Frank Reidy Research Center for Bioelectrics
Old Dominion University
Eastern Virginia Medical School
Electric Fields Open New Gateways into Biological Cells
The application of electrical pulses in the microsecond range [.000001 second or 1 µs for short] to biological cells has been a focus of studies for more than two decades. Such pulses cause the accumulation of electrical charges at the cell membrane shielding the interior of the cell from the external electrical fields. Typical charging times for the mammalian cell membrane are on the order of one microsecond. In contrast to these microsecond pulses that do not penetrate into cells, shorter pulses, in the nanosecond range [.000000001 second or 1 ns], penetrate the entire cell, nucleus and organelles, and affect cell functions.
Researchers from different disciplines; electrical engineers, cell biologists and medical professionals, work side by side at the Center for Bioelectrics to explore the cellular responses to nanosecond pulsed electric fields. High voltage pulse generators apply nanosecond pulses as high as 40,000 volts to small test chambers called cuvettes. Biological cells held in liquid suspension in these cuvettes are placed between two electrodes for pulsing. The power density in these cuvettes is up to one Gigawatt per cubic centimeter (109 W/cm3), but the energy density is rather low. Even under the most extreme conditions, it is less than 10 Joules per cubic centimeter, a value that could only increase the temperature of the suspension by about 2 degrees.
The cells in a suspension behave quite differently after pulsing. Depending on the pulse amplitude, human platelets (blood cells crucial for blood clotting) mobilize intracellular calcium and clump together. White blood cells take up calcium from the surrounding medium and become immobilized for a certain time. This process will be beneficial in wound healing.
Cancer cells have also been studied and begin dying by a process called apoptosis after being pulsed in the nanosecond range. Apoptosis is the best characterized of several programmed cell death mechanisms. The cells begin, in a very orderly fashion, to disassemble themselves. Over a period of days the fragments are recycled for use by the body. This process allows the removal of cells that are no longer needed by the body, or which pose a threat. Research on apoptosis induction in cancer cells with nanosecond pulses has many exciting potential applications. In recent lab research with melanoma, the tumor was reduced in size by ninety percent after only one treatment.
Research on the effect of nanosecond pulses on tissues began several years ago. Dr. Stephen Beebe, a Professor of Pediatrics at Eastern Virginia Medical School, injected fibrosarcoma cancer cells into mice. When the tumors were fully developed, needles were inserted around the tumor, and high voltage pulses were applied. Tumors exposed to the electric fields were found to grow much slower than the control tumors, and follow–up studies showed that many of the tumor cells died through apoptosis.
These early results indicated the possibility of affecting the growth rate of cancer cells, but major questions were not answered:
Was it possible to eliminate tumors, rather than just slow growth?
Can the healthy surrounding tissue be spared when tumor apoptosis is triggered?
Is the method cell-selective?
Research on tumors was restarted in fall of 2003, this time on a melanoma tumor line that allowed easier treatment and diagnostics. Melanoma is an extremely aggressive cancer, and if not detected and treated early, has a high mortality rate. About 50,000 people develop melanoma cancer each year with about 7,000 dying from it in the USA alone. It is also a cancer that can easily be treated with electric pulse delivery systems since it typically develops in the skin. As in the earlier experiments, the Bioelectrics Team inoculated mice with tumors, one for treatment and one for control. Again using needle electrodes, one tumor was treated with nanosecond pulses.
The results were striking.
Figure 1. Melanoma tumor treated with 100 pulses with time duration of 300 ns and an electrical field of 40 kV/cm. Transillumination images show the regression of the tumor.
There was a substantial reduction of the tumor size after a few days. In some instances, we have seen complete remission. Photographs taken by transillumination show a typical tumor before and 10 days, 20 days, and 47 days after treatment. Multiple pulse applications have eliminated this tumor completely. Experiments to determine the mechanism(s) for tumor reduction are underway. Initial results indicate not only apoptosis is involved in tumor reduction, but also necrosis, caused by destructive effects of the nanosecond pulses on blood vessels feeding the tumor.
Is the effect of ultrashort electric pulses cell-specific? Can we kill the bad cells and spare the good ones, using the same pulse? Ultrashort pulse experiments performed on different types of cells clearly show that intracellular electroeffects are cell-specific. Experiments with Jurkat cells (leukocytes cells) and normal human blood polymorphonuclear leukocytes showed quite different responses after pulsing. For a single treatment, the plasma membrane of the Jurkat cells disintegrated, whereas the membrane of leukocytes stayed intact. This was for cells in suspension. How about tissue? We expect to have different effects on different types of tissue, but data to support this hypothesis are not yet available. So far, we can only conclude there is less damage in the surrounding, healthy tissue than in the tumor. While the tumor shows striking and destructive effects after pulse applications, the healthy tissue heals rather quickly.
