Reports on cancer with pulses similar to PAPIMI
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Michael J Robey
Sep 30, 2011, 1:15:15 PM9/30/11
to Papimi UK
Zap
By: Karl H. Schoenbach, Richard Nuccitelli, and Stephen J. Beebe
40 Thousand volts, four thousand amperes, and over one hundred million
watts squeezed into a cubic centimeter. You’d think that would be
enough to vaporize just about anything, and it certainly doesn’t seem
like the kind of electricity you’d want to apply to your body. But if
our research continues to succeed as it has, years from now we’ll be
asking some cancer patients to do just that. And it might just save
their lives.
The trick is to apply that gargantuan jolt for only a few billionths
of a second. That’s so brief a time that the energy delivered is a
mere 1.6 joules per cubic centimeter—barely enough to warm a
thimbleful of water by a third of a degree Celsius. But these
powerful, ultrashort voltage pulses do something nothing else can—
harmlessly slip past a cell’s exterior to shock the vital structures
within.
The effects of such pulses of power on living tissue are profound and
varied. Malignant tumors—in mice, at least—can be completely wiped
out, even by significantly lower power levels; new genes can be
efficiently inserted into living cells in the hope of correcting
genetic defects; and immune-system cells can be marshaled to fight off
invading microbes.
A new field of research, bioelectrics, is emerging to study these
effects, as well as the naturally occurring electric fields in
biological systems. Bioelectrics relies on a curious pairing of
disciplines that until now have had almost nothing to do with each
other: high-voltage engineering and cell biology. In particular, the
new field depends on advanced pulsed power technology. That’s the
ability to switch on and off thousands of amperes of current and just
as many volts in mere nanoseconds (the kind of parameters needed to
detonate nuclear bombs, it so happens).
The use of high voltages and currents to manipulate structures inside
cells is barely five years old, but it is a fast-growing international
research endeavor. The largest R&D program at the moment is being
supported by the U.S. Air Force Office of Scientific Research, in
Arlington, Va. That program supports work at a new center established
jointly by Old Dominion University and Eastern Virginia Medical
School, where we authors are working, as well as at several other
institutions in the United States, including the Massachusetts
Institute of Technology, the University of Texas Health Science
Center, the University of Wisconsin–Madison, and Washington
University. Progress in this program has already sparked interest and
some excellent science at academic institutions in Japan, China, and
the state of California. And more institutions, notably in the UK,
France, and the state of Missouri, are planning bioelectrics research.
It’s easy to see the attractions for biologists and for engineers. For
biologists, it’s the potential scientific payoff: these strong but
exceedingly brief electric fields act as a kind of electrical probe,
letting scientists prod key structures inside cells—making the cells
expel certain vital chemicals or begin the production of others—with
the aim of understanding basic biological processes. For engineers,
it’s the opportunity to forge an important new application of pulsed
power technology, which even 10 years ago was seldom used outside the
military.
The most promising and practical result so far has been our recent
discovery that certain pulsed electric fields can wipe out skin tumors
in mice. Melanoma, the skin cancer we’ve worked with, is an extremely
aggressive disease that kills about 8000 people a year in the United
States alone. A few hundred pulses totaling just 120 microseconds of
treatment shrank tumors in mice by 90 percent. A second treatment,
days later, destroyed the tumors completely.
Biomedical science is, of course, littered with cancer cures that work
in mice but fail or are impractical in humans. And it will be many
years before we know if bioelectrics will even be worth testing in
humans. Nevertheless, even at this early stage, bioelectrics seems to
offer a totally new therapeutic avenue—one that could lead to a
therapy free of the debilitating side effects of chemotherapy drugs
and the tissue damage of radiation.
To understand what happens when a cell is hit with tens of thousands
of volts, and why it may help cure disease, you have to know something
about cells themselves. At its simplest, a cell is a pocket of water
containing a bunch of small functional units called organelles, which
are bounded by oily membranes. These organelles are the cell’s version
of internal organs: they perform the functions that keep the cell
alive, just as the brain, kidneys, and lungs, among other organs, keep
the body alive.
Cells do the things they need to do—contract if they are muscle cells,
sense light if they are retinal cells, transport oxygen if they are
blood cells—because they produce proteins with specialized functions.
The creation of proteins begins in the nucleus, the cell’s most
prominent and arguably most important organelle. It houses the cell’s
fantastically complex genetic programming apparatus, which lets the
cell repair itself and tells it how and when to reproduce, what to do
when it detects a particular hormone, and how and when to die. Errors
in these genetic programs go to the heart of most of the diseases
suffered by humankind. These errors can predispose a person to heart
disease, cancer, schizophrenia, and countless other maladies.
The programs are written into your genetic code. This code exists
physically as a set of 23 pairs of chromosomes that reside in the
nucleus. Each chromosome is a rod-shaped or threadlike structure of
deoxyribonucleic acid, or DNA, made up of a sequence of four chemical
building blocks. The sequence of these building blocks—there are tens
of millions of them on a single chromosome—is the code, and the
“words” of this code are genes. In effect, genes are segments of a
chromosome’s DNA. They are groups of many thousands of building blocks
that encode a specific protein, with each chromosome containing
thousands of genes.
These genes are the blueprints for the proteins that determine whether
you have brown or blue eyes, whether your hair is straight or curly,
whether you are tall or short, and whether you are likely ever to
suffer from depression, schizophrenia, or cancer. That’s why gaining
control of what goes on inside the nucleus—which genetic programs are
turned on or off and when—has been a primary goal in biomedical
science practically since the discovery of the structure of DNA about
50 years ago. It is the object of the long-standing, multimillion-
dollar research endeavor called gene therapy, which after decades of
work in some of the world’s foremost laboratories has had mixed
results.
Basically, our work with nanoseconds-long, high-voltage pulses offers
a way to gain access to the cell’s organelles, including its nucleus—
something that has historically bedeviled biomedical scientists.
Remember that the cell and its organelles are bound by membranes. The
main component of these 5-nanometer-thick boundaries is called a
phospholipid bilayer. It is an oily barrier that blocks the flow of
water and ions and therefore also blocks the flow of electric current.
