, a dielectric barrier at low frequencies, becomes increasingly transparent to external electric field as frequency increases. Therefore the contribution of cytoplasm conductivity is progressively added to the whole blood conductivity. Below 100 kHz, the significant values in relative permittivity (up to 4 × 104) are due to the contribution of electrode polarization, which is an electric double layer formed at the electrode/electrolyte interface. This polarization effect dominates the low frequency end of the relative permittivity spectrum and diminishes exponentially with increasing frequency. It overlaps partially with the β-dispersion therefore has to be considered in subsequent analysis. The influence of electrode polarization is less pronounced in the conductivity spectrum, mostly due to the capacitive nature of the electric double layer. The slight drop in conductivity around 10 kHz is a sign of electrode polarization. Exposure to nsPEFs resulted in significant changes in both relative permittivity and conductivity spectrum. The conductivity of exposed blood sample below 10 MHz shows a significant drop within 5 min. The decrease in conductivity ranges from 10% to 20% in this frequency

range. The conductivity spectrum above 10 MHz showed little variation after nsPEF-exposure because the plasma membrane is essentially short-circuited at high frequencies. On the other hand, the changes in relative permittivity spectrum show a more complicated pattern. Below 100 kHz the drop in permittivity after exposure can be attributed to a drop in electrode polarization intensity but the possible changes in the so-called α-dispersion cannot be totally ruled out [27]. From 100 kHz to 1 MHz a decrease in the intensity of β-dispersion is seen which can be explained by the reduction in membrane interfacial polarization. However the permittivity spectrum in this frequency range still suffers from the residual influence of electrode polarization. Further analysis is necessary in order to elucidate the potential implications of this permittivity drop. After nsPEF-exposure, the degree of dispersion in the relative permittivity spectrum between 100 kHz and 100 MHz shows a noticeable decrease, i.e., the β-dispersion becomes narrower. More specifically, the dielectric response of exposed blood sample approaches towards a Debye-type relaxation for identical spheres. Beyond 100 MHz the permittivity spectrum is mainly determined by water molecules which are not likely changed by nanosecond pulses used in this study. As a consequence no significant change is observed in this frequency range after nsPEF-exposure. 3.2. Changes in relaxation intensity The relaxation intensity (Δε) of pig whole blood after nsPEFexposures is shown in Fig. 3. In Cole–Cole model, Δε stands for the intensity of the dielectric dispersion such as the β-dispersion of pig whole blood in this study. More specifically it is a measure of the intensity of interfacial polarization along the cell membranes, mostly erythrocyte plasma membranes. For unexposed control the value of Δε is 2060 which is shown at time 0. In comparison the value of Δε for cell-free blood plasma in the β-dispersion range is around 78 due to the absence of the membrane polarization mechanism. The application of nanosecond pulses reduces Δε to 1450 after 1 min, which means a significant drop in membrane polarization. From 1 min to 30 min, Δε shows slight fluctuation over time. Statistical analysis using the Student's t-test shows the differences are not significant (P N 0.05). 3.3. Changes in relaxation time The relaxation time (τr) of Cole–Cole model is a measure of the average characteristic time that the dipoles need to “relax” from a measuring electric signal. For pig blood, the majority of dipoles responsible for the β-dispersion are formed by the interfacial polarization along the plasma membrane of erythrocytes. As shown in Fig. 4, the relaxation time of

Fig. 2. (A) Relative permittivity and (B) conductivity spectrum of control (●) and exposed (Δ) pig blood from 10 kHz to 400 MHz. Both control and exposed blood samples exhibit a β-dispersion from several hundred kHz to 400 MHz. Electrode polarization dominates the low frequency region of the relative permittivity spectrum (f b 100 kHz), while it is barely seen in the conductivity spectrum. The best fit (−) is obtained by fitting the relative permittivity spectrum and the conductivity spectrum simultaneously into a Cole–Cole relaxation model with electrode polarization modeled as a CPA element.

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Fig. 3. The temporal development of the relaxation intensity (Δε) of the Cole–Cole model after exposure to eight 30 kV/cm, 300 ns pulses. The relaxation intensity of exposed samples is significantly lower than unexposed control. Error bars are calculated by standard deviation.

