Gorazd Pucihar

University of Ljubljana, Lubliano, Ljubljana, Slovenia

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Publications (32)73.62 Total impact

  • Lea Retelj, Gorazd Pucihar, Damijan Miklavcic
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    ABSTRACT: Nanosecond (ns) electric pulses of sufficient amplitude can provoke electroporation of intracellular organelles. This paper investigates whether such pulses could provide a method for controlled intracellular release of a content of small internalized artificial lipid vesicles (liposomes). To estimate the pulse parameters needed to selectively electroporate liposomes while keeping the plasma and nuclear membranes intact, we constructed a numerical model of a biological cell containing a nucleus and liposomes of different sizes (with radii from 50 nm to 500 nm), which were placed in various sites in the cytoplasm. Our results show that under physiological conditions selective electroporation is only possible for the largest liposomes and when using very short pulses (few ns). By increasing the liposome interior conductivity and/or decreasing the cytoplasmic conductivity, selective electroporation of even smaller liposomes could be achieved. The location of the liposomes inside the cell does not play a significant role, meaning that liposomes of similar size could all be electroporated simultaneously. Our results indicate the possibility of using ns pulse treatment for liposomal drug release.
    IEEE transactions on bio-medical engineering 05/2013; · 2.15 Impact Factor
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    ABSTRACT: Electrofusion is an efficient method for fusing cells using short-duration high-voltage electric pulses. However, electrofusion yields are very low when fusion partner cells differ considerably in their size, since the extent of electroporation (consequently membrane fusogenic state) with conventionally used microsecond pulses depends proportionally on the cell radius. We here propose a new and innovative approach to fuse cells with shorter, nanosecond (ns) pulses. Using numerical calculations we demonstrate that ns pulses can induce selective electroporation of the contact areas between cells (i.e. the target areas), regardless of the cell size. We then confirm experimentally on B16-F1 and CHO cell lines that electrofusion of cells with either equal or different size by using ns pulses is indeed feasible. Based on our results we expect that ns pulses can improve fusion yields in electrofusion of cells with different size, such as myeloma cells and B lymphocytes in hybridoma technology.
    Scientific Reports 01/2013; 3:3382. · 5.08 Impact Factor
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    ABSTRACT: Simulations probing the conductivity changes of three-dimensional models of biological tissues consisting of random ternary core-shell sphere packings with different spatial scales are described. We investigate the temporal evolution of the electric conductivity of these packings during application of an electric field with magnitude either below or above the value leading to cell membrane electroporation. The fraction of electroporated cells can be described by a hyperbolic tangent function of the electric field. The collective physical processes causing the transient permeability of the cell membranes can be understood by analogy with the physics of a two-state system with an external field.
    Applied Physics Letters 04/2012; 100(14). · 3.79 Impact Factor
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    ABSTRACT: Electrical breakdown of cell membranes (electroporation) can be either reversible of irreversible, each having many applications in medicine and biotechnology. Here, we describe electroporation on the molecular and cellular level.
    IEEE Electrical Insulation Magazine 01/2012; 28(5):14-23. · 1.32 Impact Factor
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    ABSTRACT: Electroporation-based applications require the use of specific pulse parameters for a successful outcome. When recommended values of pulse parameters cannot be set, similar outcomes can be obtained by using equivalent pulse parameters. We determined the relations between the amplitude and duration/number of pulses resulting in the same fraction of electroporated cells. Pulse duration was varied from 150 ns to 100 ms, and the number of pulses from 1 to 128. Fura 2-AM was used to determine electroporation of cells to Ca(2+). With longer pulses or higher number of pulses, lower amplitudes are needed for the same fraction of electroporated cells. The expression derived from the model of electroporation could describe the measured data on the whole interval of pulse durations. In a narrower range (0.1-100 ms), less complex, logarithmic or power functions could be used instead. The relation between amplitude and number of pulses could best be described with a power function or an exponential function. We show that relatively simple two-parameter power or logarithmic functions are useful when equivalent pulse parameters for electroporation are sought. Such mathematical relations between pulse parameters can be important in planning of electroporation-based treatments, such as electrochemotherapy and nonthermal irreversible electroporation.
