Numerical study of the electroporation pulse shape effect on molecular uptake of biological cells.

BACKGROUND: In order to reduce the side-effects of chemotherapy, combined chemotherapy-electroporation (electrochemotherapy) has been suggested. Electroporation, application of appropriate electric pulses to biological cells, can significantly enhance molecular uptake of cells due to formation of transient pores in the cell membrane. It was experimentally demonstrated that the efficiency of electroporation is under the control of electric pulse parameters. However, the theoretical basis for these experimental results is not fully explained. In order to predict the outcome of experiments and optimize the efficiency of electroporation before each treatment, we developed a model to investigate the effect of pulse shape on efficiency of electroporation. RESULTS: Our model is based on a developed chemical-kinetics scheme and trapezium barrier model, while self-consistency was taken into account. This model is further supplemented with a molecular transport model to acquire the molecular uptake of cells. The investigated pulse shapes in this study were unipolar rectangular pulses with different rise and fall times, triangular, sinusoidal and bipolar rectangular pulses and also sinusoidal modulated unipolar pulses with different percentages of modulation. The obtained results from our modelling and simulations are in good agreement with previously published experimental results. CONCLUSIONS: We therefore conclude that this model can be used to predict the effects of arbitrarily shaped electroporation pulses on cell membrane conductivity and molecular transport across the cell membrane.

Variability of the minimal transmembrane voltage resulting in detectable membrane electroporation.

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.

Comparison between direct and reverse electroporation of cells in situ: a simulation study.

The discovery of the human genome has unveiled new fields of genomics, transcriptomics, and proteomics, which has produced paradigm shifts on how to study disease mechanisms, wherein a current central focus is the understanding of how gene signatures and gene networks interact within cells. These gene function studies require manipulating genes either through activation or inhibition, which can be achieved by temporarily permeabilizing the cell membrane through transfection to delivercDNAorRNAi. An efficient transfection technique is electroporation, which applies an optimized electric pulse to permeabilize the cells of interest. When the molecules are applied on top of seeded cells, it is called "direct" transfection and when the nucleic acids are printed on the substrate and the cells are seeded on top of them, it is termed "reverse" transfection. Direct transfection has been successfully applied in previous studies, whereas reverse transfection has recently gained more attention in the context of high-throughput experiments. Despite the emerging importance, studies comparing the efficiency of the two methods are lacking. In this study, a model for electroporation of cells in situ is developed to address this deficiency. The results indicate that reverse transfection is less efficient than direct transfection. However, the model also predicts that by increasing the concentration of deliverable molecules by a factor of 2 or increasing the applied voltage by 20%, reverse transfection can be approximately as efficient as direct transfection.

Development of a synthetic gene network to modulate gene expression by mechanical forces.

The majority of (mammalian) cells in our body are sensitive to mechanical forces, but little work has been done to develop assays to monitor mechanosensor activity. Furthermore, it is currently impossible to use mechanosensor activity to drive gene expression. To address these needs, we developed the first mammalian mechanosensitive synthetic gene network to monitor endothelial cell shear stress levels and directly modulate expression of an atheroprotective transcription factor by shear stress. The technique is highly modular, easily scalable and allows graded control of gene expression by mechanical stimuli in hard-to-transfect mammalian cells. We call this new approach mechanosyngenetics. To insert the gene network into a high proportion of cells, a hybrid transfection procedure was developed that involves electroporation, plasmids replication in mammalian cells, mammalian antibiotic selection, a second electroporation and gene network activation. This procedure takes 1 week and yielded over 60% of cells with a functional gene network. To test gene network functionality, we developed a flow setup that exposes cells to linearly increasing shear stress along the length of the flow channel floor. Activation of the gene network varied logarithmically as a function of shear stress magnitude.

Influence of shear stress magnitude and direction on atherosclerotic plaque composition.

The precise flow characteristics that promote different atherosclerotic plaque types remain unclear. We previously developed a blood flow-modifying cuff for ApoE-/- mice that induces the development of advanced plaques with vulnerable and stable features upstream and downstream of the cuff, respectively. Herein, we sought to test the hypothesis that changes in flow magnitude promote formation of the upstream (vulnerable) plaque, whereas altered flow direction is important for development of the downstream (stable) plaque. We instrumented ApoE-/- mice (n = 7) with a cuff around the left carotid artery and imaged them with micro-CT (39.6 µm resolution) eight to nine weeks after cuff placement. Computational fluid dynamics was then performed to compute six metrics that describe different aspects of atherogenic flow in terms of wall shear stress magnitude and/or direction. In a subset of four imaged animals, we performed histology to confirm the presence of advanced plaques and measure plaque length in each segment. Relative to the control artery, the region upstream of the cuff exhibited changes in shear stress magnitude only (p < 0.05), whereas the region downstream of the cuff exhibited changes in shear stress magnitude and direction (p < 0.05). These data suggest that shear stress magnitude contributes to the formation of advanced plaques with a vulnerable phenotype, whereas variations in both magnitude and direction promote the formation of plaques with stable features.