However, the membranes are also studded with proteins, some of which
form nanometer-scale channels designed to allow specific ions to flow
in a direction useful to the cell. In a way, a cell’s membranes are
like leaky capacitors. (Some, such as the one surrounding the nucleus,
leak more than others.) To extend this analogy, the briny fluid within
the membranes, the cytosol, is conductive and can be thought of as a
resistor [see illustration, “Cellular Circuit”].
Now consider what happens when you apply a pulse to the cell. In
general, there are four important characteristics that determine the
precise effects. These are how fast the pulse comes on, or its rise
time; how long the pulse lasts; how many pulses there are; and, of
course, how great the peak voltage is. Different values for each
produce a range of effects, but it’s a very fast rise time that makes
it possible to electrically manipulate organelles.
To see why rise time is critical, imagine a long voltage pulse applied
to the cell that comes on rather slowly, in milliseconds, say. This
slow-rising pulse will set up an electric field across the cell
membrane. In response, ions dissolved in the cell’s cytosol will
stream to the cell membrane, charging it up to counteract the applied
field. Because the voltage is rising rather slowly, the ions have
enough time to accumulate at the cell membrane and cancel out the
electric field, thereby shielding internal structures, such as the
nucleus, from the voltage.
Now, as with any capacitor, if too much charge gathers at the cell
membrane, the electric field there breaks the membrane down. In a
cell, this means large holes, or pores, form in the membrane and allow
ions to pour across, short-circuiting the cell. This effect is called
electroporation, appropriately enough, and it is generally reversible
and even useful. Scientists hoping to kill tumors more efficiently,
use electroporation experimentally, for instance, to increase the
amount of chemotherapy drugs that tumors take up. In fact, San Diego–
based Inovio Biomedical Corp. is in the late stages of clinical tests
on such a cancer treatment for tumors of the head and neck.
To manipulate a cell’s internal structures, we want instead to
generate a strong electric field inside the cell, and do it before too
much charge has accumulated at the cell membrane and turned it into
Swiss cheese. Take the case of a brief pulse with a fast rise time,
reaching its full force in a matter of nanoseconds. With so brief a
rise time, not enough ions will have time to reach the cell membrane
to counteract the sudden electric field, so the nucleus and other
organelles will feel the field’s full effect.
For pulses with a fast rise time, then, the electric field charges up
the membranes of both the cell and its organelles. Generally, the
cell’s plasma membrane doesn’t fully charge to the point where large
pores form in it until it’s been exposed to at least a microsecond and
typically tens of microseconds of voltage. Because the organelles are
much smaller than the cell itself, however, they reach their maximum
charge much more quickly. Ending the pulse after the organelles are
charged up, within a few hundred nanoseconds but before large pores
appear in the cell’s own membrane, lets you focus the electric field’s
effects on the organelles, such as the nucleus, while leaving the cell
membrane relatively untouched. That, in turn, lets you do the complex
and varied things medical science is interested in, such as killing
tumor cells or triggering an immune system response.
This new ability to electrically tweak a cell’s insides would not
exist without pulsed power technology: generating, measuring, and
using extremely high-power electric pulses. Developed initially to
power radar in World War II, pulsed power technology now drives X-ray
imagers, particle accelerators, and nuclear weapons, to name a few
applications.
The kinds of pulses that work best in bioelectrics are simple
rectangular waves. There are a few ways to make such a pulse. The
simplest is to discharge a capacitor. Provided that the time it takes
the capacitor to discharge is long in comparison to the length of the
pulse, you get a roughly rectangular pulse. The problem is that the
pulse length is determined by the closing and opening of a switch. And
no high-voltage mechanical switch can open and close in the few
nanoseconds we need.
Certain types of transistors can do the trick, but they can switch
only 1 kilovolt or less, and we usually need 10 kilovolts or more.
Switches that can handle that kind of voltage can reliably close in
just nanoseconds, but they can’t open so quickly.
What’s needed is a way to separate the length of the pulse from the
speed of the switch. A transmission line pulse generator does just
that. In electric power, transmission lines are generally paired
conductors, such as coaxial cables, that are long in comparison to the
wavelength of the signal they carry. In particular, we make use of
transmission line generators in a Blumlein configuration, named for
the British stereo recording and radar pioneer Alan Blumlein.
Picture two long rectangular conductors sandwiching a thin layer of
insulation [see illustration, “Power Pulse”]. One conductor is divided
into two pieces of equal length, and the load—in our case, a small
tube of cells or a patch of tumor-riddled skin—is placed between them.
The other conductor is charged up because it’s connected at one end to
a high voltage. And the bisected conductor is grounded at the same
end.
A Blumlein generator produces brief high-voltage pulses when
electromagnetic waves change polarity and collide.
Closing a switch connects the two conductors, discharging the device
and setting up waves of voltage that rocket along it [see bioelectric
researcher Juergen F. Kolb's animated clip of the waves in the online
version of this article athttp://www.spectrum.ieee.org/blumlein].
These waves travel in a way not unlike a wave that you’d set up on a
length of rope by holding one end and snapping it.
When the switch closes, waves travel both toward and away from the
load. For those initially traveling toward the load, some portion
reflects off it, and the rest transmits right across it. For those
waves traveling away from the load—including the portions that have
now transmitted across—what happens depends on which end of the device
they encounter.
Taking the rope analogy again, note that if you send a wave down a
rope, the wave will reflect off the end and head back toward you. If
the end is hanging loose, the reflection will be of the same phase as
the initial wave. That is, if the voltage change was positive, the
reflection will be, too. But secure the loose end and the wave will
invert when it reflects. The unsecured rope is analogous to the end of
the transmission line opposite the switch. The secured end, on the
other hand, is like the end at the closed switch.