    IEEE transactions on bio-medical engineering 09/2011; 58(11):3279-88. · 2.15 Impact Factor
  • Mojca Pavlin, Gorazd Pucihar, Maša Kandušer
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    ABSTRACT: Gene electrotransfer is an established method for transfer of genes into cells, however, the mechanism of transfer of DNA across the cell membrane is still not known. Some studies suggest that DNA is translocated through membrane pores while others propose that DNA enters the cell via electro-endocytosis, but no direct observation was performed. In this paper we investigated the second hypothesis. Cells were stained with membrane dye FM 1-43FX, which is used for observation of endocytosis, and then exposed to electric pulses. We analyzed if endocytosis was stimulated by applying electric pulses with intensities below and above the threshold value for gene electrotransfer. No increase in endocytosis from 20 min or even up to 2h after the pulse delivery was observed, regardless of the electric field strength. These observations do not correlate with electrotransfer efficiency, which increases with field strength and is observed only above the threshold value. Our results suggest that electro-endocytosis is not a crucial mechanism for gene electrotransfer and that the hypothesis of DNA entry by translocation through permeabilized membrane is more plausible. The presented results are important for better understanding of the mechanisms of gene electrotransfer and for its optimization for clinical applications.
    Bioelectrochemistry (Amsterdam, Netherlands) 08/2011; 83:38-45. · 2.65 Impact Factor
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    IEEE Trans. Biomed. Engineering. 01/2011; 58:3279-3288.
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    ABSTRACT: An exposure of a cell to an external electric field results in the induced transmembrane voltage (ΔΨm) that superimposes to the resting voltage. This can have a range of effects, from modification of the activity of voltage-gated channels to membrane electroporation, and accurate knowledge of spatial distribution and time course of ΔΨm is important for the understanding of these effects. In this chapter, we present the analytical, numerical, and experimental methods of determination of ΔΨm, and combine them with the monitoring of electroporation-induced transmembrane molecular transport (TMT) in Chinese Hamster Ovary (CHO) cells. Potentiometric measurements are performed using di-8-ANEPPS, and TMT is monitored using propidium iodide. In isolated cells, we combine analytical derivation (for spherical cells) and numerical computation of ΔΨm (for irregularly shaped cells) with potentiometric measurements to show that the latter are accurate and reliable. Monitoring of TMT in these same cells shows that it is confined to the regions with the highest |ΔΨm|. We then review other parameters influencing electroporation of isolated cells, and proceed, through the intermediate case of dense suspensions, to cells in direct contact with each other. We use the scrape-loading test to show that the CHO cells in a monolayer are interconnected, and then study ΔΨm and TMT in a cluster of four such cells. With low pulse amplitudes, the cluster behaves as one big cell, with ΔΨm continuous along its outer boundary, reflecting the interconnections. With interconnections inhibited, the cells start to behave as individual entities, with ΔΨm continuous along the plasma membrane of each cell. With the cluster exposed to porating (higher amplitude) pulses, TMT occurs in the membrane regions for which computations predict the highest |ΔΨm| if the cells are modeled as insulated, suggesting that the interconnections are blocked by supraphysiological ΔΨm, either directly by voltage gating or indirectly through changes in ionic concentrations caused by electroporation.
    01/2011: pages 19-29; , ISBN: 978-1-4419-8362-6
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    Tadej Kotnik, Gorazd Pucihar, Damijan Miklavcic
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    ABSTRACT: Exposure of a cell to an electric field results in inducement of a voltage across its membrane (induced transmembrane voltage, DeltaPsi (m)) and, for sufficiently strong fields, in a transient increase of membrane permeability (electroporation). We review the analytical, numerical and experimental methods for determination of DeltaPsi (m) and a method for monitoring of transmembrane transport. We then combine these methods to investigate the correlation between DeltaPsi (m) and molecular transport through an electroporated membrane for isolated cells of regular and irregular shapes, for cells in dense suspensions as well as for cells in monolayer clusters. Our experiments on isolated cells of both regular and irregular shapes confirm the theoretical prediction that the highest absolute values of DeltaPsi (m) are found in the membrane regions facing the electrodes and that electroporation-mediated transport is confined to these same regions. For cells in clusters, the location of transport regions implies that, at the field strengths sufficient for electroporation, the cells behave as electrically insulated (i.e., as individual) cells. In contrast, with substantially weaker, nonelectroporating fields, potentiometric measurements show that the cells in these same clusters behave as electrically interconnected cells (i.e., as one large cell). These results suggest that sufficiently high electric fields affect the intercellular pathways and thus alter the electric behavior of the cells with respect to their normal physiological state.