Enhancement of non-invasive trans-membrane drug delivery using ultrasound and microbubbles during physiologically relevant flow.

Sonoporation has been associated with drug delivery across cell membranes and into target cells, yet several limitations have prohibited further advancement of this technology. Higher delivery rates were associated with increased cellular death, thus implying a safety-efficacy trade-off. Meanwhile, there has been no reported study of safe in vitro sonoporation in a physiologically relevant flow environment. The objective of our study was not only to evaluate sonoporation under physiologically relevant flow conditions, such as fluid velocity, shear stress and temperature, but also to design ultrasound parameters that exploit the presence of flow to maximize sonoporation efficacy while minimizing or avoiding cellular damage. Human umbilical vein endothelial cells (EA.hy926) were seeded in flow chambers as a monolayer to mimic the endothelium. A peristaltic pump maintained a constant fluid velocity of 12.5 cm/s. A focused 0.5 MHz transducer was used to sonicate the cells, while an inserted focused 7.5 MHz passive cavitation detector monitored microbubble-seeded cavitation emissions. Under these conditions, propidium iodide, which is normally impermeable to the cell membrane, was traced to determine whether it could enter cells after sonication. Meanwhile, calcein-AM was used as a cell viability marker. A range of focused ultrasound parameters was explored, with several unique bioeffects observed: cell detachment, preservation of cell viability with no membrane penetration, cell death and preservation of cell viability with sonoporation. The parameters were then modified further to produce safe sonoporation with minimal cell death. To increase the number of favourable cavitation events, we lowered the ultrasound exposure pressure to 40 kPapk-neg and increased the number of cavitation nuclei by 50 times to produce a trans-membrane delivery rate of 62.6% ± 4.3% with a cell viability of 95% ± 4.2%. Furthermore, acoustic cavitation analysis showed that the low pressure sonication produced stable and non-inertial cavitation throughout the pulse sequence. To our knowledge, this is the first study to demonstrate a high drug delivery rate coupled with high cell viability in a physiologically relevant in vitro flow system.

Systems biology of the functional and dysfunctional endothelium.

This review provides an overview of the effect of blood flow on endothelial cell (EC) signalling pathways, applying microarray technologies to cultured cells, and in vivo studies of normal and atherosclerotic animals. It is found that in cultured ECs, 5-10% of genes are up- or down-regulated in response to fluid flow, whereas only 3-6% of genes are regulated by varying levels of fluid flow. Of all genes, 90% are regulated by the steady part of fluid flow and 10% by pulsatile components. The associated gene profiles show high variability from experiment to experiment depending on experimental conditions, and importantly, the bioinformatical methods used to analyse the data. Despite this high variability, the current data sets can be summarized with the concept of endothelial priming. In this concept, fluid flows confer protection by an up-regulation of anti-atherogenic, anti-thrombotic, and anti-inflammatory gene signatures. Consequently, predilection sites of atherosclerosis, which are associated with low-shear stress, confer low protection for atherosclerosis and are, therefore, more sensitive to high cholesterol levels. Recent studies in intact non-atherosclerotic animals confirmed these in vitro studies, and suggest that a spatial component might be present. Despite the large variability, a few signalling pathways were consistently present in the majority of studies. These were the MAPK, the nuclear factor-κB, and the endothelial nitric oxide synthase-NO pathways.

Lucifer Yellow uptake by CHO cells exposed to magnetic and electric pulses.

BACKGROUND: The cell membrane acts as a barrier that hinders free entrance of most hydrophilic molecules into the cell. Due to numerous applications in medicine, biology and biotechnology, the introduction of impermeant molecules into biological cells has drawn considerable attention in the past years. One of the most famous methods in this field is electroporation, in which electric pulses with high intensity and short duration are applied to the cells. The aim of our study was to investigate the effect of time-varying magnetic field with different parameters on transmembrane molecular transport. MATERIALS AND METHODS: 'Moreover, a comparison was made between the uptake results due to magnetic pulse exposure and electroporation mediated uptake.' at the end of Background part. The Chinese hamster ovary (CHO) cells were exposed to magnetic pulses of 2.2 T peak strength and 250 µs duration delivered by Magstim stimulator and double 70 mm coil. Three different frequencies of 0.25, 1 and 10 Hz pulses with 112, 56 and 28 number of pulses were applied (altogether nine experimental groups) and Lucifer Yellow uptake was measured in each group. Moreover, maximum uptake of Lucifer Yellow obtained by magnetic pulses was compared to the measured uptake due to electroporation with typical parameters of 8 pulses of 100 µs, repetition frequency of 1 Hz and electric field intensities of 200 to 600 V/cm. RESULTS AND CONCLUSIONS: Our results show that time-varying magnetic field exposure increases transmembrane molecular transport and this uptake is greater for lower frequencies and larger number of pulses. Besides, the comparison shows that electroporation is more effective than pulsed magnetic field, but the observed uptake enhancement due to magnetic exposure is still considerable.