Nanosecond electric pulses are equally effective in electrochemotherapy with cisplatin as microsecond pulses.

BACKGROUND: Nanosecond electric pulses showed promising results in electrochemotherapy, but the underlying mechanisms of action are still unexplored. The aim of this work was to correlate cellular cisplatin amount with cell survival of cells electroporated with nanosecond or standardly used 8 × 100 µs pulses and to investigate the effects of electric pulses on cisplatin structure. MATERIALS AND METHODS: Chinese hamster ovary CHO and mouse melanoma B16F1 cells were exposed to 1 × 200 ns pulse at 12.6 kV/cm or 25 × 400 ns pulses at 3.9 kV/cm, 10 Hz repetition rate or 8 × 100 µs pulses at 1.1 (CHO) or 0.9 (B16F1) kV/cm, 1 Hz repetition rate at three cisplatin concentrations. Cell survival was determined by the clonogenic assay, cellular platinum was measured by inductively coupled plasma mass spectrometry. Effects on the structure of cisplatin were investigated by nuclear magnetic resonance spectroscopy and high-resolution mass spectrometry. RESULTS: Nanosecond pulses equivalent to 8 × 100 µs pulses were established in vitro based on membrane permeabilization and cell survival. Equivalent nanosecond pulses were equally efficient in decreasing the cell survival and accumulating cisplatin intracellularly as 8 × 100 µs pulses after electrochemotherapy. The number of intracellular cisplatin molecules strongly correlates with cell survival for B16F1 cells, but less for CHO cells, implying the possible involvement of other mechanisms in electrochemotherapy. The high-voltage electric pulses did not alter the structure of cisplatin. CONCLUSIONS: Equivalent nanosecond pulses are equally effective in electrochemotherapy as standardly used 8 × 100 µs pulses.

Electroporation with nanosecond pulses and bleomycin or cisplatin results in efficient cell kill and low metal release from electrodes.

Nanosecond electric pulses have several potential advantages in electroporation-based procedures over the conventional micro- and millisecond pulses including low level of heating, reduced electrochemical reactions and reduced muscle contractions making them alluring for use in biomedicine and food industry. The aim of this study was to evaluate if nanosecond pulses can enhance the cytotoxicity of chemotherapeutics bleomycin and cisplatin in vitro and to quantify metal release from electrodes in comparison to 100 µs pulses commonly used in electrochemotherapy. The effects of nanosecond pulse parameters (voltage, pulse duration, number of pulses) on cell membrane permeabilization, resealing and on cell survival after electroporation only and after electrochemotherapy with bleomycin and cisplatin were evaluated on Chinese hamster ovary cells. Application of permeabilizing nanosecond pulses in combination with chemotherapeutics resulted in successful cell kill. Higher extracellular concentrations of bleomycin - but not cisplatin - were needed to achieve the same decrease in cell survival with nanosecond pulses as with eight 100 µs pulses, however, the tested bleomycin concentrations were still considerably lower compared to doses used in clinical practice. Decreasing the pulse duration from microseconds to nanoseconds and concomitantly increasing the amplitude to achieve the same biological effect resulted in reduced release of aluminum ions from electroporation cuvettes.

Effect of interphase and interpulse delay in high-frequency irreversible electroporation pulses on cell survival, membrane permeabilization and electrode material release.

To achieve high efficiency of electroporation and to minimize unwanted side effects, the electric field parameters must be optimized. Recently, it was suggested that biphasic high-frequency irreversible electroporation (H-FIRE) pulses reduce muscle contractions. However, it was also shown for sub-microsecond biphasic pulses that the opposite polarity phase of the pulse cancels the effect of the first phase if the interphase delay is short enough. We investigated the effect of interphase and interpulse delay (ranging from 0.5 to 10,000 µs) of 1 µs biphasic H-FIRE pulses on cell membrane permeabilization, on survival of four mammalian cell lines and determined metal release from aluminum, platinum and stainless steel electrodes. Biphasic H-FIRE pulses were compared to 8 × 100 µs monophasic pulses. We show that a longer interphase and interpulse delay results in lower cell survival, while the effects on cell membrane permeabilization are ambiguous. The cancellation effect was observed only for the survival of one cell line. Application of biphasic H-FIRE pulses results in lower metal release from electrodes but the interphase and interpulse delay does not have a large effect. The electrode material, however, importantly influences metal release - the lowest release was measured from platinum and the highest from aluminum electrodes.

Biosafety of biotechnologically important microalgae: intrinsic suicide switch implementation in cyanobacterium Synechocystis sp. PCC 6803.

In recent years, photosynthetic autotrophic cyanobacteria have attracted interest for biotechnological applications for sustainable production of valuable metabolites. Although biosafety issues can have a great impact on public acceptance of cyanobacterial biotechnology, biosafety of genetically modified cyanobacteria has remained largely unexplored. We set out to incorporate biocontainment systems in the model cyanobacteriumSynechocystissp. PCC 6803. Plasmid-encoded safeguards were constructed using the nonspecific nuclease NucA fromAnabaenacombined with different metal-ion inducible promoters. In this manner, conditional lethality was dependent on intracellular DNA degradation for regulated autokilling as well as preclusion of horizontal gene transfer. In cells carrying the suicide switch comprising thenucAgene fused to a variant of thecopMpromoter, efficient inducible autokilling was elicited. Parallel to nuclease-based safeguards, cyanobacterial toxin/antitoxin (TA) modules were examined in biosafety switches. Rewiring ofSynechocystisTA pairsssr1114/slr0664andslr6101/slr6100for conditional lethality using metal-ion responsive promoters resulted in reduced growth, rather than cell killing, suggesting cells could cope with elevated toxin levels. Overall, promoter properties and translation efficiency influenced the efficacy of biocontainment systems. Several metal-ion promoters were tested in the context of safeguards, and selected promoters, including anrsBvariant, were characterized by beta-galactosidase reporter assay.