Temperature Modulation of Electric Fields in Biological Matter

Pulsed electric fields (PEF) have become an important minimally invasive surgical technology for various applications including genetic engineering, electrochemotherapy and tissue ablation. This study explores the hypothesis that temperature dependent electrical parameters of tissue can be used to modulate the outcome of PEF protocols, providing a new means for controlling and optimizing this minimally invasive surgical procedure. This study investigates two different applications of cooling temperatures applied during PEF. The first case utilizes an electrode which simultaneously delivers pulsed electric fields and cooling temperatures. The subsequent results demonstrate that changes in electrical properties due to temperature produced by this configuration can substantially magnify and confine the electric fields in the cooled regions while almost eliminating electric fields in surrounding regions. This method can be used to increase precision in the PEF procedure, and eliminate muscle contractions and damage to adjacent tissues. The second configuration considered introduces a third probe that is not electrically active and only applies cooling boundary conditions. This second study demonstrates that in this probe configuration the temperature induced changes in electrical properties of tissue substantially reduce the electric fields in the cooled regions. This novel treatment can potentially be used to protect sensitive tissues from the effect of the PEF. Perhaps the most important conclusion of this investigation is that temperature is a powerful and accessible mechanism to modulate and control electric fields in biological tissues and can therefore be used to optimize and control PEF treatments

High-frequency irreversible electroporation (H-FIRE) for non-thermal ablation without muscle contraction

<p>Abstract</p> <p>Background</p> <p>Therapeutic irreversible electroporation (IRE) is an emerging technology for the non-thermal ablation of tumors. The technique involves delivering a series of unipolar electric pulses to permanently destabilize the plasma membrane of cancer cells through an increase in transmembrane potential, which leads to the development of a tissue lesion. Clinically, IRE requires the administration of paralytic agents to prevent muscle contractions during treatment that are associated with the delivery of electric pulses. This study shows that by applying high-frequency, bipolar bursts, muscle contractions can be eliminated during IRE without compromising the non-thermal mechanism of cell death.</p> <p>Methods</p> <p>A combination of analytical, numerical, and experimental techniques were performed to investigate high-frequency irreversible electroporation (H-FIRE). A theoretical model for determining transmembrane potential in response to arbitrary electric fields was used to identify optimal burst frequencies and amplitudes for <it>in vivo </it>treatments. A finite element model for predicting thermal damage based on the electric field distribution was used to design non-thermal protocols for <it>in vivo </it>experiments. H-FIRE was applied to the brain of rats, and muscle contractions were quantified via accelerometers placed at the cervicothoracic junction. MRI and histological evaluation was performed post-operatively to assess ablation.</p> <p>Results</p> <p>No visual or tactile evidence of muscle contraction was seen during H-FIRE at 250 kHz or 500 kHz, while all IRE protocols resulted in detectable muscle contractions at the cervicothoracic junction. H-FIRE produced ablative lesions in brain tissue that were characteristic in cellular morphology of non-thermal IRE treatments. Specifically, there was complete uniformity of tissue death within targeted areas, and a sharp transition zone was present between lesioned and normal brain.</p> <p>Conclusions</p> <p>H-FIRE is a feasible technique for non-thermal tissue ablation that eliminates muscle contractions seen in IRE treatments performed with unipolar electric pulses. Therefore, it has the potential to be performed clinically without the administration of paralytic agents.</p

Development of an In Vitro Compartmentalization Screen for High-Throughput Directed Evolution of [FeFe] Hydrogenases

BACKGROUND: [FeFe] hydrogenase enzymes catalyze the formation and dissociation of molecular hydrogen with the help of a complex prosthetic group composed of common elements. The development of energy conversion technologies based on these renewable catalysts has been hindered by their extreme oxygen sensitivity. Attempts to improve the enzymes by directed evolution have failed for want of a screening platform capable of throughputs high enough to adequately sample heavily mutated DNA libraries. In vitro compartmentalization (IVC) is a powerful method capable of screening for multiple-turnover enzymatic activity at very high throughputs. Recent advances have allowed [FeFe] hydrogenases to be expressed and activated in the cell-free protein synthesis reactions on which IVC is based; however, IVC is a demanding technique with which many enzymes have proven incompatible. METHODOLOGY/PRINCIPAL FINDINGS: Here we describe an extremely high-throughput IVC screen for oxygen-tolerant [FeFe] hydrogenases. We demonstrate that the [FeFe] hydrogenase CpI can be expressed and activated within emulsion droplets, and identify a fluorogenic substrate that links activity after oxygen exposure to the generation of a fluorescent signal. We present a screening protocol in which attachment of mutant genes and the proteins they encode to the surfaces of microbeads is followed by three separate emulsion steps for amplification, expression, and evaluation of hydrogenase mutants. We show that beads displaying active hydrogenase can be isolated by fluorescence-activated cell-sorting, and we use the method to enrich such beads from a mock library. CONCLUSIONS/SIGNIFICANCE: [FeFe] hydrogenases are the most complex enzymes to be produced by cell-free protein synthesis, and the most challenging targets to which IVC has yet been applied. The technique described here is an enabling step towards the development of biocatalysts for a biological hydrogen economy

The study of atmospheric ice-nucleating particles via microfluidically generated droplets

