High-Voltage Electrical Pulses in Oncology: Irreversible Electroporation, Electrochemotherapy, Gene Electrotransfer, Electrofusion, and Electroimmunotherapy.

This review summarizes the use of high-voltage electrical pulses (HVEPs) in clinical oncology to treat solid tumors with irreversible electroporation (IRE) and electrochemotherapy (ECT). HVEPs increase the membrane permeability of cells, a phenomenon known as electroporation. Unlike alternative ablative therapies, electroporation does not affect the structural integrity of surrounding tissue, thereby enabling tumors in the vicinity of vital structures to be treated. IRE uses HVEPs to cause cell death by inducing membrane disruption, and it is primarily used as a radical ablative therapy in the treatment of soft-tissue tumors in the liver, kidney, prostate, and pancreas. ECT uses HVEPs to transiently increase membrane permeability, enhancing cellular cytotoxic drug uptake in tumors. IRE and ECT show immunogenic effects that could be augmented when combined with immunomodulatory drugs, a combination therapy the authors term electroimmunotherapy. Additional electroporation-based technologies that may reach clinical importance, such as gene electrotransfer, electrofusion, and electroimmunotherapy, are concisely reviewed. HVEPs represent a substantial advancement in cancer research, and continued improvement and implementation of these presented technologies will require close collaboration between engineers, interventional radiologists, medical oncologists, and immuno-oncologists.

Ultra-thin and ultra-porous nanofiber networks as a basement-membrane mimic.

Current basement membrane (BM) mimics used for modeling endothelial and epithelial barriers in vitro do not faithfully recapitulate key in vivo physiological properties such as BM thickness, porosity, stiffness, and fibrous composition. Here, we use networks of precisely arranged nanofibers to form ultra-thin (∼3 µm thick) and ultra-porous (∼90%) BM mimics for blood-brain barrier modeling. We show that these nanofiber networks enable close contact between endothelial monolayers and pericytes across the membrane, which are known to regulate barrier tightness. Cytoskeletal staining and transendothelial electrical resistance (TEER) measurements reveal barrier formation on nanofiber membranes integrated within microfluidic devices and transwell inserts. Further, significantly higher TEER values indicate a biological benefit for co-cultures formed on the ultra-thin nanofiber membranes. Our BM mimic overcomes critical technological challenges in forming co-cultures that are in proximity and facilitate cell-cell contact, while still being constrained to their respective sides. We anticipate that our nanofiber networks will find applications in drug discovery, cell migration, and barrier dysfunction studies.

Engineering high post-electroporation viabilities and transfection efficiencies for elongated cells on suspended nanofiber networks.

The impact of cell shape on cell membrane permeabilization by pulsed electric fields is not fully understood. For certain applications, cell survival and recovery post-treatment is either desirable, as in gene transfection, electrofusion, and electrochemotherapy, or is undesirable, as in tumor and cardiac ablations. Understanding of how morphology affects cell viability post-electroporation may lead to improved electroporation methods. In this study, we use precisely aligned nanofiber networks within a microfluidic device to reproducibly generate elongated cells with controlled orientations to an applied electric field. We show that cell viability is significantly dependent on cell orientation, elongation, and spread. Further, these trends are dependent on the external buffer conductivity. Additionally, we see that cell survival for elongated cells is still supported by the standard pore model of electroporation. Lastly, we see that manipulating the cell orientation and shape can be leveraged for increased transfection efficiencies when compared to spherical cells. An improved understanding of cell shape and pulsation buffer conductivity may lead to improved methods for enhancing cell viability post-electroporation by engineering the cell morphology, cytoskeleton, and electroporation buffer conditions.

A Multiplexed Microfluidic Device to Measure Blood-Brain Barrier Disruption by Pulsed Electric Fields.

Local disruption of the blood-brain barrier (BBB) by pulsed electric fields shows significant potential for treating neurological conditions. Microfluidic BBB models can provide low-cost, controlled experiments with human cells and test a range of parameters for preclinical studies. We developed a multiplexed BBB device that can test a three-fold range of electric field magnitudes. A tapered channel creates a linear gradient of the electric field within the device, and an asymmetric branching channel enables an on-chip control. We monitored BBB permeability in real-time using the diffusion of a fluorescent marker across an endothelial monolayer to determine BBB disruption after high-frequency bipolar electrical pulses (HFIRE). We show that HFIRE pulses can transiently open the BBB. Unexpectedly, electrofusion of cells resulted in decreased permeability for some conditions. Our multiplexed device can efficiently probe treatment variables for efficient preclinical testing of optimal parameters for reversible BBB disruption.Clinical Relevance-This in vitro model of the BBB can inform preclinical studies by investigating a range of electroporation parameters for BBB disruption.

Frequency-specific, valveless flow control in insect-mimetic microfluidic devices.