The voltage pulse comes about when the inverted reflection and the
noninverted reflection crash into each other at the load. The pulse
ends when the trailing edge of each wave has completed its trip down
the transmission line to the load. Therefore, it is not necessary to
open a switch to terminate the pulse; it simply ends abruptly when
there is no energy left in the line. What’s more, you can easily
adjust the duration of the pulse by either adding or subtracting
length from the transmission line.
So what happens to a cell when you zap its innards with so much power?
We’re still working out the biological details, but experiments using
cancer cells suspended in liquid or even growing as tumors in mice
have yielded a good deal of insight.
In our most recently reported experiments, we injected melanoma cancer
cells under the skin of 120 mice and allowed tumors to form [see
photo, “Diminished”]. We then used a Blumlein pulse generator to
subject the tumors to electric field pulses 300 nanoseconds long—too
short to cause classical electroporation—that reached 90 percent of
their peak of 40 kilovolts per centimeter in just 30 ns. We hit the
tumors with a total of 400 pulses, one every other second. Over the
course of two weeks, the tumors shrank by 90 percent. Then they began
to grow again. But in a few experiments, we subjected the tumors to
subsequent sets of pulses, and they were destroyed completely and did
not grow back.
We believe our ultrashort electric pulses killed the tumor cells by
kick-starting a cellular phenomenon called apoptosis, but proving that
theory beyond a doubt is difficult. Apoptosis is also called cell
suicide or programmed cell death. In apoptosis the cell disassembles
itself in an orderly fashion in minutes or hours, leaving behind only
fragments useful as recycling material for the body. It is a process
that allows the removal of cells that are no longer needed by the
organism or of cells that pose a threat to it. As part of the
apoptosis system, cells can sense if they are too badly damaged to
reproduce correctly. Almost by definition, in cancers, the apoptosis
system is off-line, allowing a dangerously aberrant cell not only to
survive but also to multiply.
We saw two nearly immediate effects of the pulses in the mouse tumors
that could indicate apoptosis. First, within just a few minutes, the
tumor cell nuclei had shrunk to half their original sizes, suggesting
that the electric field had either directly or indirectly damaged the
cell’s DNA.
Also, separate experiments done on cells in a liquid suspension showed
that similar pulses resulted in broken DNA and that genetic programs
involved in DNA repair became more active in the pulses’ aftermath.
DNA damage can trigger apoptosis, but such damage also occurs during
apoptosis. However, a classic experiment to prove that apoptosis is in
progress, measuring the amount of a chemical called caspase in cells,
showed no change and no apoptosis.
We think that’s because of the second immediate effect we observed:
within minutes of treatment, blood stopped flowing to the tumor. It
takes energy for a cell to kill itself—in other words, apoptosis can’t
happen without a steady supply of nutrients and oxygen from the blood.
Though we don’t know the exact reason blood stops flowing, stopping
the flow is clearly helpful in destroying tumors. Malignant tumors can
grow to dangerous proportions only because they have the ability to
trick the body into growing new blood vessels to feed them. Developing
drugs to starve tumors by disrupting their blood supply and their
ability to build a new supply is a major goal of many pharmaceutical
firms. And it appears that disrupting the blood supply is something
that nanoseconds-long pulsed electric fields can do.
A key measure of how useful a cancer treatment might be is if it is
more harmful to tumors than to normal tissue. When we shocked vials
containing both cancer cells and normal cells, the pulses killed only
the cancer cells. However, in the mouse experiments, our pulses did
some damage to healthy skin surrounding the mouse tumors. But this
blackening was temporary, and within a couple of weeks, the skin had
healed. Minor tissue damage is common in cancer therapies. In fact,
most treatments, such as chemotherapy, are damaging to tumors and
healthy tissue alike, but they rely on the fact that healthy tissue
has working genetic programs that allow it to survive the chemical
attack and tumors do not.
We are probably years away from performing a similar test of
ultrashort high-power pulses on human cancer patients. But even if
those tests are successful, there will be many hurdles to overcome for
nanosecond pulsed electric fields to become a viable treatment in the
clinic. For one thing, we must be able to deliver gigawatts of power
accurately to sites deep within the body—not just at the skin surface
where we can pinch the tumor between two parallel plates or poke it
with pin electrodes. And we must be able to do so with little or no
harm to the surrounding healthy tissue.
So this summer we are working with antenna expert Carl E. Baum, at the
University of New Mexico, in Albuquerque, to build a device to let us
beam the pulses at cells deeper inside the body. When pressed against
the skin, such a pulse generator’s half-ellipsoid antenna should focus
an electric field pulse to a small volume several centimeters inside
the body. The antenna is only at the modeling stage, but using our
existing laboratory equipment we have begun to examine what sorts of
pulses it would create and what those pulses would do to living cells.
For the time being, though, and notwithstanding the fact that we’ve
made a lot of progress observing the effect of this high-voltage
treatment on tumors in mice, we know far too little about it now to
move on to experiments in humans. It’s important to keep in mind that
the majority of new therapies that show promise in the lab never
develop into approved treatments. We hope nanosecond pulses will, but
the road ahead will be twisty and difficult.
Cancer cells are just one target of ultrashort pulsed electric fields.
By lowering the power and altering their target, for example, we can
also use the pulses in gene therapy. For instance, in proof-of-
principle experiments, we used the pulses to insert new genes into
chromosomes in the nuclei of cells—one of the key challenges of gene
therapy.
For various reasons, the enormous potential of gene therapy has
largely eluded medical researchers. Basically, the techniques have
proven difficult for physicians to execute and dangerous for patients
subjected to them. A prominent example of gene therapy in humans was a
trial in Europe in the 1990s to treat severe combined immune
deficiency syndrome. Commonly called “bubble boy” syndrome, the
disease is caused by an inherited defect in a single gene, which
cripples the body’s defense against infection.
To combat the disease, doctors introduced a corrected copy of the gene
into the nuclei of the children’s immune-cell-generating tissue.
Encouragingly, the therapy defeated the disease, but unfortunately,
three of the first 11 patients developed leukemia, caused by the way
the new gene inserted itself into their existing DNA. Despite the
setbacks, medical scientists have not given up on gene therapy for
bubble-boy syndrome and are also trying it out for nerve damage from
diabetes, heart failure, hemophilia, and a host of other diseases.
