Discoveries in Travels from Nanovolts to Kilovolts

In 1957 the Soviet Union launched Sputnik I, the world’s first artificial satellite and that was a wake-up call for US science. While I was only in the 4^th grade at the time, I remember many newspaper and magazine articles urging our country to increase our scientific output and that is when I first decided that science would be a good path for me. This was reinforced by an excellent physics laboratory at Serra High School in San Mateo California followed by the outstanding physics courses and laboratories at the University of Santa Clara in Silicon Valley. The laws of physics lay the foundation for all of the sciences, and I had a gift for explaining how these laws worked in plain language. That led me to investigate a teaching career and I realized that graduate work would be very beneficial. After applying to several graduate programs, I accepted an offer from Purdue of full support as a graduate teaching assistant. The first year included course work in advanced electricity and magnetism and quantum mechanics along with rotations through professors’ laboratories to help students choose a major professor to guide graduate research.

Lionel Jaffe, a Guiding Light

One of the professors who described his work to the incoming students was Lionel Jaffe who was actually in the Department of Biological Sciences. He described his measurements of the ionic currents in germinating algal eggs that were forming rhizoid outgrowths. I thought that measuring electrical currents in living cells would be much more interesting than studying them in inert solids, so I chose a rotation through his laboratory. I really enjoyed that work so made the first life-changing decision in my career, to apply the laws of physics to living systems. One problem was that as a physics major, I had never taken any biology courses, so I had to play catch-up, taking biological sciences, physical chemistry, and organic chemistry and genetics. Those courses were less challenging than my physics experience and I completed them in two semesters while working in Lionel’s laboratory.

Lionel Jaffe (Fig. 1) was a brilliant biologist/mathematician who trained at Cal Tech and was a world authority on the development of the growth axis in plants. He had incredible physical intuition and his studies of the environmental factors that influence the plant growth direction, such as gradients of light, pH, and temperature, led him to believe that ionic currents were probably involved in the polarization mechanism.

FIG. 1.

Lionel Jaffe. Photograph (courtesy of Mike Levin), taken ca. 2006, and reproduced with the permission of his daughter, Laurinda Jaffe.

Richard Nuccitelli, PhD

I cannot overemphasize what a stretch this was. Most biologists receive very little course work in electricity, and bioelectricity is far from the typical biologist’s thought process. Yet, Lionel decided to spend months of his time devising an experiment to determine if there were ion currents traversing the polarizing algal eggs. He decided to place 200 fertilized eggs of the seaweed, Pelvetia, in a fine glass capillary tube and shine light from one end of the tube to stimulate them all to grow toward the opposite end (Fig. 2). He predicted that if current flowed through the germinating eggs, aligning them in a capillary would be equivalent to placing 200 small batteries in series and this would amplify the voltage across a single egg 200-fold to generate a measurable voltage difference between the two ends of the capillary.² After much trial and error, he actually measured a voltage gradient on the order of hundreds of microvolts between the two ends of the capillary, a brilliant example of bioelectricity in action.² This provided proof of concept, but he still wanted to measure the current flowing through a single polarizing egg, which is when I came along.

FIG. 2.

Lionel Jaffe’s experiment to detect transcellular current in germinating algal eggs. (A) Photograph of eggs in part of a 100 µm bore capillary 6 h after fertilization and before germination. (B) Photograph of same eggs 26 h after fertilization. All eggs have germinated in the same direction due to light shining down left end of capillary. (C) Schematic view of inferred current pattern in a tube. (D) Schematic graph of inferred change of potential along the tube. Reproduced with permission.¹