    Journal of Membrane Biology 07/2010; 236(1):3-13. · 2.48 Impact Factor
  • G. Pucihar, D. Miklavčič
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    ABSTRACT: In this paper we examine the influence of gap junction inhibition on the amplitude and distribution of the induced transmembrane voltage (ITV) and electroporation of cells in clusters. Cell clusters were used as they represent simple models of cells in tissues and thus enable the study of the effects of the external electric field on the level of individual cells. We demonstrated that cells in clusters respond differently to the electric field exposure, depending on the field parameters. Namely, when exposed to long, low voltage pulses (such as during the measurements of the ITV) cells in clusters behave as one giant, single cell. At short, high voltage pulses (such as during electroporation) they behave as individual cells. Different response of cells in clusters was attributed to the changes in the properties of gap junctions, specifically, their opening and closing. This was demonstrated by pre-treating the cells with gap junction inhibitor, which caused the cells in clusters to respond as individual cells, regardless of the pulse parameters. KeywordsGap junctions–Transmembrane Potential–Electropermeabilization–Lucifer Yellow–di-8-Anepps
    01/2010: pages 74-77;
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    Tadej Kotnik, Gorazd Pucihar
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    ABSTRACT: 3.1 The Cell and the Induced Transmembrane Voltage 3.2 Analytical Derivation Laplace’s Equation • Spherical Cells • Spheroidal, Ellipsoidal, and Cylindrical Cells • High Frequencies and Very Short Pulses 3.3 Numerical Computation Computational Methods • Irregularly Shaped Cells • Cells in Dense Suspensions and Tissues 3.4 Experimental Determination Potentiometric Dyes • Image Acquisition and Data Processing
    01/2010: pages 51-70; , ISBN: 978-1-4398-1906-7
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    Gorazd Pucihar, Damijan Miklavcic, Tadej Kotnik
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    ABSTRACT: We describe a finite-element model of a realistic irregularly shaped biological cell in an external electric field that allows the calculation of time-dependent changes of the induced transmembrane voltage (ΔΨ) and simulation of cell membrane electroporation. The model was first tested by comparing its results to the time-dependent analytical solution for ΔΨ on a nonporated spherical cell, and a good agreement was obtained. To simulate electroporation, the model was extended by introducing a variable membrane conductivity. In the regions exposed to a sufficiently high ΔΨ, the membrane conductivity rapidly increased with time, leading to a modified spatial distribution of ΔΨ. We show that steady-state models are insufficient for accurate description of ΔΨ, as well as determination of electroporated regions of the membrane, and time-dependent models should be used instead. Our modeling approach also allows direct comparison of calculations and experiments. As an example, we show that calculated regions of electroporation correspond to the regions of molecular transport observed experimentally on the same cell from which the model was constructed. Both the time-dependent model of ΔΨ and the model of electroporation can be exploited further to study the behavior of more complicated cell systems, including those with cell-to-cell interactions.
    IEEE Transactions on Biomedical Engineering 05/2009; 56(5):1491-1501. · 2.35 Impact Factor
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    Gorazd Pucihar, Tadej Kotnik, Damijan Miklavcic
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    ABSTRACT: Placement of a cell into an external electric field causes a local charge redistribution inside and outside of the cell in the vicinity of the cell membrane, resulting in a voltage across the membrane. This voltage, termed the induced membrane voltage (also induced transmembrane voltage, or induced transmembrane potential difference) and denoted by DeltaPhi, exists only as long as the external field is present. If the resting voltage is present on the membrane, the induced voltage superimposes (adds) onto it. By using one of the potentiometric fluorescent dyes, such as di-8-ANEPPS, it is possible to observe the variations of DeltaPhi on the cell membrane and to measure its value noninvasively. di-8-ANEPPS becomes strongly fluorescent when bound to the lipid bilayer of the cell membrane, with the change of the fluorescence intensity proportional to the change of DeltaPhi. This video shows the protocol for measuring DeltaPhi using di-8-ANEPPS and also demonstrates the influence of cell shape on the amplitude and spatial distribution of DeltaPhi.