Another reason to insert new genes into people is to immunize them
against a particular disease. Ordinary vaccines provide immunity
because they are made up of crippled or dead versions of a disease-
causing microbe. Exposure to a neutered version of the microbe enables
our immune systems to recognize the chemical characteristics of the
weakened microbe and to mount a fast, effective defense against the
real version. However, the vaccine must be refrigerated, and if the
microbe is not weakened enough—and this only very rarely occurs—it can
cause rather than protect against the disease.
Partly because of these drawbacks, researchers have become intrigued
with the idea of injecting a person with the DNA that codes for one of
the infectious bug’s proteins. Some of the person’s own cells take up
the DNA, produce the protein, and trigger the immune system to learn
to recognize and defend against any microbe carrying that protein.
Among the chief technical difficulties with these DNA vaccines, as
well as with gene therapies, is getting the DNA into cells. Simply
injecting a dollop of DNA into someone is not good enough, because the
cell membrane is such a strong barrier against DNA. One popular
solution is to actually genetically engineer the DNA into a virus.
Viruses infect us by “sneaking” their genetic material through the
cell membrane and tricking the cell into copying it. So scientists
have sought to include the DNA they want into harmless viruses, with
which they then infect the patient in the hopes that the virus will
deliver the new gene to the place it needs to go.
The problem is that the virus can stitch the new gene into a bad spot
in the cell’s own DNA, disrupting an important chemical program and
causing disease, as happened when the immune-deficient children
developed leukemia. Or the virus itself can cause a runaway immune
system reaction that can kill the patient, as seems to have happened
in a gene therapy trial several years ago at the University of
Pennsylvania, in Philadelphia.
Pulsed electricity may offer a safer solution. First we can use
strong, but rather long-lasting, electric fields to induce
electroporation, the state we mentioned earlier in which the cell’s
outer membrane temporarily becomes porous. This works, to a point,
because although the new DNA can now enter the cell, it must still get
past the nucleus’s membrane for the cell to decode it.
Because the ultrashort pulses we’ve worked with appear to affect
subcellular membranes, such as the double membrane that bounds the
nucleus, we figured they might help genes make it through that last
step of their journey by opening pores in the nuclear membrane. As a
test, we tried to insert a certain gene from a jellyfish into bone
marrow cells in a test tube. If this gene makes it into the nucleus
and is decoded, it produces a protein that glows green.
By itself, electroporation improved the amount of the gene that was
taken into the cells’ nuclei by 260 percent, as measured by the number
of cells glowing and the strength of the green glow. But following
electroporation with a nanoseconds-long pulse aimed at opening the
cells’ nuclei increased gene uptake by a whopping 900 percent—
potentially enough to improve the efficiency and safety of gene
therapies or DNA vaccinations.
The list of effects that scientists have achieved using nanoseconds-
long pulses is growing rapidly, though their actual use as a medical
treatment is still years away. For example, brief pulses cause
platelets, cellular fragments in the blood, to begin the complicated
cascade of steps needed to form clots. Though the experiments were
performed in a test tube rather than on a human being, we hope the
effect might one day be used in healing wounds.
In other research, E. Stephen Buescher, a professor of pediatrics at
Eastern Virginia Medical School, did a fascinating set of experiments
with white blood cells that also might ultimately help heal wounds. In
it, he observed the effect of ultrashort pulses on the release of
calcium inside cells from internal stores. Calcium acts as a kind of
signal transducer in many cells, translating an external signal such
as a hormone into some cellular action, such as manufacturing a
protein.
In a type of white blood cell whose purpose is to seek out foreign
material and digest it, for example, the release of calcium allows the
cell to follow an invader’s chemical trail. When Buescher subjected
these cells, called leukocytes, to nanoseconds-long, 12-kV/cm electric
fields, the cells immediately, but briefly, spilled calcium from their
internal stores into their own cytosol. In experiments where the cells
were actively crawling over a microscope slide, hot on the simulated
trail of an invader, pulsing stopped them in their tracks and then
sent them marching off in the direction of the electric field. One day
doctors might use such an effect to recruit immune cells to the site
of an infection.
The list of cells and the effects of pulsed power on them goes on and
will only get longer as more laboratories begin work in bioelectrics.
Scientists at Kumamoto University, in Japan, for example, are studying
the subcellular effects of high-power RF pulses. Those at Karlsruhe
University, in Germany, are testing nanopulses for killing bacteria.
And researchers at the University of Southern California are studying
how the pulses cause dying cells to signal other cells to consume
them. Whether or not pulsed power becomes a cancer treatment, a gene
therapy technology, or an infection fighter, ultrashort electric
fields have already proved a powerful research tool. And the mark they
ultimately make on medicine may be in allowing scientists
unprecedented access to the internal workings of cells.
Still, we hope for more practical—and potentially lucrative—
possibilities. While treatments for cancer and genetic diseases would
be revolutionary, somewhat more prosaic applications are in the
offing. We at Old Dominion University have recently used nanosecond
pulsed electric fields to destroy fat cells. Think of it as electric
liposuction. Hey, if it helps pay for the research needed to fight
dread diseases, we’re all for it.
ABOUT THE AUTHORS
Karl H. Schoenbach, an IEEE Fellow, holds the Frank Batten Endowed
Chair in Bioelectric Engineering at Old Dominion University, in
Norfolk, Va. There he directs the Frank Reidy Research Center for
Bioelectrics.
Richard Nuccitelli is a biophysicist at Frank Reidy who has studied
the role of ion currents and ion concentration changes in the
regulation of cell physiology for 30 years. He was the lead
investigator on the melanoma project.
Steven J. Beebe is a faculty member in the department of physiological
sciences and pediatrics at Eastern Virginia Medical School, in
Norfolk, and is on the staff at Frank Reidy. He has studied mechanisms
for signal transduction and apoptosis regulation for decades.