PhD Research

For my PhD thesis I worked closely with Lionel developing a new technique for measuring the tiny currents in the nanoamp range traversing a single polarizing seaweed egg of Pelvetia. This egg starts out as a perfectly symmetric sphere and, based on environmental cues such as sunlight, selects a growth axis to guide the vesicle secretion that results in the outgrowth of a rhizoid. My project involved measuring this small current generated by a single growing egg 100 μm in diameter. To do this, we decided to measure the small voltage gradient generated by the transcellular current as it flowed through the seawater from the thallus end to the rhizoid end. We vibrated a platinum-tipped electrode between two points 30 μm apart and used a lock-in amplifier to average thousands of measurements and detect the nanovolt difference between the two points^1,3 (Fig. 3). This ultrasensitive “vibrating probe” or “self-referencing” technique has since been, as shown in Figure 3, used by dozens of other scientists to measure the transcellular currents characteristic of essentially all polarized cells in plants and animals.^4–6 These electrical signals are typically in the 10–100 μA/cm² range and are usually associated with a growth axis such as the midline of the 7-day mouse embryo⁷ or a child’s regenerating fingertip.⁸ These transembryonic currents have been shown to play a causal role in development in the chicken, frog, and axolotl embryos^9–12 in that disrupting the internal electric fields results in striking tail abnormalities. These studies of the bioelectricity in embryos were further advanced at the Marine Biological Laboratory in Woods Hole, Massachusetts, when Lionel moved there in 1982 to open the National Vibrating Probe Facility. In addition to measuring bioelectric currents, he published many seminal articles on the role of Ca²⁺ waves in egg activation.^13–16

FIG. 3.

Photograph of the growing tip of a 1-day-old Pelvetia embryo (P). The extracellular potential near the tip was measured using a probe (indicated by an asterisk) vibrating at 200 Hz near the tip, between two points 30 μm apart.

The Jaffe laboratory at Purdue was unique in its composition with six students who would go on to be very productive professors in bioelectricity. These included Mu-ming Poo who is now a member of the National Academy of Sciences at UC Berkeley studying neural circuit development, Ken Robinson who returned to Purdue as a professor to show the causal role of transembryonic currents in chick embryos, Richard Borgens who returned to Purdue as a professor to conduct research stimulating nerve cord regeneration with applied electric fields, Benjamin Peng now a department chair at Hong Kong University studying acetylcholine receptor dynamics, TH Chen who is a professor in Taiwan, and myself. I loved working there and stayed on after my PhD for a couple of years as a postdoc when I studied the transcellular ion currents in the amoeba¹⁷ and growing pollen tubes.¹⁸ This work provided additional examples of the general observation that all cells drive steady currents through themselves associated with cell polarity, and in these two cases it was associated with vesicle secretion or lamellipodium extension, respectively.

Postdoctoral Research with Susumu Hagiwara

After receiving my PhD from Purdue, I took a postdoctoral position with Susumu Hagiwara at UCLA. Dr. Hagiwara was a famous electrophysiologist who discovered the calcium action potential. Known as “Hagi”, his approach was pretty “hands-off,” leaving his postdocs to work independently. However, he was always available to advise as needed and provided most of the equipment required for a given project. There I learned how to clamp the transmembrane voltage in cells and showed that the egg of the freshwater fish, Medaka, did not use the electrical block to polyspermy exhibited by many other marine species but instead used the mechanical approach of secreting material into the micropyle to block further sperm entry.

Time at UC Davis

In 1978, I took a position as assistant professor at UC Davis where I spent 23 years studying the ionic changes involved in egg activation as well as the role of physiological electric fields in directed cell migration during development. I had fruitful collaborations with Carol Erickson and Rivkah Isseroff documenting the galvanotaxis of fibroblasts¹⁹ and keratinocytes^20,21 and with Lynn Wiley measuring the 0.2 μA/cm² (inward apical and outward basal) transcellular currents in the mouse blastomere.²² I was also very lucky to work with some outstanding graduate students and postdocs during that period.

My first postdoc, Dennis Webb, and I used nuclear magnetic resonance (NMR) to detect the small pH increase that occurred in the frog egg at fertilization.^23,24 This required collecting thousands of eggs to fill an NMR tube 1 inch in diameter . We had earlier measured the pH increase at fertilization with pH-sensitive electrodes but an artifact due to electrode impalement damage could not be ruled out, so we needed another method to confirm it.²⁵ We went to the chemistry department’s NMR facility and Gerald Matson helped us to use the pH-dependence of the internal P_i peak of ³¹P NMR. NMR required obtaining eggs from 3–6 Xenopus females to fill a large tube with 30 mL of eggs to be placed in the superconducting magnetic field required to detect the pH-dependent ³¹P NMR spectral shift. We were pleasantly surprised to find very close agreement between the electrode and NMR methods, with both measuring the internal pH to be 7.4 in unfertilized eggs and 7.7 in both fertilized and ionophore-activated eggs.^26,27 This increase in internal pH is a very common way cells control their metabolism as pointed out in a seminal review²⁸ by my second postdoc, Bill Busa.

