Ultrashort Electric Pulse Effects in Biology and Medicine

The effects of intense electrical pulses on cell membranes have been studied extensively since the second half of the twentieth century, and have found multiple applications, ranging from bacterial decontamination to medical therapies. The first studies on this effect, named electroporation, rarely extended beyond microsecond pulse effects on cells. In the past two decades, however, the use of nanosecond and even shorter pulses gained interest, since electrical circuit models of biological cells indicated that not only the plasma membrane, but also the subcellular structures of mammalian cells could be affected by such extremely short pulses. The first experimental study published in 2001 confirmed this hypothesis. It was followed by a large number of publications which showed that such ultrashort, high electric field, but low electrical energy pulses, affect cell functions, such as programmed cell death, and a lower intensity, calcium mobilization from intracellular structures. This chapter, after a short introduction to electroporation, provides an overview of the progress of basic studies on nano- and picosecond pulse effects on cells, tissues, and organisms over the past two decades.

Receptors, channels, glycoprotein, and transport proteins, among other integral proteins, load the plasma membrane lipid bilayer, which receives reinforcement from cytoskeletal filaments as the boundaries of cellular life. Early studies suggested that usEPs did not induce plasma membrane permeabilization until relatively high amplitude conditions. As will be discussed here and elsewhere, this was because the usEP-induced plasma membrane pores were so small that they did not allow entry of propidium iodide, a commonly used marker for permeability, or calcein exit from the cell. The plasma membrane contained nanoelectropores or nanopores, so-called because they were ~1 nm in diameter. These lipid nanopore structures exhibited complex conductances similar to classic pores, which are common in protein structures. Another finding presented some confounding information regarding plasma membrane phosphatidylserine (PS) externalization, commonly used as a marker for apoptosis. usEPs “pulled” PS through these nanopores. Now PS externalization was an ambiguous marker for apoptosis. Although plasma membrane lipids were clear usEP targets, plasma membrane channels were also targets for usEPs. As will be discussed, these effects could be due indirectly to nanopore formation or possibly directly on the channels themselves, although more evidence for direct effects is necessary. In any event, patch-clamp techniques provided new, exciting, and valuable information about usEP effects on plasma membranes. As might be expected, cells have mechanisms that confront nanopore formation and other possible usEP-induced injuries, which will be presented. Finally, in relatively uncharted territory, usEPs were also shown to affect redox systems in plasma membranes as electron carriers. Although usEPs are unique for their effects on intracellular structures and functions, their impact on the plasma membrane has provided a wealth of information about how they can be used as tools in biology and medicine.

Externally applied nanosecond electric pulses are useful to trigger and tailor bioeffects in cells and tissues. However, the parameter space is large given the different types of cells, and the wide range of potential electrical parameters (such as pulse durations and waveforms, field intensities, and number of pulses) that are available for use. To maximize benefits, tailor a desired response, and devise an efficient system, it becomes necessary to first understand and quantify the biological behavior driven by the electrical input. Model development based on the inherent biophysical processes is an elegant and cost-effective option to help advance this technology. Given the merits and need for modeling then, this chapter focuses on the various schemes for analysis and simulations of the electrically driven bioeffects. Schemes ranging from molecular level descriptions to an averaged continuum analyses are presented and discussed in this chapter. Relevant examples and illustrative result are also given in this context of cellular bioelectrics.

The utility of electric pulsing for attaining and driving a range of bio-effects have already been discussed. This chapter focuses and probes the role of pulse shape (e.g., monopolar-vs.-bipolar), multiple electrode scenarios, and serial-versus-simultaneous pulsing. A possible analysis based on a three-dimensional time-dependent continuum model is discussed. Our results indicate that monopolar pulsing always leads to higher and stronger cellular uptake. This prediction is in agreement with experimental reports and observations. It is also demonstrated that multipronged electrode configurations influence and increase the degree of cellular uptake.