Acknowledgments
The authors would like to thank Peter F. Blackmore, E. Stephen
Buescher, Ravindra P. Joshi, Juergen F. Kolb, and R. James Swanson.
TO PROBE FURTHER
Proceedings of the IEEE devoted its July 2004 issue to pulsed power
technology and its applications. The issue includes a more detailed
look at bioelectrics: “Ultrashort Electrical Pulses Open a New Gateway
Into Biological Cells,” by Karl H. Schoenbach et al., pp. 1122–37.
For more on the effects of nanosecond pulses on cell biology, see
“Nanosecond Pulsed Electric Fields Modulate Cell Function Through
Intracellular Signal Transduction Mechanisms,” by Stephen J. Beebe et
al., Physiological Measurements, Vol. 25, 2004, pp. 1077–93.
For the latest on the authors’ experiments on melanoma, see
“Nanosecond Pulsed Electric Fields Cause Melanomas to Self-destruct,”
by Richard Nuccitelli et al.,Biochemical and Biophysical Research
Communications, 5 May 2006, pp. 351–60.
Cellular Circuit: A cell can be thought of as a circuit made up of
capacitors and resistors. Its membrane and those of its organelles,
such as the nucleus, act like capacitors. The briny liquid encased
within the membranes, the cytosol and nucleoplasm, is conductive and
so can be modeled as resistors.
Diminished: A skin tumor in a mouse before [top] and 16 days after
[bottom] treatmentwith nanoseconds-long pulses of voltage.
Images: Frank Reidy Research Center for Bioelectrics
Source www.papimi.gr
http://papimiuk.blogspot.com
By: Karl H. Schoenbach, Richard Nuccitelli, and Stephen J. Beebe
40 Thousand volts, four thousand amperes, and over one hundred million
watts squeezed into a cubic centimeter. You’d think that would be
enough to vaporize just about anything, and it certainly doesn’t seem
like the kind of electricity you’d want to apply to your body. But if
our research continues to succeed as it has, years from now we’ll be
asking some cancer patients to do just that. And it might just save
their lives.
The trick is to apply that gargantuan jolt for only a few billionths
of a second. That’s so brief a time that the energy delivered is a
mere 1.6 joules per cubic centimeter—barely enough to warm a
thimbleful of water by a third of a degree Celsius. But these
powerful, ultrashort voltage pulses do something nothing else can—
harmlessly slip past a cell’s exterior to shock the vital structures
within.
The effects of such pulses of power on living tissue are profound and
varied. Malignant tumors—in mice, at least—can be completely wiped
out, even by significantly lower power levels; new genes can be
efficiently inserted into living cells in the hope of correcting
genetic defects; and immune-system cells can be marshaled to fight off
invading microbes.
A new field of research, bioelectrics, is emerging to study these
effects, as well as the naturally occurring electric fields in
biological systems. Bioelectrics relies on a curious pairing of
disciplines that until now have had almost nothing to do with each
other: high-voltage engineering and cell biology. In particular, the
new field depends on advanced pulsed power technology. That’s the
ability to switch on and off thousands of amperes of current and just
as many volts in mere nanoseconds (the kind of parameters needed to
detonate nuclear bombs, it so happens).
The use of high voltages and currents to manipulate structures inside
cells is barely five years old, but it is a fast-growing international
research endeavor. The largest R&D program at the moment is being
supported by the U.S. Air Force Office of Scientific Research, in
Arlington, Va. That program supports work at a new center established
jointly by Old Dominion University and Eastern Virginia Medical
School, where we authors are working, as well as at several other
institutions in the United States, including the Massachusetts
Institute of Technology, the University of Texas Health Science
Center, the University of Wisconsin–Madison, and Washington
University. Progress in this program has already sparked interest and
some excellent science at academic institutions in Japan, China, and
the state of California. And more institutions, notably in the UK,
France, and the state of Missouri, are planning bioelectrics research.
It’s easy to see the attractions for biologists and for engineers. For
biologists, it’s the potential scientific payoff: these strong but
exceedingly brief electric fields act as a kind of electrical probe,
letting scientists prod key structures inside cells—making the cells
expel certain vital chemicals or begin the production of others—with
the aim of understanding basic biological processes. For engineers,
it’s the opportunity to forge an important new application of pulsed
power technology, which even 10 years ago was seldom used outside the
military.
The most promising and practical result so far has been our recent
discovery that certain pulsed electric fields can wipe out skin tumors
in mice. Melanoma, the skin cancer we’ve worked with, is an extremely
aggressive disease that kills about 8000 people a year in the United
States alone. A few hundred pulses totaling just 120 microseconds of
treatment shrank tumors in mice by 90 percent. A second treatment,
days later, destroyed the tumors completely.
Biomedical science is, of course, littered with cancer cures that work
in mice but fail or are impractical in humans. And it will be many
years before we know if bioelectrics will even be worth testing in
humans. Nevertheless, even at this early stage, bioelectrics seems to
offer a totally new therapeutic avenue—one that could lead to a
therapy free of the debilitating side effects of chemotherapy drugs
and the tissue damage of radiation.
To understand what happens when a cell is hit with tens of thousands
of volts, and why it may help cure disease, you have to know something
about cells themselves. At its simplest, a cell is a pocket of water
containing a bunch of small functional units called organelles, which
are bounded by oily membranes. These organelles are the cell’s version
of internal organs: they perform the functions that keep the cell
alive, just as the brain, kidneys, and lungs, among other organs, keep
the body alive.
Cells do the things they need to do—contract if they are muscle cells,
sense light if they are retinal cells, transport oxygen if they are
blood cells—because they produce proteins with specialized functions.
The creation of proteins begins in the nucleus, the cell’s most
prominent and arguably most important organelle. It houses the cell’s
fantastically complex genetic programming apparatus, which lets the
cell repair itself and tells it how and when to reproduce, what to do
when it detects a particular hormone, and how and when to die. Errors
in these genetic programs go to the heart of most of the diseases
suffered by humankind. These errors can predispose a person to heart
disease, cancer, schizophrenia, and countless other maladies.