For his main research project, Bill Busa used multiple Ca²⁺-sensitive electrode impalements to detect the wave of elevated free Ca²⁺ following fertilization of the frog egg. These electrodes were very difficult to make and had to be very sharp, made from borosilicate glass and calibrated before use. He then showed that inositol-tris phosphate (IP₃) injection would also activate the frog egg by releasing Ca²⁺ from intracellular stores.^29,30 This was the first demonstration of this IP₃-induced Ca²⁺ release from the endoplasmic reticulum (ER) in eggs and required the development of a double-barreled IP₃-iontophoresis/Ca²⁺-measurement electrode.

Several excellent students contributed to our understanding of the ionic changes involved in egg activation. Jin-Kwan Han used the trick of stratifying organelles in the frog egg by centrifugation to show that the endoplasmic reticulum was the major organelle of the IP₃-sensitive Ca²⁺ store in the egg.³² This very delicate work required fabricating double-barreled electrodes using theta tubing similar to those made by Bill Busa. Doug Kline used the vibrating probe to detect the wave of activation current that passes over the frog egg following fertilization (Fig. 4) and measured the ion currents in the cleaving frog egg.^33,34 James Ferguson further clarified the role of the different isomers of IP₃ in Ca²⁺ release and showed that IP₄ can also release Ca²⁺ but is only 20% as effective.³⁵ Glen Winkel worked on mouse embryos and identified the large ionic currents leaving the primitive streak of the 7-day-old embryo as well as studying octoploid mouse embryos produced by electrofusion.^7,31 This work is an example of how bioelectricity is intimately involved in development and can also be used to fuse blastomeres by electrofusion to explore the role (or lack thereof) of the genome in development.

FIG. 4.

Time course of the activation potential in frog egg along with extracellular current measurements at two positions showing the propagation of the activation current wave of Cl^- efflux responsible for the change in membrane potential. A ring of channel activation slowly moves from the IP₃ injection site to the opposite side of the egg. Once it reaches the opposite side, the wave stops, Cl channels close, and the membrane potential returns to normal. Reproduced from Kline and Nuccitelli³¹ where further details can be found.

In the 1990s, Roger Tsien developed new tools for imaging intracellular Ca²⁺ and I took a brief sabbatical in his laboratory to learn how to use Fura-2 to image the calcium wave at activation in frog eggs.³⁶ We reproduced his imaging system in my laboratory and analyzed the Ca²⁺ wave following fertilization in the frog egg and found that it involved lipid hydrolysis in a wave along the cortex of the egg.^36–38

Moving into Industry

About this time, a second marriage contributed to a decision to leave academia for industry, the second life-changing decision. My wife, Pam, and I formed a research company, RPN Research, and moved to the Marine Biological Lab at Woods Hole to work in the Vibrating Probe Center, which had changed its name to the Biocurrents Research Center. There we helped to develop a new aerial probe to detect the electric field outside of skin and applied for a Small Business Innovative Research (SBIR) grant from the NIH. This device vibrated a capacitor normal to the skin and used capacitative coupling to measure the voltage at the skin surface. We reasoned that if there is current flow along the skin surface, the surface potential would be modified along the current path. We began making measurements on wounds in mouse skin using this approach when I was offered a half-time position at the Center for Bioelectrics at Old Dominion University (ODU) by Karl Schoenbach. Karl had discovered some amazing effects of ultrashort kilovolt pulses on cells, so I made a third life-changing decision to explore this new tool of kilovolt pulses at the Center for Bioelectrics.

We were successful in obtaining SBIR funding and could move our small company to the Center under a new ODU program encouraging academic–industry collaboration. We had two engineers and one biologist working with Pam and me to both continue work studying wound currents and looking at fields around melanoma tumors in mice. We built a prototype of an instrument we called the Dermacorder to measure the electric field at the skin surface and obtained a second SBIR grant to develop an electrical diagnostic for melanoma. The wound healing data in mice led us to begin a clinical trial at the nearby Eastern Virginia Medical School to look at skin wounds in humans.

This trial was very productive and we found that the wound current decreased with age and provided a noninvasive indicator of wound healing.³⁹ This is another great example of the integral role of bioelectricity in our physiology. Our skin generates a voltage of about 100 mV across the epidermis, inside positive. When we damage our skin, the wound creates a low resistance pathway through which the transepithelial potential drives out positive current to generate an electric field along the inner side of the epidermis (Fig. 5). That wound current makes the wound negative with respect to all regions surrounding it and this electric field guides keratinocytes and white blood cells to the wound by galvanotaxis because they both migrate toward the negative pole of an electric field.

