Pulsed Electric Fields Promote Liposome Buddings.

Background: Liposomes have been a useful tool to analyze membrane behavior. Various studies have attempted to induce biological activities, for example, buddings, divisions, and endocytosis, on liposomes, focusing on lipid rafts that move along electric fields. Materials and Methods: Liposomes consisting of soybean lecithin, phosphatidylcholine, and cholesterol were prepared, with inner and outer liquid conductivities of 0.595 and 1.564 S/m, respectively. Results: We tried to induce buddings by pulsed electric fields (PEFs) on liposomes. Results demonstrated that 1.248 kV/cm, 400 µs PEF promoted postpulse liposome buddings, which were preceded by a membrane relaxation. Although a transient thick area (a lipid raft-like area) on the membrane just after PEF application preceded buddings, it was not the sufficient factor for buddings. Conclusion: We established a brief model as follows: 1.248 kV/cm, 400 µs PEF induced the lipid membrane relaxation without electroporation to trigger buddings. The current results could be a new frontier in bioelectrics.

Author Correction: 1.2 MV/cm pulsed electric fields promote transthyretin aggregate degradation.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

1.2 MV/cm pulsed electric fields promote transthyretin aggregate degradation.

Numerous theoretical studies have been conducted on the effects of high-voltage electric fields on proteins, but few have produced experimental evidence. To acquire experimental data for the amyloid disassemble theory, we exposed transthyretin aggregates to 1,000 ns 1.26 MV/cm pulsed electric fields (PEFs) to promote transthyretin degradation. The process produced no changes in pH, and the resulting temperature increases were < 1 °C. We conclude that the physical effects of PEFs, rather than thermal or chemical effects, facilitate aggregate degradation.

Intense Pulsed Electric Fields Denature Urease Protein.

Background: This article describes the effects of nanosecond pulsed electric fields (nsPEFs) on the structure and enzyme activity of three types of proteins. Materials and Methods: Intense (up to 300 kV/cm) 5-ns-long electrical pulses were applied for 500 times at 3 Hz to solutions of lysozyme, albumin, and urease. We analyzed covalent bonds (peptide bonds and disulfide bonds) of lysozyme and albumin, and also the tertiary and quaternary structures of urease as well as urease activity. Results: The results indicated deformation of both the quaternary and tertiary structures of urease upon exposure to an electric field with an amplitude of 250 kV/cm or higher, whereas no structural changes were observed in lysozyme or albumin, even at 300 kV/cm. The enzyme activity of urease also decreased at field strengths of 250 kV/cm or higher. Conclusion: Our experiments demonstrated that intense nsPEFs physically affected the conformation and function of some types of proteins. Such intense electric fields often occur in cell membranes when exposed to a moderate pulsed electric field.

Variations of Intracellular Ca2+ Mobilization Initiated by Nanosecond and Microsecond Electrical Pulses in HeLa Cells.

GOAL: Herein, the variations in transient Ca2+ mobilizations in HeLa cells exposed to a single, non-thermal pulsed electric field (PEF) are described. METHODS: Three PEF waveforms categorized by pulse duration and intensity were used to deduce the kinetics involved in Ca2+ mobilization. A fast microscopic fluorescent imaging system and a fluorescent molecular probe were used to observe transient intracellular Ca2+ mobilization after pulse exposure. The sources and pathways in the transient Ca2+ mobilizations were investigated using an inhibitor of inositol-1,4,5-trisphosphate receptor (IP3R) on the endoplasmic reticulum (ER) along with a Ca2+-free buffer. RESULTS: When exposed to the 10-µs-long PEF, the Ca2+ concentration increased mainly at the cathodic region near the membrane. However, Ca2+ concentration increased at both anodic and cathodic regions when Na+ concentration in the buffer was reduced. Ca2+ concentration increased only in the presence of extracellular Ca2+. CONCLUSION: These results suggest that the 10-µs PEF takes a large amount of extracellular Na+ into the cell through the electropermeabilized plasma membrane, especially at the anodic side, resulting in the suppression of the Ca2+ influx. On the contrary, the 20-ns-long PEF increased Ca2+ concentration in the surrounding region of the nucleus only in the presence of extracellular Ca2+. The PEF exposure with inhibition of the IP3R indicates that increased Ca2+ ions are released from the ER via the activated IP3R. SIGNIFICANCE: These mechanisms could induce specific cell responses, such as Ca2+ oscillations, Ca2+ waves, and Ca2+ puffs.

Spatio-temporal dynamics of calcium electrotransfer during cell membrane permeabilization.

Pulsed electric fields (PEFs) are applied as physical stimuli for DNA/drug delivery, cancer therapy, gene transformation, and microorganism eradication. Meanwhile, calcium electrotransfer offers an interesting approach to treat cancer, as it induces cell death easier in malignant cells than in normal cells. Here, we study the spatial and temporal cellular responses to 10 µs duration PEFs; by observing real-time, the uptake of extracellular calcium through the cell membrane. The experimental setup consisted of an inverted fluorescence microscope equipped with a color high-speed framing camera and a specifically designed miniaturized pulsed power system. The setup allowed us to accurately observe the permeabilization of HeLa S3 cells during application of various levels of PEFs ranging from 0.27 to 1.80 kV/cm. The low electric field experiments confirmed the threshold value of transmembrane potential (TMP). The high electric field observations enabled us to retrieve the entire spatial variation of the permeabilization angle (θ). The temporal observations proved that after a minimal permeabilization of the cell membrane, the ionic diffusion was the prevailing mechanism of the delivery to the cell cytoplasm. The observations suggest 0.45 kV/cm and 100 pulses at 1 kHz as an optimal condition to achieve full calcium concentration in the cell cytoplasm. The results offer precise levels of electric fields to control release of the extracellular calcium to the cell cytoplasm for inducing minimally invasive cancer calcium electroporation, an interesting affordable method to treat cancer patients with minimum side effects.

Activation of the JNK pathway by nanosecond pulsed electric fields.

Nanosecond pulsed electric fields (nsPEFs) are increasingly recognized as a novel and unique tool in various life science fields, including electroporation and cancer therapy, although their mode of action in cells remains largely unclear. Here, we show that nsPEFs induce strong and transient activation of a signaling pathway involving c-Jun N-terminal kinase (JNK). Application of nsPEFs to HeLa S3 cells rapidly induced phosphorylation of JNK1 and MKK4, which is located immediately upstream of JNK in this signaling pathway. nsPEF application also elicited increased phosphorylation of c-Jun protein and dramatically elevated c-jun and c-fos mRNA levels. nsPEF-inducible events downstream of JNK were markedly suppressed by the JNK inhibitor SP600125, which confirmed JNK-dependency of these events in this pathway. Our results provide novel mechanistic insights into the mode of nsPEF action in human cells.