The endoplasmic reticulum (ER) is a tubular membranous labyrinth throughout the cytoplasm that is continuous with the nuclear membrane, exhibits contact sites with mitochondria and the plasma membrane, and displays domains for specific functions. It is smooth or rough with ribosomes for protein synthesis and is the home for folding proteins in their proper tertiary structures. The ER includes cellular stress response sensors that can lead to unfolded protein response (UPR), leading to regulated cell death (RCD). The ER is also a primary storage site for Ca²⁺ that can be used for scores of Ca²⁺ mediated signal transduction responses such as neurotransmitter release, muscle contraction, or contributors to RCD, among others. Since usEPs were unique for intracellular electric field effects, usEP-induced Ca²⁺ release was an excellent way to define these intracellular effects, albeit not without caveats. Because usEPs also induced plasma membrane permeabilization for Ca²⁺ influx, which occurred at lower charging conditions than ER-induced nanopore formation and Ca²⁺ release; because some cells expressed voltage-gated Ca²⁺ channels (VGCC), which could be directly activated or activated due to usEP-induced plasma membrane depolarization; because capacitative Ca²⁺ increases could increase intracellular Ca²⁺; and Ca²⁺ increases from Ca²⁺-induced Ca²⁺ release, significant care, and experimental manipulations were required to be sure that increases in intracellular Ca²⁺ were due to release from internal stores. Also, because there were other intracellular stores for Ca²⁺, other approaches were needed to ensure that the source of intracellular Ca²⁺ release was from the ER. This chapter provides details from several studies using many different experimental techniques that lead to the conclusion that usEPs could induce Ca²⁺ release from the ER by forming ER nanopores. Notably, another series of experimental studies supported theoretical evidence that shorter pulse durations lead to more significant increases in intracellular Ca²⁺ than longer pulse durations.

Permeabilization of cell membranous structures by nanosecond electric field pulses triggers a transient rise of cytosolic calcium with multifarious downstream effects. Electroporation of intracellular membranes (such as those of the Endoplasmic Reticulum) are likely responsible for the calcium release. This is an important application of pulsed electric fields, since calcium is known as a ubiquitous second messenger molecule that regulates several responses in cell signaling, including enzyme activation, gene transcription, neurotransmitter release, secretion, muscle contraction etc. In this chapter, a model based analysis of the dynamical calcium release in response to an external electric pulse is discussed. The results obtained are shown to match experimental data fairly well.

As the largest intracellular structure in mammalian cells, the nucleus, its double phospholipid nuclear envelope, and its chromatin/DNA content were suspected targets for usEPs. Many different methods were used to determine DNA/ nuclear damage including analyses with the comet assay, DNA migration on agarose gels, mitotic indices, and chromatid structures, fluorescent in situ hybridization (FISH). In Jurkat cells exposed to (3.6 × 10⁻³ Vs/cm), the telomers were displaced from the nucleus, and nuclear membranes were sheared from the nucleus. SV40 fibroblasts did not show this apparent telomer and nuclear membrane damage, indicating cell-type differences., It was also shown as adherent cells were less susceptible to usEP-induced damage. Other studies showed that usEP induced significant physical damage to the nuclear membrane, cytoskeleton, and telomers, which form protein–protein or protein-DNA interactions with the nuclear envelope. Some usEP-induced nuclear/DNA damages were suspected due to effects similar to ionizing radiation caused by reactive oxygen species (ROS). Other studies using stably transfected cells with fluorescently labeled Histone-2b (H2B), which is tightly wound with DNA, and PCNA (proliferating cell nuclear antigen), which is loosely associated with DNA, indicated that H2B remained in the nucleus. In contrast, translocation of PCNA from the nucleus to the cytoplasm showed permeabilization of the nuclear membrane. So, usEPs had relatively severe and seemly rapid effects on nuclear structures. Yet, when phosphorylated Histone 2AX (γH2AX) was used as an early indicator of DNA damage in Jurkat cells, the damage appeared to be related to apoptosis's end stages since it was caspase-dependent. While all these methods are valid indicators of effects on DNA and/or the nucleus, the results between the comet assay and γH2AX under similar conditions with the same cell type are not readily reconcilable. One other study demonstrated that usEP also had effects on nuclear substructures called nuclear speckles, which are part of splicing factors and small nuclear ribonucleoproteins (snRNPs) that exhibit roles to provide splicing factors at transcription sites. So, there are apparent effects of usEPs on DNA, the nucleus, and subnuclear factors; the full extent of these effects requires additional experimentation.