The programs are written into your genetic code. This code exists
physically as a set of 23 pairs of chromosomes that reside in the
nucleus. Each chromosome is a rod-shaped or threadlike structure of
deoxyribonucleic acid, or DNA, made up of a sequence of four chemical
building blocks. The sequence of these building blocks—there are tens
of millions of them on a single chromosome—is the code, and the
“words” of this code are genes. In effect, genes are segments of a
chromosome’s DNA. They are groups of many thousands of building blocks
that encode a specific protein, with each chromosome containing
thousands of genes.
These genes are the blueprints for the proteins that determine whether
you have brown or blue eyes, whether your hair is straight or curly,
whether you are tall or short, and whether you are likely ever to
suffer from depression, schizophrenia, or cancer. That’s why gaining
control of what goes on inside the nucleus—which genetic programs are
turned on or off and when—has been a primary goal in biomedical
science practically since the discovery of the structure of DNA about
50 years ago. It is the object of the long-standing, multimillion-
dollar research endeavor called gene therapy, which after decades of
work in some of the world’s foremost laboratories has had mixed
results.
Basically, our work with nanoseconds-long, high-voltage pulses offers
a way to gain access to the cell’s organelles, including its nucleus—
something that has historically bedeviled biomedical scientists.
Remember that the cell and its organelles are bound by membranes. The
main component of these 5-nanometer-thick boundaries is called a
phospholipid bilayer. It is an oily barrier that blocks the flow of
water and ions and therefore also blocks the flow of electric current.
However, the membranes are also studded with proteins, some of which
form nanometer-scale channels designed to allow specific ions to flow
in a direction useful to the cell. In a way, a cell’s membranes are
like leaky capacitors. (Some, such as the one surrounding the nucleus,
leak more than others.) To extend this analogy, the briny fluid within
the membranes, the cytosol, is conductive and can be thought of as a
resistor [see illustration, “Cellular Circuit”].
Now consider what happens when you apply a pulse to the cell. In
general, there are four important characteristics that determine the
precise effects. These are how fast the pulse comes on, or its rise
time; how long the pulse lasts; how many pulses there are; and, of
course, how great the peak voltage is. Different values for each
produce a range of effects, but it’s a very fast rise time that makes
it possible to electrically manipulate organelles.
To see why rise time is critical, imagine a long voltage pulse applied
to the cell that comes on rather slowly, in milliseconds, say. This
slow-rising pulse will set up an electric field across the cell
membrane. In response, ions dissolved in the cell’s cytosol will
stream to the cell membrane, charging it up to counteract the applied
field. Because the voltage is rising rather slowly, the ions have
enough time to accumulate at the cell membrane and cancel out the
electric field, thereby shielding internal structures, such as the
nucleus, from the voltage.
Now, as with any capacitor, if too much charge gathers at the cell
membrane, the electric field there breaks the membrane down. In a
cell, this means large holes, or pores, form in the membrane and allow
ions to pour across, short-circuiting the cell. This effect is called
electroporation, appropriately enough, and it is generally reversible
and even useful. Scientists hoping to kill tumors more efficiently,
use electroporation experimentally, for instance, to increase the
amount of chemotherapy drugs that tumors take up. In fact, San Diego–
based Inovio Biomedical Corp. is in the late stages of clinical tests
on such a cancer treatment for tumors of the head and neck.
To manipulate a cell’s internal structures, we want instead to
generate a strong electric field inside the cell, and do it before too
much charge has accumulated at the cell membrane and turned it into
Swiss cheese. Take the case of a brief pulse with a fast rise time,
reaching its full force in a matter of nanoseconds. With so brief a
rise time, not enough ions will have time to reach the cell membrane
to counteract the sudden electric field, so the nucleus and other
organelles will feel the field’s full effect.
For pulses with a fast rise time, then, the electric field charges up
the membranes of both the cell and its organelles. Generally, the
cell’s plasma membrane doesn’t fully charge to the point where large
pores form in it until it’s been exposed to at least a microsecond and
typically tens of microseconds of voltage. Because the organelles are
much smaller than the cell itself, however, they reach their maximum
charge much more quickly. Ending the pulse after the organelles are
charged up, within a few hundred nanoseconds but before large pores
appear in the cell’s own membrane, lets you focus the electric field’s
effects on the organelles, such as the nucleus, while leaving the cell
membrane relatively untouched. That, in turn, lets you do the complex
and varied things medical science is interested in, such as killing
tumor cells or triggering an immune system response.
This new ability to electrically tweak a cell’s insides would not
exist without pulsed power technology: generating, measuring, and
using extremely high-power electric pulses. Developed initially to
power radar in World War II, pulsed power technology now drives X-ray
imagers, particle accelerators, and nuclear weapons, to name a few
applications.
The kinds of pulses that work best in bioelectrics are simple
rectangular waves. There are a few ways to make such a pulse. The
simplest is to discharge a capacitor. Provided that the time it takes
the capacitor to discharge is long in comparison to the length of the
pulse, you get a roughly rectangular pulse. The problem is that the
pulse length is determined by the closing and opening of a switch. And
no high-voltage mechanical switch can open and close in the few
nanoseconds we need.
Certain types of transistors can do the trick, but they can switch
only 1 kilovolt or less, and we usually need 10 kilovolts or more.
Switches that can handle that kind of voltage can reliably close in
just nanoseconds, but they can’t open so quickly.
What’s needed is a way to separate the length of the pulse from the
speed of the switch. A transmission line pulse generator does just
that. In electric power, transmission lines are generally paired
conductors, such as coaxial cables, that are long in comparison to the
wavelength of the signal they carry. In particular, we make use of
transmission line generators in a Blumlein configuration, named for
the British stereo recording and radar pioneer Alan Blumlein.
Picture two long rectangular conductors sandwiching a thin layer of
insulation [see illustration, “Power Pulse”]. One conductor is divided
into two pieces of equal length, and the load—in our case, a small
tube of cells or a patch of tumor-riddled skin—is placed between them.