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Fig. 5. Changes in the broadening parameter (α) of Cole–Cole model as a function of time after exposure to eight 30 kV/cm, 300 ns pulses. Error bars are calculated by standard deviation.

3.5. Changes in dc conductivity unexposed pig blood is about 67 ns. A quasi-exponential decay in relaxation time can be observed after nsPEF-exposures. Within 1 min τr decreases to 42 ns and stabilizes around 32 ns after 5 min. This decrease is a manifestation of the fact that it takes less time to fully discharge the plasma membrane of red cells, which corresponds to possible changes in the dielectric properties of intra-/extra-cellular environment or plasma membrane. After 30 min, τr recovers to 33 ns. The difference in relaxation time between 5 min and 30 min is not statistically significant (P N 0.05).

3.4. Changes in broadening parameter The broadening parameter (α) of the Cole–Cole model is a scalar introduced as the distribution in the relaxation time (τr) of individual dipolar entities or the degree of deviation from a Debye-type dispersion where α equals 1. As shown in Fig. 5, the value for unexposed pig whole blood is 0.87 (0 min) which is typical for mammalian blood. The deviation from 1, representing identical spherical cells, is due to the biconcave shape of red cells. Within 1 min following exposure, α increases to 0.90 and reaches 0.92 at 2 min, which means that the distribution of relaxation time becomes narrower than control. The changes from 2 min to 10 min are not statistically significant (P N 0.05). After 20 min, α drops to 0.90 which is significantly lower than the value at 2 min (P b 0.05).

Fig. 4. The temporal development of the relaxation time (τr) of Cole–Cole model for pig whole blood after exposure to eight 30 kV/cm, 300 ns pulses. After application of pulses, the relaxation time shows an exponential decrease. Error bars are calculated by standard deviation.

Fig. 6 shows the changes in the dc conductivity (σ) of pig blood samples after nsPEF-exposure. Here the dc conductivity of Cole–Cole model represents a blood sample's ability to conduct ionic (dc) current. The value of this parameter is primarily determined by the conductivity of the blood plasma (supernatant), the plasma membrane conductivity and the hematocrit of erythrocytes. As shown in Fig. 6, the dc conductivity of control is 0.65 S/m which is much lower than that of blood plasma (~ 1.2 S/m). This is due to the fact that the high volume fraction of erythrocytes covered by poorly conductive membrane impedes the conduction of ionic current. The change in blood dc conductivity is not significant at 1 min after exposure. Starting from 1 min, the time course of dc conductivity exhibits a 22% quasi-linear decrease till 6 min. No major changes are observed between 6 and 10 min. At 30 min, the dc conductivity increases to 0.58 S/m which is 12% higher than the minimum at 6 min but is still significantly lower than that of control (P b 0.05).

4. Discussion The dielectric characteristics of biological cells and tissues are essential information for understanding PEF-cell interactions because the immediate response, i.e., charging of membranes, is determined by these values. In return, PEF-induced conformational and functional changes in biological matter will lead to modifications of these intrinsic dielectric properties. Dielectric characterization of pulsed cells and tissues may help understand the fundamental PEF-membrane interaction

Fig. 6. The temporal development of the dc conductivity (σ) of Cole–Cole model for pig whole blood after exposure to eight 30 kV/cm, 300 ns pulses. Error bars are calculated by standard deviation.