Ice-nucleating particles (INPs) play a significant role in the climate and hydrological cycle by triggering ice formation in supercooled clouds, thereby causing precipitation and affecting cloud lifetimes and their radiative properties. However, despite their importance, INP often comprise only 1 in 10³–10⁶ ambient particles, making it difficult to ascertain and predict their type, source, and concentration. The typical techniques for quantifying INP concentrations tend to be highly labour-intensive, suffer from poor time resolution, or are limited in sensitivity to low concentrations. Here, we present the application of microfluidic devices to the study of atmospheric INPs via the simple and rapid production of monodisperse droplets and their subsequent freezing on a cold stage. This device offers the potential for the testing of INP concentrations in aqueous samples with high sensitivity and high counting statistics. Various INPs were tested for validation of the platform, including mineral dust and biological species, with results compared to literature values. We also describe a methodology for sampling atmospheric aerosol in a manner that minimises sampling biases and which is compatible with the microfluidic device. We present results for INP concentrations in air sampled during two field campaigns: (1) from a rural location in the UK and (2) during the UK’s annual Bonfire Night festival. These initial results will provide a route for deployment of the microfluidic platform for the study and quantification of INPs in upcoming field campaigns around the globe, while providing a benchmark for future lab-on-a-chip-based INP studies

Irreversible Electroporation of Human Primary Uveal Melanoma in Enucleated Eyes

Uveal melanoma (UM) is the most common primary intraocular tumor in adults and is characterized by high rates of metastatic disease. Although brachytherapy is the most common globe-sparing treatment option for small- and medium-sized tumors, the treatment is associated with severe adverse reactions and does not lead to increased survival rates as compared to enucleation. The use of irreversible electroporation (IRE) for tumor ablation has potential advantages in the treatment of tumors in complex organs such as the eye. Following previous theoretical work, herein we evaluate the use of IRE for uveal tumor ablation in human ex vivo eye model. Enucleated eyes of patients with uveal melanoma were treated with short electric pulses (50–100 µs, 1000–2000 V/cm) using a customized electrode design. Tumor bioimpedance was measured before and after treatment and was followed by histopathological evaluation. We found that IRE caused tumor ablation characterized by cell membrane disruption while sparing the non-cellular sclera. Membrane disruption and loss of cellular capacitance were also associated with significant reduction in total tumor impedance and loss of impedance frequency dependence. The effect was more pronounced near the pulsing electrodes and was dependent on time from treatment to fixation. Future studies should further evaluate the potential of IRE as an alternative method of uveal melanoma treatment

Cryosurgery with Pulsed Electric Fields

This study explores the hypothesis that combining the minimally invasive surgical techniques of cryosurgery and pulsed electric fields will eliminate some of the major disadvantages of these techniques while retaining their advantages. Cryosurgery, tissue ablation by freezing, is a well-established minimally invasive surgical technique. One disadvantage of cryosurgery concerns the mechanism of cell death; cells at high subzero temperature on the outer rim of the frozen lesion can survive. Pulsed electric fields (PEF) are another minimally invasive surgical technique in which high strength and very rapid electric pulses are delivered across cells to permeabilize the cell membrane for applications such as gene delivery, electrochemotherapy and irreversible electroporation. The very short time scale of the electric pulses is disadvantageous because it does not facilitate real time control over the procedure. We hypothesize that applying the electric pulses during the cryosurgical procedure in such a way that the electric field vector is parallel to the heat flux vector will have the effect of confining the electric fields to the frozen/cold region of tissue, thereby ablating the cells that survive freezing while facilitating controlled use of the PEF in the cold confined region. A finite element analysis of the electric field and heat conduction equations during simultaneous tissue treatment with cryosurgery and PEF (cryosurgery/PEF) was used to study the effect of tissue freezing on electric fields. The study yielded motivating results. Because of decreased electrical conductivity in the frozen/cooled tissue, it experienced temperature induced magnified electric fields in comparison to PEF delivered to the unfrozen tissue control. This suggests that freezing/cooling confines and magnifies the electric fields to those regions; a targeting capability unattainable in traditional PEF. This analysis shows how temperature induced magnified and focused PEFs could be used to ablate cells in the high subzero freezing region of a cryosurgical lesion

Asymmetric Waveforms Decrease Lethal Thresholds in High Frequency Irreversible Electroporation Therapies

Irreversible electroporation (IRE) is a promising non-thermal treatment for inoperable tumors which uses short (50–100 μs) high voltage monopolar pulses to disrupt the membranes of cells within a well-defined volume. Challenges with IRE include complex treatment planning and the induction of intense muscle contractions. High frequency IRE (H-FIRE) uses bursts of ultrashort (0.25–5 μs) alternating polarity pulses to produce more predictable ablations and alleviate muscle contractions associated with IRE. However, H-FIRE generally ablates smaller volumes of tissue than IRE. This study shows that asymmetric H-FIRE waveforms can be used to create ablation volumes equivalent to standard IRE treatments. Lethal thresholds (LT) of 505 V/cm and 1316 V/cm were found for brain cancer cells when 100 μs IRE and 2 μs symmetric H-FIRE waveforms were used. In contrast, LT as low as 536 V/cm were found for 2 μs asymmetric H-FIRE waveforms. Reversible electroporation thresholds were 54% lower than LTs for symmetric waveforms and 33% lower for asymmetric waveforms indicating that waveform symmetry can be used to tune the relative sizes of reversible and irreversible ablation zones. Numerical simulations predicted that asymmetric H-FIRE waveforms are capable of producing ablation volumes which were 5.8–6.3x larger than symmetric H-FIRE waveforms indicating that in vivo investigation of asymmetric waveforms is warranted