The endocytotic and symbiotic inclusion of a prokaryote by an early eukaryote, its subsequent evolution as mitochondria, and its collaboration with the nucleus provided these new symbiotes with enough ATP to evolve a new world of extraordinarily diverse organisms. Mitochondria assumed roles for lives replete with energy from ATP and control over the death of cells when their usefulness was finished or when they malfunctioned or were injured beyond repair. The outer mitochondrial membrane (OMM) protects the electron transport chain (ETC) in the inner mitochondrial membrane and the mitochondria’s DNA, which is used for some of the proteins in the ETC. The ETC is supplied with electrons from NADH, FADH2 produced by oxidative phosphorylation (OXPHOS) as Complexes I, III, and IV pump proton (H +) out of the matrix to generate a proton motive force and a mitochondrial membrane potential (ΔΨm). H + reenter the matrix through ATP synthase for the production of ATP. All this complexity provides usEPs with multiple targets for effects on cell life and death. UsEP’s role in cytochrome c release in apoptosis and other regulated cell death (RCD) mechanisms in cancer ablation has been a significant application with clinical medicine, which is still in developmental stages in clinical trials. UsEPs increase reactive oxygen species (ROS) and dissipate the ΔΨm, which can occur without permeabilization of the IMM, especially in the presence of Ca²⁺ that enters cells through nanopores in the plasma membrane. This loss of ΔΨm is facilitated by usEP effects on the Ca²⁺-dependent and redox-sensitive protein cyclophilin D (CypD). CypD regulates the mitochondrial permeability transition pore (mPTP) that dissipates the ΔΨm, leading to regulated cell death and apoptosis if mitochondria release cytochrome c into the cytoplasm to activate caspases. We also discuss the possible identity of the mPTP as ATP synthase. Experiments continue to test this hypothesis. Experiments here also show that usEPs with a shorter (faster) rise-fall time are more effective to dissipate ΔΨm than usEPs with a longer (slower) rise-fall time. It also appears that over-expression of BCL-xl and BCL2 cannot protect the mitochondria from the effects of usEPs. Experiments measuring oxygen consumption in cells treated or not with usEPs indicate that the usEPs attenuate oxygen consumption in Complexes I and IV of the ETC. These results suggest that usEPs inhibit electron transport in the ETC. We also show that usEPs that ultimately lead to cell death in 4T1-luc mammary cancer cells up-regulates essential subunits in the ETC. Thus, usEPs target several mitochondrial components, including those that regulate ΔΨm and electron transport in the ETC.

Cells employs many different mechanisms for their expiration. The best known and studied regulated cell death mechanism is apoptosis. Cell death subtypes, including regulated cell death (RCD), programmed cell death (PCD), and accidental cell death, are discussed. A discussion on and caution for the use of the term “necrosis” is included. Apoptosis was the earliest RCD mechanism shown in Jurkat cell responses to usEPs as determined by cytochrome c release and caspase activation. This apoptotic cell death was enhanced by usEP-induced supraelectroporation, as electric fields with nanosecond durations and short(fast) rise-fall times passed through the cell, while pulses with microsecond duration go around cells. However, using Jurkat clones that did and did not express APAF-1, which is an essential protein for apoptosome formation as a platform for caspase-9 and caspase-3 activation, it was also shown that usEPs induced caspase-dependent and caspase-independent cell death. A role for caspases depended on the usEP charging intensity with lower usEP impact causing caspase-dependent cell death while higher charging caused caspase-independent cell death. Although a full discussion of all RCD mechanisms is not included, evidence is presented that not all cell types responded to usEP by apoptotic cell death. The presence or absence of Ca²⁺ has an impact on the RCD mechanisms. Human triple-negative breast cancer cells expressed either or both necroptosis and parthanatos. Necroptosis is sometimes considered regulated necrosis because plasma membrane pores form from intracellular proteins. Finally, usEPs are also shown to induce cell responses downstream of toll-like receptors (TLRs). Considerations for immunogenic cell death (ICD) are also considered.