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mechanisms and provide new diagnostic modalities for PEF-related biomedical applications, which is the motivation of this study. The raw dielectric spectrum obtained from TDR dielectric spectroscopy (Fig. 2) includes permittivity and conductivity of the blood sample under test in the β-dispersion range. Direct analysis with the raw dielectric spectrum is a straightforward approach but unfortunately the underlying relationship between the frequency-dependent values and cell structure and morphology is ambiguous. Fitting the raw spectrum into an appropriate dielectric model with dielectric parameters of more explicit physical meaning is a widely adopted procedure and sometimes crucial for successful for interpreting the measurement results. In pig whole blood, the volume fraction of erythrocytes is more than 38% while other blood cells are much less than 1%. In terms of the contribution to the overall bulk dielectric properties, it is reasonable to approximate pig whole blood as a suspension of erythrocytes in blood plasma. For dielectric modeling of a cell suspension, two strategies are widely employed. The first strategy is based on the effective medium theory (EMT) which is developed by averaging the dielectric contribution of all entities in a heterogeneous system. Typical models are the Maxwell–Wagner model for diluted cell suspensions [28] and the Hanai model for concentrated cells [29]. Cells in suspension can be further model as shelled particles to derive the dielectric parameters of different layers such as plasma membrane and cytoplasm [22,30]. The other strategy is based on phenomenological models such as the Debye model and the Cole–Cole model which describe the bulk dielectric behavior of the cell suspension through a collection of dielectric parameters with no direct relationship to the dielectric properties of cellular structures [31,32]. In this study we used the Cole–Cole relaxation model which falls into the scope of the second strategy. The Cole–Cole model has a number of unique advantages in modeling the dielectric properties of exposed blood samples. In comparison with the Maxwell–Wagner model or the Hanai model, the Cole–Cole model does not require detailed microscopic structural information of the blood sample under test, such as cell size, cell shape and volume fraction which are essential parameters in the first 2 models. For nsPEF-exposed erythrocytes the above parameters are extremely difficult to measure due to continuous osmotic swelling/shrinking of cells. By fitting Eqs. (1) and (2) simultaneously into the raw dielectric spectrum measured through TDR dielectric spectroscopy, the Cole– Cole dielectric parameters were derived for control and exposed blood samples. The relaxation intensity (Δε) for unexposed pig whole blood is 2060, which lies in the range of literature results for other animals (900 to 4000) [33]. The values of relaxation time (τr) and broadening parameter (α) are about 67 ns and 0.87, which are also in good agreement with values reported for many types of mammalian erythrocytes [21,32]. Exposure to nsPEFs induced significant changes in these dielectric parameters over the entire measuring time. In the following sections the underlying relationship of these changes with the kinetics of membrane poration is discussed. It is generally accepted that the kinetics of PEF-induced reversible membrane poration can be split into three stages: pore creation, pore expansion and pore recovery. There are experimental evidences showing that the pore recovery process may further consist of several phases ranging from microseconds to hours [34,35]. The creation and expansion of membrane pores happen during pulse application. The fast relaxation of pores can start immediately following the removal of pulsed electric fields with a characteristic relaxation time on the order of milliseconds or shorter [36,37]. These fast resealing pores are usually called short-lived pores. On the other hand, the lifetime of some membrane pores may last from milliseconds to days, depending on many factors such as cell type, pulse parameters, medium composition and temperature [5,11,15]. Characterization of these long-lived pores under physiological conditions is thus critical for nsPEF-related biomedical applications, e.g., designing nsPEFs as a novel technique for controlled drug release from carrier erythrocytes in vivo. The exposure parameter of nsPEFs used in this study is typical for inducing supra-