FIG. 5.

Typical Dermacorder scan of a mouse skin wound. Pink bars mark the wound location on the scan.

Meanwhile, we could not resist treating our mouse melanoma tumors with the new nanosecond kilovolt electric pulses (nsPEF) that Karl discovered. When we placed a needle electrode around the tumor and applied 10 pulses 300 ns long at 20 kV/cm, the tumors stopped growing and when we increased the number to100 pulses, the tumors actually shrunk and disappeared over a period of 2 to 3 weeks! We were only applying a fraction of a joule and increasing the skin temperature less than 3°C to cause melanomas to self-destruct! I was completely blown away by this result so repeated it many times to find that it was extremely reproducible. This discovery was a fourth life-changing event, which changed my research focus completely. Up to this point, we had been studying the small endogenous electric fields of 10–100 mV/mm associated with development and wound healing. After discovering that nonthermal electrical signals 10 million times larger could convince tumor cells to self-destruct, I had to focus on this powerful tool.

Pulsed Electric Fields and Applications

How is it that 10s of kilovolts can be applied across cells without blowing them up? The key reason here is pulse duration. The energy delivered by a pulse is determined by the product of the voltage gradient times current and duration. So if the 20 kV is only applied for 10⁻⁷s, only fractions of a joule will be delivered. By applying the field as a brief pulse, work can be done without significant heat delivery. But how much work can we expect to do in only some billionths of a second?

The key targets of these electric pulses are the cellular membrane bilayers that limit transport into both cells and organelles. Electrophysiologists learned 50 years ago that an electric pulse greater than 400 mV across a bilayer would transiently generate water-filled pores in the membrane to permeabilize it.⁴⁰ Pulses in the microsecond domain open pores large enough to introduce toxic drugs or DNA into the cell through the plasma membrane making electrochemotherapy possible.⁴¹ The amplitude of these pulses is limited to a few kV/cm to avoid significant heating and that limits the size of the membrane-bound compartment across which the minimum 400 mV can be generated to 5–10 μm. The shorter pulses in the nanosecond domain can be applied at 10–50 kV/cm without significant heating and this allows the permeabilization of smaller membrane-bound compartments such as the mitochondria. By permeabilizing the mitochondria and endoplasmic reticulum, nanosecond pulses can disrupt cellular functions that can lead to regulated cell death.

Killing cells without hyperthermia or drugs⁴²

Eliminating tumors was much more exciting than simply detecting their presence so we changed the name of our company to BioElectroMed and applied for another SBIR grant to treat tumors with nanosecond pulses. During the next two years at the Center for Bioelectrics, we studied the tumor’s response to nanosecond pulses. We measured the immediate increase in intracellular Ca²⁺and the 54% shrinkage (pyknosis) of the nuclei within a few minutes after pulsing along with a 90% shrinkage of the tumor by two weeks.⁴⁴ We observed the reduction in blood flow to the tumor within 15 min after pulsing using Doppler ultrasound, and histology confirmed that local blood vessels became leaky and allowed red blood cells to escape into the surrounding tissues. We measured the fragmentation of the DNA from comet assay data. We also showed that these nsPEF effects are highly localized to the region between the bipolar electrodes.⁴⁵ All of these characteristics are steps in the “programmed cell death” or “regulated cell death” pathway that had been studied extensively and is used by all cells when they reach the end of their useful life.

In 2007, we moved BioElectroMed back to California with the goal of commercializing the nsPEF technology. By applying this energy to other murine tumor models, we learned that nsPEF could ablate every type of tumor that we treated, including basal cell carcinoma,⁴⁶ pancreatic carcinoma^43,47 (Fig. 6), and hepatocellular carcinoma.⁴⁸ We showed that the mechanism involves the initiation of immunogenic cell death,⁴⁹ which triggers an immune response to any foreign antigens found in the treated tumor.⁵⁰

FIG. 6.