    Journal of Visualized Experiments 01/2009;
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    ABSTRACT: For an effective tissue controlled electropermeabilization as requested for electrochemotherapy and electrogenotherapy, it is very important to have informations about the electric field distribution provided by a defined set of electrodes. Computer simulations using the finite element models approach predicted the associated field distributions and currents. Phantoms made of gels with well-defined electrical conductance were used to measure the current responses of a new electrode geometry (wires), A good agreement between the measured and predicted currents was observed supporting the validity of the prediction for the field distribution. Field distribution was observed to be very localized and highly homogeneous with the new concept of contact wire electrodes. They allowed to focus the field effect along the surface of the tissue to induce a controlled release of drugs or plasmids. Non invasive (contact) electrodes can be moved rapidly on the body and avoid puncturing the skin and the tissue. They can be used for large surface effects, to treat the skin and subcutaneous tumors. The use of contact electrodes after drug or DNA intradermal injection were validated by clinical treatment of large surface skin tumors and by in vivo imaging of permeabilization or of gene expression.
    Journal of Controlled Release 12/2008; 134(2):125-31. · 7.63 Impact Factor
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    ABSTRACT: Electrochemotherapy is a local drug delivery approach aimed at treatment with palliative intent of cutaneous and subcutaneous tumour nodules of different histologies. Electrochemotherapy, via cell membrane permeabilising electric pulses, potentiates the cytotoxicity of non-permeant or poorly permeant anticancer drugs with high intrinsic cytotoxicity, such as bleomycin or cisplatin, at the site of electric pulse application. An overview of preclinical and clinical studies is presented, and the treatment procedure is further critically evaluated. In clinical studies electrochemotherapy has proved to be a highly efficient and safe approach for treating cutaneous and subcutaneous tumour nodules. The treatment response for various tumours (predominantly melanoma) was approximately 75% complete and 10% partial response of the treated nodules. Electrochemotherapy is a new, clinically acknowledged method for the treatment of cutaneous and subcutaneous tumours. Its advantages are high effectiveness on tumours with different histologies, simple application, minimal side effects and the possibility of effective repetitive treatment.
    European journal of surgical oncology: the journal of the European Society of Surgical Oncology and the British Association of Surgical Oncology 03/2008; 34(2):232-40. · 2.56 Impact Factor
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    ABSTRACT: We present a study of the variability of the minimal transmembrane voltage resulting in detectable electroporation of the plasma membrane of spherical and irregularly shaped CHO cells (we denote this voltage by ITVc). Electroporation was detected by monitoring the influx of Ca(2+), and the transmembrane voltage was computed on a 3D finite-elements model of each cell constructed from its cross-section images. We found that ITVc was highly variable, particularly in irregularly shaped cells, where it ranged from 512-1028 mV. We show that this range is much too large to be an artifact due to numerical errors and experimental inaccuracies, implying that for cells of the same type and exposed to the same number of pulses with the same duration, the value of ITVc can differ considerably from one cell to another. We also observed that larger cells are in many cases characterized by a higher ITVc than a smaller one. This is in qualitative agreement with the reports that higher membrane curvature facilitates electroporation, but quantitative considerations suggest that the observed variability of ITVc cannot be attributed entirely to the differences in membrane curvature.
    Electromagnetic Biology and Medicine 02/2008; 27(4):372-85. · 0.81 Impact Factor
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    ABSTRACT: The transport of propidium iodide into electropermeabilized Chinese hamster ovary cells was monitored with a photomultiplier tube during and after the electric pulse. The influence of pulse amplitude and duration on the transport kinetics was investigated with time resolutions from 200 ns to 4 ms in intervals from 400 µs to 8 s. The transport became detectable as early as 60 micros after the start of the pulse, continued for tens of seconds after the pulse, and was faster and larger for higher pulse amplitudes and/or longer pulse durations. With fixed pulse parameters, transport into confluent monolayers of cells was slower than transport into suspended cells. Different time courses of fluorescence increase were observed during and at various times after the pulse, reflecting different transport mechanisms and ongoing membrane resealing. The data were compared to theoretical predictions of the Nernst-Planck equation. After a delay of 60 µs, the time course of fluorescence during the pulse was approximately linear, supporting a mainly electrophoretic solution of the Nernst-Planck equation. The time course after the pulse agreed with diffusional solution of the Nernst-Planck equation if the membrane resealing was assumed to consist of three distinct components, with time constants in the range of tens of milliseconds, hundreds of milliseconds, and tens of seconds, respectively.