Apoptosis is one of the most complex signaling pathways, and can be triggered by a number of factors, radiation, chemotherapeutic drugs, electric field application, etc. The apoptotic pathways can generally be divided into signaling via the death receptors (extrinsic pathway) or the mitochondria effects (intrinsic pathway), and each pathway implies caspases as effector molecules. The role and extent of electric field modifications to the apoptotic pathways remains unclear. Here in this chapter, we attempt to qualitatively probe some of the issues pertaining to apoptotic cell death based on simple model simulations. The objectives of the present discussions are: (i) to determine if a pulse-number threshold might suitably apply to cell killing by nano-second, high-intensity pulses, and (ii) to assess whether the intrinsic or the extrinsic pathway is more dominant following electric pulsing.

Electroporation is considered a nonthermal process, and when used in medical therapies great care is taken to ensure that temperature effects due to Joule heating are avoided by limiting the average electrical power of the electroporation pulse train. However, moderate temperature increases, far below those which are used in hyperthermia therapies, have shown to increase safety and efficacy of electroporation-based therapies. This chapter provides an introduction into the heating mechanisms by applied electrical pulses, Joule heating and heating due to dielectric relaxion, followed by a short introduction of external heat sources, with emphasis on infrared light sources. The second part describes basic thermal effects on cells, particularly on cell membranes. This is followed by an overview of recent in vitro and in vivo studies on the effects of thermal assistance in electrotherapies with emphasis on its application in cancer treatments. Moderate heating of tumors in combination with electrical pulse treatments was found, for a wide range of pulse durations, to cause a significantly higher rate of complete tumor regression compared to the use of pulsed electric fields alone.

Generally, in studies of the bioelectric effects of nanosecond pulses, thermal effects are not considered. While this is certainly true for the delivery of single or a small number of pulses, developments in medical treatment based on tissue ablation have been based pulse trains applied at a high repetition rate. In fact, temperature increases may trigger thermally activated bioeffects. This suggests technological opportunities for electro-manipulation that takes advantage of synergisms between thermal and electrically driven processes. Even with modest temperature changes, large thermal gradients could be established, which in itself can lead to additive electric field creation. The focus in this chapter is on thermal aspects and the possible synergies with electrical stimulation for bio-effects.

The application of pulsed electric fields and discharges can lead to the generation of shockwaves in aqueous media. The interaction of shockwaves with biological cells and membranes is a relative recent area of research, with applications ranging from water purification and desalination, bacterial decontamination, and extracorporeal shock wave lithotripsy. Here, the possible role and impact of shockwaves on membrane pore creation is discussed. Model results based on Molecular Dynamic simulations are presented. The results also touch upon synergistic benefits of a scenario that combines the application of shockwave with an electric field.

Membrane pore creation by applying voltage pulses has been used for various applications including gene electrotransfer, electrochemotherapy, and tissue ablation. Electric pulse stimulation, however, requires insertion of electrodes into tissue, which may not always be conducive or convenient. On the other hand, time-varying magnetic fields can also induce time-dependent electric fields based on Lenz’s law and Maxwell’s equations, and thus be an alternative to creating transmembrane voltages across membranes. Pulsed magnetic fields would be a contactless, noninvasive technique allowing clinicians to affect any target within the body. This chapter discusses the concept of magnetically induced voltages, presents model results, with further elaboration of electromagnetic bio-stimulation.

This chapter presents an overview of commonly used pulsed generators in bioelectrics. The concept of pulsed power is discussed in Sect. 15.1. One of the key components of ultrashort pulse generator is the switch that is capable of closing in time of nanoseconds or subnanoseconds. A variety of switches, including gas spark gap switches and MOSFETs, can be used (Sect. 15.2). Besides the switches, the circuits that are often used in producing ultrashort pulses are discussed in Sect. 15.3. Finally, when ultra-high voltage pulses are needed, an air-core transformer or a Marx generator can be used, which are discussed in Sect. 15.4.

This chapter discusses how electric pulses are delivered to a biological target using electrodes and a transmission line. The electric fields in the tissue near the electrodes can be determined through analytical approach or numerical calculation (Sect. 16.2). In the time domain, the pulses seen at the target are largely affected by the pulse duration and the electrical characteristics of the electrodes in the tissue, especially the associate capacitance (Sect. 16.3). It is also important to treat the electrode configuration as part of the transmission line for the delivery of ultrashort pulses when the exact waveform is determined. Two types of electrodes that can deliver ultrafast pulses for in vitro studies are discussed in Sect. 16.4.