Human pancreatic tumor in Nu/Nu mouse after treating with 500 pulses of nsPEF. Each row shows a bright field image on the left and a transillumination image on the right taken on given days after treatment (1–49) indicated on the far left.⁴³

We expanded our R&D to include three engineers and built improved pulse generators with microprocessor controls and developed a catheter applicator to target pancreatic carcinoma imaged with an ultrasound gastroscope. Our first application of the catheter in a pig successfully treated 1 cc of normal pancreas with the treated tissue exhibiting apoptosis in the histological sections obtained 3 days after treatment. These data convinced the NIH to approve another Phase II SBIR award to commercialize this application and also convinced ODU to license their IP to us for treating internal tumors. However, ODU decided to license the use of nsPEF to treat skin to a competitor at USC, despite the fact that BioElectroMed had been issued the patent for treating melanoma. This set up a competition between two new start-ups applying the same new energy to human disease.

Along came MDB Capital, an investment group specializing in funding disruptive technologies, pointing out this competition problem and proposing that we all join forces, merging all of the nsPEF IP from ODU, USC, and BioElectroMed into one new company in exchange for shares in the company. It took two years to convince ODU to go along with that plan, but in 2014, Electroblate Corp. (quickly renamed Pulse Biosciences) was formed with an $8M investment. I was named Chief Science Officer while the Board searched for a CEO. BioElectroMed was the only company with a working physical plant so the new company was located in our space. Within a year, Darrin Uecker was selected as CEO and began enlarging the engineering team and building a new pulse generator under Design Controls that were required for obtaining an FDA clearance for treating human disease. MDB Capital generally took their companies public very early to reward their investors, so in 2016 they listed an IPO at $4 per share on the Nasdaq, raising about $20M.

We built a unique nsPEF generator called the CellFX with adjustable pulse width, amplitude, and frequency, and conducted several clinical trials treating benign skin lesions, receiving FDA clearance for that application in 2021. After two years supporting the dermatology applications, including over 5000 successful lesion ablations, we found that this application generated insufficient income, so we switched our efforts to two internal targets for our energy, thyroid nodule ablation and treating atrial fibrillation (A-fib). Both of these applications have a much larger market that we expect to be more profitable.

Thyroid nodule ablation

Radio frequency ablation (RFA) delivered by a percutaneous needle is already being used as an alternative to surgery for removing thyroid nodules. We designed a similarly shaped single-needle bipolar applicator to apply nanosecond pulsed field ablation (nsPFA) and have just received FDA clearance for its use to ablate soft tissue. The first results from clinical trials treating thyroid nodules have been very promising and due to its nonthermal nature, nsPFA should be a safer alternative to RFA.

Treating atrial fibrillation

A-fib is thought to be due to the conduction of electrical signals that trigger contractions into the atrium through the pulmonary veins. This conductive pathway can be eliminated by ablating the atrial tissue at the base of the four pulmonary veins either during open heart surgery or less invasively with catheter ablation of the inner atrial wall. We have been developing applicators for both of these approaches. For surgery we have developed a parallel clamp that sandwiches a region of the atrial wall at the base of a pulmonary vein between two finger-like electrodes to apply nsPFA. It is much faster and generates better transmural lesions than the currently used RFA electrode that relies on heat to ablate the tissue.

We have also developed a catheter that generates a ring-shaped ablation zone at the base of each pulmonary vein to electrically isolate it completely. Our preliminary clinical trial results have been very good but the FDA will require a large pivotal trial with data analyzed after one-year follow-up, so this product will take some time for clearance.

A Concluding Comment

The takeaway from all of this is that all of our body’s physiology utilizes bioelectricity for the control of function. Whether it is the transcellular currents associated with cell polarity, determining the direction of rhizoid or root outgrowth, the detection of light by the retinal rod, the action potential triggering the contraction of our muscles, or transmission of the feeling of touch to our brain, it is all based on bioelectricity. It is a fascinating field of study that I have been lucky enough to explore for my entire life and have enjoyed immensely. PFA has become the most popular current treatment for A-fib because it is so much safer than thermal modalities that risk damaging structures far from the applicator. As you will see from perusing the articles in this special issue, pulsed electric fields are now being used to introduce DNA into cells and tissues to modify their gene expression, introduce poisons into tumors to treat cancer, to ablate tumors directly by triggering regulated cell death, and to remove all types of skin lesions without leaving a scar. Another interesting development is sequencing of the whole transcriptome resulting from nanosecond pulsed electric fields. The hope of stimulating the immune system to attack metastases and replace chemotherapy is also becoming more feasible thanks to advances in immunotherapy and regulated cell death. There is no doubt, therefore, that future prospects of using bioelectricity as a tool for treating disease are very bright indeed.