The other conductor is charged up because it’s connected at one end to
a high voltage. And the bisected conductor is grounded at the same
end.
A Blumlein generator produces brief high-voltage pulses when
electromagnetic waves change polarity and collide.
Closing a switch connects the two conductors, discharging the device
and setting up waves of voltage that rocket along it [see bioelectric
researcher Juergen F. Kolb's animated clip of the waves in the online
version of this article athttp://www.spectrum.ieee.org/blumlein].
These waves travel in a way not unlike a wave that you’d set up on a
length of rope by holding one end and snapping it.
When the switch closes, waves travel both toward and away from the
load. For those initially traveling toward the load, some portion
reflects off it, and the rest transmits right across it. For those
waves traveling away from the load—including the portions that have
now transmitted across—what happens depends on which end of the device
they encounter.
Taking the rope analogy again, note that if you send a wave down a
rope, the wave will reflect off the end and head back toward you. If
the end is hanging loose, the reflection will be of the same phase as
the initial wave. That is, if the voltage change was positive, the
reflection will be, too. But secure the loose end and the wave will
invert when it reflects. The unsecured rope is analogous to the end of
the transmission line opposite the switch. The secured end, on the
other hand, is like the end at the closed switch.
The voltage pulse comes about when the inverted reflection and the
noninverted reflection crash into each other at the load. The pulse
ends when the trailing edge of each wave has completed its trip down
the transmission line to the load. Therefore, it is not necessary to
open a switch to terminate the pulse; it simply ends abruptly when
there is no energy left in the line. What’s more, you can easily
adjust the duration of the pulse by either adding or subtracting
length from the transmission line.
So what happens to a cell when you zap its innards with so much power?
We’re still working out the biological details, but experiments using
cancer cells suspended in liquid or even growing as tumors in mice
have yielded a good deal of insight.
In our most recently reported experiments, we injected melanoma cancer
cells under the skin of 120 mice and allowed tumors to form [see
photo, “Diminished”]. We then used a Blumlein pulse generator to
subject the tumors to electric field pulses 300 nanoseconds long—too
short to cause classical electroporation—that reached 90 percent of
their peak of 40 kilovolts per centimeter in just 30 ns. We hit the
tumors with a total of 400 pulses, one every other second. Over the
course of two weeks, the tumors shrank by 90 percent. Then they began
to grow again. But in a few experiments, we subjected the tumors to
subsequent sets of pulses, and they were destroyed completely and did
not grow back.
We believe our ultrashort electric pulses killed the tumor cells by
kick-starting a cellular phenomenon called apoptosis, but proving that
theory beyond a doubt is difficult. Apoptosis is also called cell
suicide or programmed cell death. In apoptosis the cell disassembles
itself in an orderly fashion in minutes or hours, leaving behind only
fragments useful as recycling material for the body. It is a process
that allows the removal of cells that are no longer needed by the
organism or of cells that pose a threat to it. As part of the
apoptosis system, cells can sense if they are too badly damaged to
reproduce correctly. Almost by definition, in cancers, the apoptosis
system is off-line, allowing a dangerously aberrant cell not only to
survive but also to multiply.
We saw two nearly immediate effects of the pulses in the mouse tumors
that could indicate apoptosis. First, within just a few minutes, the
tumor cell nuclei had shrunk to half their original sizes, suggesting
that the electric field had either directly or indirectly damaged the
cell’s DNA.
Also, separate experiments done on cells in a liquid suspension showed
that similar pulses resulted in broken DNA and that genetic programs
involved in DNA repair became more active in the pulses’ aftermath.
DNA damage can trigger apoptosis, but such damage also occurs during
apoptosis. However, a classic experiment to prove that apoptosis is in
progress, measuring the amount of a chemical called caspase in cells,
showed no change and no apoptosis.
We think that’s because of the second immediate effect we observed:
within minutes of treatment, blood stopped flowing to the tumor. It
takes energy for a cell to kill itself—in other words, apoptosis can’t
happen without a steady supply of nutrients and oxygen from the blood.
Though we don’t know the exact reason blood stops flowing, stopping
the flow is clearly helpful in destroying tumors. Malignant tumors can
grow to dangerous proportions only because they have the ability to
trick the body into growing new blood vessels to feed them. Developing
drugs to starve tumors by disrupting their blood supply and their
ability to build a new supply is a major goal of many pharmaceutical
firms. And it appears that disrupting the blood supply is something
that nanoseconds-long pulsed electric fields can do.
A key measure of how useful a cancer treatment might be is if it is
more harmful to tumors than to normal tissue. When we shocked vials
containing both cancer cells and normal cells, the pulses killed only
the cancer cells. However, in the mouse experiments, our pulses did
some damage to healthy skin surrounding the mouse tumors. But this
blackening was temporary, and within a couple of weeks, the skin had
healed. Minor tissue damage is common in cancer therapies. In fact,
most treatments, such as chemotherapy, are damaging to tumors and
healthy tissue alike, but they rely on the fact that healthy tissue
has working genetic programs that allow it to survive the chemical
attack and tumors do not.
We are probably years away from performing a similar test of
ultrashort high-power pulses on human cancer patients. But even if
those tests are successful, there will be many hurdles to overcome for
nanosecond pulsed electric fields to become a viable treatment in the
clinic. For one thing, we must be able to deliver gigawatts of power
accurately to sites deep within the body—not just at the skin surface
where we can pinch the tumor between two parallel plates or poke it
with pin electrodes. And we must be able to do so with little or no
harm to the surrounding healthy tissue.
So this summer we are working with antenna expert Carl E. Baum, at the
University of New Mexico, in Albuquerque, to build a device to let us
beam the pulses at cells deeper inside the body. When pressed against
the skin, such a pulse generator’s half-ellipsoid antenna should focus
an electric field pulse to a small volume several centimeters inside
the body. The antenna is only at the modeling stage, but using our
existing laboratory equipment we have begun to examine what sorts of
pulses it would create and what those pulses would do to living cells.