Another weapon against cancer based on this use of ultrashort electrical pulses, is gene therapy. Because ultrashort pulses appear to have predominant effects on subcellular membranes, they could be used to modify the nuclear membrane and possibly enhance gene delivery to the nucleus after classical plasma membrane electroporation allows the entry of plasmid DNA through the cell membrane. Gene therapy allows the replacement of defective genes by introducing the normal genes into the nucleus of cells. Experiments where HL60 (leukaemia) cells were exposed, in the presence of a green fluorescent protein (GFP) reporter gene, to a combination of a long pulse followed 30 min later by a short pulse showed a dramatic increase in expression compared to the results with only an electroporation pulse applied. Essentially all of the cells expressed GFP with greater fluorescence intensity than cells exposed to classical electroporation conditions. The results suggest the potential to increase gene expression by combining electroporation pulses with ultrashort pulses. The mechanism(s) that leads to increased gene expression is not yet known. Alternatively, it is possible that the ultrashort pulses could promote the expression of genes though other unidentified mechanisms.
Apoptosis is just one effect that is caused by ultrashort pulses. It is the predominant effect at high electric fields. If a lower electric field is used with the same pulse duration, a host of nonlethal effects emerge. In some cell types, even increased proliferation was observed. When the pulse amplitude was lowered, aggregation of platelets was observed, an effect that might be employed in wound healing. Another set of studies performed at Old Dominion University/ Eastern Virginia Medical School and at the University of Southern California, on secondary effects of ultrashort pulses, deals with calcium release from intracellular stores. Steve Buescher, at EVMS, did a fascinating set of experiments with white blood cells in which he observed the effect of ultrashort pulses on calcium release. When modest electric fields below the threshold for apoptosis, were applied to leukocytes, immediate, but brief, rises in the intracellular calcium concentration occurred with pulse amplitudes as low as 12 kV/cm. In experiments where the cells were actively moving over a slide surface, with associated fluctuations in [Ca++] prior to pulse application (observed by using Fluo-3, a calcium indicator), pulsing caused an abrupt loss of mobility that correlated to the rise in intracellular calcium. The immobilization phase of the cells was dependent on the amplitude of the field. Lowering the electric field caused the cells to recover more quickly.
The new field of intracellular electromanipulation would not exist without pulse power technology using extremely high, pulsed electrical field. As discussed before, intracellular electromanipulation, depending on the desired effect, requires high electric fields at nanosecond pulse duration. The field required to cause a specific effect is strongly dependent on the cell type. For single pulses or pulse trains with only a few pulses, typical electric fields necessary to cause apoptosis range from tens to hundreds of kV/cm. The highest electric field requirements for 10 ns pulses were 300 kV/cm. They are lower for pulse trains with a much greater number of pulses. Using smaller electrode gaps, e.g. 100 micrometer, and longer pulses, or exploring effects which require lower electric fields allows us to use pulse generators of one kV or less.
The first peer-reviewed publication on Intracellular Electromanipulation appeared just four years ago, in 2001. So this part of Bioelectrics, the effect of ultrashort pulse effects on cells and tissue, is still in its infancy. Understanding the pathways that lead to apoptosis in cells following the application of such pulses requires extensive experimental and theoretical studies. Fortunately, with support of the US Air Force Office of Scientific Research [AFOSR], a consortium has been established, which is looking into the basic processes of intracellular electromanipulation. Members of the MURI (Multidisciplinary University Research Initiative) consortium, which is administered by Old Dominion University, include scientists at Old Dominion University, Eastern Virginia Medical School, the Harvard/ MIT Health Science Center, University of Texas Health Sciences Center, Washington University, and the University of Wisconsin at Madison. Purdue Calumet scientists are working on similar studies under another MURI, and a team at the University of Southern California is performing similar research under yet a different program. There is increasing interest outside the United States in this emerging field. In November of 2005, a research consortium for Bioelectrics was established with Old Dominion University and Kumamoto University in Japan and Karlsruhe in Germany as charter members. Beijing University in China and ONERA in France are exploring ways to enter this new field.
There are two reasons for this increasing interest. One is scientific. By utilizing electrical pulses higher than the “built-in” electric fields in membranes, and using pulse durations long enough to charge membranes of subcellular structures, but short enough to avoid thermal effects; we have created an electrical probe, which allows us to expand our knowledge about cells.
The second is the exciting applications of this technology. Research on cell manipulation with applications in tumor treatment, gene therapy and wound healing has the potential to end up in your doctor’s office. Research in the cosmetic area is looking into the effects on tissue and the removal of warts and skin funguses. The effects that have been observed thus far, some of which have been discussed in this manuscript, are only the tip of the iceberg. There is more to come, with many opportunities for electrical engineers and biologists and the medical profession to participate in this new field of bioelectrics.