    Biophysical Journal 01/2008; 95(6):2837-48. · 3.67 Impact Factor
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    ABSTRACT: This paper investigates the influence of cell density on cell membrane electropermeabilization. The experiments were performed on dense cell suspensions (up to 400 x 10(6) cells/ml), which represent a simple model for studying electropermeabilization of tissues. Permeabilization was assayed with a fluorescence test using Propidium iodide to obtain the mean number of permeabilized cells (i.e. fluorescence positive) and the mean fluorescence per cell (amount of loaded dye). In our study, as the cell density increased from 10 x 10(6) to 400 x 10(6) cells/ml, the fraction of permeabilized cells decreased by approximately 50%. We attributed this to the changes in the local electric field, which led to a decrease in the amplitude of the induced transmembrane voltage. To obtain the same fraction of cell permeabilization in suspensions with 10 x 10(6) and 400 x 10(6) cells/ml, the latter suspension had to be permeabilized with higher pulse amplitude, which is in qualitative agreement with numerical computations. The electroloading of the cells also decreased with cell density. The decrease was considerably larger than expected from the differences in the permeabilized cell fractions alone. The additional decrease in fluorescence was mainly due to cell swelling after permeabilization, which reduced extracellular dye availability to the permeabilized membrane and hindered the dye diffusion into the cells. We also observed that resealing of cells appeared to be slower in dense suspensions, which can be attributed to cell swelling resulting from electropermeabilization.
    European Biophysics Journal 04/2007; 36(3):173-85. · 2.27 Impact Factor
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    Gorazd Pucihar, Tadej Kotnik, Damijan Miklavcic
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    ABSTRACT: Despite the increasing use of electroporation its mechanisms are still not completely understood. This is especially the case in tissues, due to its complicated structure. To study the electric field interaction with tissues on a single cell level, we performed our study on cell clusters. We calculated the induced transmembrane voltage on numerical models of cell clusters, compared the calculations with measurements of the induced voltage, and monitored the course of electroporation. Our results show that cells in clusters can behave differently when exposed to electric field, depending on the parameters of the field. During the measurements of the voltage (long, low voltage pulses), cells in clusters behaved as one giant electrically connected cell. In contrast, during electroporation (short, high voltage pulses), cells behaved as electrically insulated and were electroporated individually. Different responses of cells in clusters to the electric field exposure could be attributed to the changes in the properties of gap junctions.
    01/2007: pages 639-642; , ISBN: 978-3-540-73043-9
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    ABSTRACT: The paper presents an approach that reduces several difficulties related to the determination of induced transmembrane voltage (ITV) on irregularly shaped cells. We first describe a method for constructing realistic models of irregularly shaped cells based on microscopic imaging. This provides a possibility to determine the ITV on the same cells on which an experiment is carried out, and can be of considerable importance in understanding and interpretation of the data. We also show how the finite-thickness, nonzero-conductivity membrane can be replaced by a boundary condition in which a specific surface conductivity is assigned to the interface between the cell interior (the cytoplasm) and the exterior. We verify the results obtained using this method by a comparison with the analytical solution for an isolated spherical cell and a tilted oblate spheroidal cell, obtaining a very good agreement in both cases. In addition, we compare the ITV computed for a model of two irregularly shaped CHO cells with the ITV measured on the same two cells by means of a potentiometric fluorescent dye, and also with the ITV computed for a simplified model of these two cells.
    Annals of Biomedical Engineering 01/2006; 34(4):642-652. · 3.23 Impact Factor

Publication Stats

770 Citations
73.62 Total Impact Points

Institutions

  • 2001–2013
    • University of Ljubljana
      • • Faculty of Electrical Engineering
      • • Department of Biomedical Engineering
      • • Laboratory of Biocybernetics
      Lubliano, Ljubljana, Slovenia
  • 2008
    • Tarbiat Modares University
      • Department of Medical Physics
      Tehrān, Ostan-e Tehran, Iran