Inexpensive, portable lab-on-a-chip devices would revolutionize fields like environmental monitoring and global health, but current microfluidic chips are tethered to extensive off-chip hardware. Insects, however, are self-contained and expertly manipulate fluids at the microscale using largely unexplored methods. We fabricated a series of microfluidic devices that mimic key features of insect respiratory kinematics observed by synchrotron-radiation imaging, including the collapse of portions of multiple respiratory tracts in response to a single fluctuating pressure signal. In one single-channel device, the flow rate and direction could be controlled by the actuation frequency alone, without the use of internal valves. Additionally, we fabricated multichannel chips whose individual channels responded selectively (on with a variable, frequency-dependent flow rate, or off) to a single, global actuation frequency. Our results demonstrate that insect-mimetic designs have the potential to drastically reduce the actuation overhead for microfluidic chips, and that insect respiratory systems may share features with impedance-mismatch pumps.

Single Cell Forces after Electroporation.

Exogenous high-voltage pulses increase cell membrane permeability through a phenomenon known as electroporation. This process may also disrupt the cell cytoskeleton causing changes in cell contractility; however, the contractile signature of cell force after electroporation remains unknown. Here, single-cell forces post-electroporation are measured using suspended extracellular matrix-mimicking nanofibers that act as force sensors. Ten, 100 µs pulses are delivered at three voltage magnitudes (500, 1000, and 1500 V) and two directions (parallel and perpendicular to cell orientation), exposing glioblastoma cells to electric fields between 441 V cm-1 and 1366 V cm-1. Cytoskeletal-driven force loss and recovery post-electroporation involves three distinct stages. Low electric field magnitudes do not cause disruption, but higher fields nearly eliminate contractility 2-10 min post-electroporation as cells round following calcium-mediated retraction (stage 1). Following rounding, a majority of analyzed cells enter an unusual and unexpected biphasic stage (stage 2) characterized by increased contractility tens of minutes post-electroporation, followed by force relaxation. The biphasic stage is concurrent with actin disruption-driven blebbing. Finally, cells elongate and regain their pre-electroporation morphology and contractility in 1-3 h (stage 3). With increasing voltages applied perpendicular to cell orientation, we observe a significant drop in cell viability. Experiments with multiple healthy and cancerous cell lines demonstrate that contractile force is a more dynamic and sensitive metric than cell shape to electroporation. A mechanobiological understanding of cell contractility post-electroporation will deepen our understanding of the mechanisms that drive recovery and may have implications for molecular medicine, genetic engineering, and cellular biophysics.

Generation of Tumor-activated T cells Using Electroporation.

Expansion of cytotoxic T lymphocytes (CTLs) is a crucial step in almost all cancer immunotherapeutic methods. Current techniques for expansion of tumor-reactive CTLs present major limitations. This study introduces a novel method to effectively produce and expand tumor-activated CTLs using high-voltage pulsed electric fields. We hypothesize that utilizing high-voltage pulsed electric fields may be an ideal method to activate and expand CTLs due to their non-thermal celldeath mechanism. Tumor cells were subjected to high-frequency irreversible electroporation (HFIRE) with various electric field magnitudes (1250, 2500 V/cm) and pulse widths (1, 5, and 10 µs), or irreversible electroporation (IRE) at 1250 V/cm. The treated tumor cells were subsequently cocultured with CD4+ and CD8+ T cells along with antigen-presenting cells. We show that tumor-activated CTLs can be produced and expanded when exposed to treated tumor cells. Our results suggest that CTLs are more effectively expanded when pulsed with HFIRE conditions that induce significant cell death (longer pulse widths and higher voltages). Activated CD8+ T cells demonstrate cytotoxicity to untreated tumor cells suggesting effector function of the activated CTLs. The activated CTLs produced with our technique could be used for clinical applications with the goal of targeting and eliminating the tumor.

Cytoskeletal Disruption after Electroporation and Its Significance to Pulsed Electric Field Therapies.

Pulsed electric fields (PEFs) have become clinically important through the success of Irreversible Electroporation (IRE), Electrochemotherapy (ECT), and nanosecond PEFs (nsPEFs) for the treatment of tumors. PEFs increase the permeability of cell membranes, a phenomenon known as electroporation. In addition to well-known membrane effects, PEFs can cause profound cytoskeletal disruption. In this review, we summarize the current understanding of cytoskeletal disruption after PEFs. Compiling available studies, we describe PEF-induced cytoskeletal disruption and possible mechanisms of disruption. Additionally, we consider how cytoskeletal alterations contribute to cell-cell and cell-substrate disruption. We conclude with a discussion of cytoskeletal disruption-induced anti-vascular effects of PEFs and consider how a better understanding of cytoskeletal disruption after PEFs may lead to more effective therapies.

Seasonal changes in morphology govern wettability of Katsura leaves.

Deciduous broad-leaf trees survive and prepare for winter by shedding their leaves in fall. During the fall season, a change in a leaf's wettability and its impact on the leaf-fall are not well understood. In this study, we measure the surface morphology and wettability of Katsura leaves from the summer to winter, and reveal how leaf structural changes lead to wettability changes. The averaged contact angle of leaves decreases from 147° to 124° while the contact-angle hysteresis significantly increases by about 35°, which are attributed to dehydration and erosion of nano-wax. Due to such wettability changes, fall brown leaves support approximately 17 times greater water volume than summer leaves.

Correction: Seasonal changes in morphology govern wettability of Katsura leaves.

[This corrects the article DOI: 10.1371/journal.pone.0202900.].