Ultrafast pulses can be measured by a resistive divider or a capacitive divider (Sect. 17.1). In terms of the ease of construction and characterization, the resistive divider is a better choice. The divider is typically consisted of a high value resistor and a 50 Ω resistor, designed to match the impedance of a standard coaxial cable. The configuration and related issues of such a divider is discussed in Sect. 17.2. With robust resistors, the divider can be used to measure high voltage, nanosecond pulses. For shorter pulses, a coaxial structure that houses resistors with low inductance can be used. One such a resistor divider for subnanosecond pulses is shown in Sect. 17.3.

One of the earliest possible medical applications for usEPs was to ablate tumors. Many laboratories carried out many studies to investigate the potential for usEPs to serve as a cancer therapy. Like simulations with cells in suspension, usEPs also passed through cells in tumor tissues forming high-density nanopores in all cell membranes as supraelectroporation, distinct from conventional electroporation. The first studies were smaller in scope, but showed proof of principle that usEPs could reduce fibrosarcoma tumor volume in mice. More extensive studies followed, showing that usEPs could treat B16f10 melanoma tumors in mice, although some tumors required more than one and as many as  six 300 ns, 40 kV/cm treatment (6 × 1.2 Vs/cm). Later studies showed that as many as 5–6 Vs/cm was required to eliminate tumors completely. The blood supply to these melanoma tumors was also  reduced,  as was revascularization, as shown using endothelial markers. Other studies showed that usEPs ablated mouse liver tumors. While all of these studies  investigated ectopic tumors within mice's flanks, studies also showed that usEPs could also completely ablate orthotopic rat liver tumors, using a 5-needle array with heterogeneous electric fields. Other studies demonstrated that usEPs could eliminate many different tumor types in mice, including some human tumors in immunodeficient mice. One study showed decreased tumor sizes in dog osteosarcoma.

That usEPs could ablate tumors was a significant finding in the use of this technology for cancer therapy. The finding that usEPs could also induce immunity was a bonus for this treatment as possible immunotherapy. However, the data to support this immune induction by usEPs had several different sets of suggestive evidence. One approach showed slower tumor growth in immunocompetent mice vs. growth in immunodeficient mice. A second approach showed the slower growth of a secondary tumor after ablation of a primary tumor, suggesting that the primary treatment caused an immune response that slowed secondary tumor growth. The presence of CD4+ T-cells in the primary treated tumors and CD4+ cells in the untreated secondary tumor was used as evidence. However, the CD4+ cells require further characterization to differentiate CD4+  CD25+ Foxp3+ T-regulatory immunosuppressor cells (Tregs) from CD4+ CD44+ with the presence or absence of CD62L+ as T-central or T effector memory cells, respectively. The strongest evidence for usEP-induced immunity indicated the complete absence of secondary tumor growth after primary tumor treatment. Such responses were present in cancer models in the ectopic mouse liver, orthotopic mouse breast, and rat liver cancers. The absence of secondary tumor growth is called vaccine effects or in situ vaccination. Thus, the treatment of the primary tumor induces immunity and vaccinates the animals by the usEP treatment. The latter two cancers exhibited early decreases in immunosuppressor Tregs and myeloid-derived suppressor cells (MDSC), which resolve suppression of immune responses, and increases in dendritic cells (DCs) in the TME that could identify antigens and induced immunity. The rat liver cancer model also showed activation of the innate immune natural killer (NK) cells with specific activation markers in its TME and the presence of effector and central memory cells in the mouse breast and rat liver (TME), which were cytotoxic. An ectopic mouse pancreatic cancer model that did not show a vaccine effect failed to show a decrease in Tregs and MDSC in the TME and blood and did not show activated T-cells, suggesting immunosuppression prevented an immune response. Continued studies will determine immunity, cell death mechanisms, and ICD factors (calreticulin, ATP, and HMGB1) in other immunogenic cancer models.