electroporation on various types of cells [24]. By recording the I–V waveforms of applied pulses (data not shown here), we observed significant increase in the in-pulse conductivity of blood sample which guarantees that poration of erythrocyte membranes has been achieved. One aim of this study is to detect and track the presence of long-lived pores using TDR dielectric spectroscopy. Using 20 μs pulses, Saulis found that the lifetime of long-lived pores in human erythrocytes was about 20–40 min at 37 °C. Similar time periods were reported by other groups [15,38]. Pores created by nsPEFs were found to last for minutes [5,6,20], which could be attributed to the existence of a significant energy barrier [34]. The poration of membrane increases membrane conductivity, and in the meantime reduces the intensity of membrane interfacial polarization. As a consequence the relaxation intensity of β-dispersion (Δε), which is the summary of membrane interfacial polarization, is sensitive to the existence of membrane pores. As shown in Fig. 2, significant changes in the β-dispersion of exposed samples were always observed in comparison with unexposed control, which is a strong indication that the erythrocytes had not fully recovered from membrane poration. In Fig. 3, the values of Δε for exposed blood samples from 1 min to 30 min are significantly lower (~30%) than that of unexposed control (P b 0.01). A plausible explanation is that the membrane conductivities of exposed erythrocytes are always higher than that of control, indicating existence of membrane pores. Further support can be derived from the time course of the dc conductivity (σ) which is strongly influenced by membrane poration. When a physiological medium was used as the supernatant, many studies found that the conductivity of cell suspension showed long-lasting decrease after exposure to intense pulsed electric fields [18,38,39]. This phenomenon was explained by osmotic swelling of cells. The long-lived pores were responsible for the unbalance osmotic pressure between intra-cellular environment and extra-cellular environment. In Fig. 6, the temporal development of dc conductivity shows a quasi-linear decrease within 6 min after nsPEFexposure. This is a strong evidence of long-lived pores. Later changes indicate that net ion efflux from erythrocytes overtakes swelling as the dominating factor affecting dc conductivity, suggesting existence of membrane pores up to 30 min. Major contributions from increasing membrane conductivity or decreasing hematocrit to the changes in dc conductivity after 6 min are very unlikely. In either case, the relaxation intensity (Δε) would decrease significantly which was not seen in our experiments. Monitoring the changes in erythrocyte morphology is another target of interest. Hayashi et al. found that the time course of the relaxation time (τr) and the broadening parameter (α) are sensitive to the morphology of erythrocytes [32]. By controlling the composition of the extracellular medium, values of τr and α were obtained for discocytes (64 ns, 0.85), enlarged erythrocytes (34 ns, 0.92) and spherocytes (31 ns, 0.94). In this study, τr decreased from 67 ns to 32 ns (Fig. 4) and α increased from 0.87 to 0.92 (Fig. 5) within 5 min after exposure. According to Hayashi's report such changes indicate that the morphology of exposed erythrocytes has transformed from discocytes towards enlarged erythrocytes. Microscopy examination further confirmed the morphology change. Within 5 min after nsPEF-exposure, all observed erythrocytes swelled from discocytes to enlarged erythrocytes or spherocytes. Under our experimental condition, these changes reflect the colloidal osmotic swelling of erythrocytes due to nsPEF-induced long-lived pores in plasma membrane. The size of long-lived pores is another parameter which has been extensively investigated. According to theoretical predictions, the size of pores created by nanosecond pulses is on the order 1 nm [3]. It is worth nothing that this value is reported for pores created during nanosecond-pulse application instead of minutes thereafter. The size of long-lived pores is usually estimated experimentally by measuring the membrane permeability to test molecules of different sizes [6,38,40,41]. To the best of our knowledge, the correlation between the Cole–Cole dielectric parameters and the size of PEF-induced membrane pores have not been established yet. This is partially due to the

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fact that the Cole–Cole parameters are derived from a phenomenological model hence they do not represent the dielectric characteristics of any specific cellular structures such as plasma membrane. It is also not clear if the time course of Cole–Cole parameters may provide insight into the temporal development of pore size. In this study, microscopy examination showed that about 5% erythrocytes became ghosts from 5 to 10 min after exposure, indicating remarkable release of hemoglobin molecules. The diameter of a single hemoglobin molecule is about 5 nm therefore it is reasonable to assume that these erythrocyte ghosts may have membrane pores of comparable or larger size. For nsPEF-exposed cells, similar delayed formation of larger pores has been reported by other researchers [42,43]. On the other hand, no statistically significant changes were found in the corresponding time course of Δε, τr and α (P N 0.05). The changes in σ after 5 min only indicate that osmotic swelling is no longer the dominating factor. Hence no conclusion can be drawn for the size of membrane pores from the Cole–Cole parameters derived in this study.

5. Conclusion In this study, TDR dielectric spectroscopy has been applied to record nsPEF-induced changes in the dielectric properties of pig whole blood under physiological conditions. By analyzing the temporal development of selected Cole–Cole parameters of exposed pig blood, long-lived pores in erythrocyte membrane with a lifetime up to 30 min were confirmed. In addition the transformation of erythrocytes from discocytes to enlarged erythrocytes could be detected. These results suggest that TDR dielectric spectroscopy is a promising technique for monitoring the conformational and morphological changes of cells induced by nsPEFs under physiological conditions.

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Time domain dielectric spectroscopy of nanosecond pulsed electric field induced changes in dielectric properties of pig whole blood.

The dielectric spectra of fresh pig whole blood in the β-dispersion range after exposure to 300-nanosecond pulsed electric fields (nsPEFs) with amplit...
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