For the time being, though, and notwithstanding the fact that we’ve
made a lot of progress observing the effect of this high-voltage
treatment on tumors in mice, we know far too little about it now to
move on to experiments in humans. It’s important to keep in mind that
the majority of new therapies that show promise in the lab never
develop into approved treatments. We hope nanosecond pulses will, but
the road ahead will be twisty and difficult.
Cancer cells are just one target of ultrashort pulsed electric fields.
By lowering the power and altering their target, for example, we can
also use the pulses in gene therapy. For instance, in proof-of-
principle experiments, we used the pulses to insert new genes into
chromosomes in the nuclei of cells—one of the key challenges of gene
therapy.
For various reasons, the enormous potential of gene therapy has
largely eluded medical researchers. Basically, the techniques have
proven difficult for physicians to execute and dangerous for patients
subjected to them. A prominent example of gene therapy in humans was a
trial in Europe in the 1990s to treat severe combined immune
deficiency syndrome. Commonly called “bubble boy” syndrome, the
disease is caused by an inherited defect in a single gene, which
cripples the body’s defense against infection.
To combat the disease, doctors introduced a corrected copy of the gene
into the nuclei of the children’s immune-cell-generating tissue.
Encouragingly, the therapy defeated the disease, but unfortunately,
three of the first 11 patients developed leukemia, caused by the way
the new gene inserted itself into their existing DNA. Despite the
setbacks, medical scientists have not given up on gene therapy for
bubble-boy syndrome and are also trying it out for nerve damage from
diabetes, heart failure, hemophilia, and a host of other diseases.
Another reason to insert new genes into people is to immunize them
against a particular disease. Ordinary vaccines provide immunity
because they are made up of crippled or dead versions of a disease-
causing microbe. Exposure to a neutered version of the microbe enables
our immune systems to recognize the chemical characteristics of the
weakened microbe and to mount a fast, effective defense against the
real version. However, the vaccine must be refrigerated, and if the
microbe is not weakened enough—and this only very rarely occurs—it can
cause rather than protect against the disease.
Partly because of these drawbacks, researchers have become intrigued
with the idea of injecting a person with the DNA that codes for one of
the infectious bug’s proteins. Some of the person’s own cells take up
the DNA, produce the protein, and trigger the immune system to learn
to recognize and defend against any microbe carrying that protein.
Among the chief technical difficulties with these DNA vaccines, as
well as with gene therapies, is getting the DNA into cells. Simply
injecting a dollop of DNA into someone is not good enough, because the
cell membrane is such a strong barrier against DNA. One popular
solution is to actually genetically engineer the DNA into a virus.
Viruses infect us by “sneaking” their genetic material through the
cell membrane and tricking the cell into copying it. So scientists
have sought to include the DNA they want into harmless viruses, with
which they then infect the patient in the hopes that the virus will
deliver the new gene to the place it needs to go.
The problem is that the virus can stitch the new gene into a bad spot
in the cell’s own DNA, disrupting an important chemical program and
causing disease, as happened when the immune-deficient children
developed leukemia. Or the virus itself can cause a runaway immune
system reaction that can kill the patient, as seems to have happened
in a gene therapy trial several years ago at the University of
Pennsylvania, in Philadelphia.
Pulsed electricity may offer a safer solution. First we can use
strong, but rather long-lasting, electric fields to induce
electroporation, the state we mentioned earlier in which the cell’s
outer membrane temporarily becomes porous. This works, to a point,
because although the new DNA can now enter the cell, it must still get
past the nucleus’s membrane for the cell to decode it.
Because the ultrashort pulses we’ve worked with appear to affect
subcellular membranes, such as the double membrane that bounds the
nucleus, we figured they might help genes make it through that last
step of their journey by opening pores in the nuclear membrane. As a
test, we tried to insert a certain gene from a jellyfish into bone
marrow cells in a test tube. If this gene makes it into the nucleus
and is decoded, it produces a protein that glows green.
By itself, electroporation improved the amount of the gene that was
taken into the cells’ nuclei by 260 percent, as measured by the number
of cells glowing and the strength of the green glow. But following
electroporation with a nanoseconds-long pulse aimed at opening the
cells’ nuclei increased gene uptake by a whopping 900 percent—
potentially enough to improve the efficiency and safety of gene
therapies or DNA vaccinations.
The list of effects that scientists have achieved using nanoseconds-
long pulses is growing rapidly, though their actual use as a medical
treatment is still years away. For example, brief pulses cause
platelets, cellular fragments in the blood, to begin the complicated
cascade of steps needed to form clots. Though the experiments were
performed in a test tube rather than on a human being, we hope the
effect might one day be used in healing wounds.
In other research, E. Stephen Buescher, a professor of pediatrics at
Eastern Virginia Medical School, did a fascinating set of experiments
with white blood cells that also might ultimately help heal wounds. In
it, he observed the effect of ultrashort pulses on the release of
calcium inside cells from internal stores. Calcium acts as a kind of
signal transducer in many cells, translating an external signal such
as a hormone into some cellular action, such as manufacturing a
protein.
In a type of white blood cell whose purpose is to seek out foreign
material and digest it, for example, the release of calcium allows the
cell to follow an invader’s chemical trail. When Buescher subjected
these cells, called leukocytes, to nanoseconds-long, 12-kV/cm electric
fields, the cells immediately, but briefly, spilled calcium from their
internal stores into their own cytosol. In experiments where the cells
were actively crawling over a microscope slide, hot on the simulated
trail of an invader, pulsing stopped them in their tracks and then
sent them marching off in the direction of the electric field. One day
doctors might use such an effect to recruit immune cells to the site
of an infection.
The list of cells and the effects of pulsed power on them goes on and
will only get longer as more laboratories begin work in bioelectrics.
Scientists at Kumamoto University, in Japan, for example, are studying
the subcellular effects of high-power RF pulses. Those at Karlsruhe
University, in Germany, are testing nanopulses for killing bacteria.
And researchers at the University of Southern California are studying
how the pulses cause dying cells to signal other cells to consume
them. Whether or not pulsed power becomes a cancer treatment, a gene
therapy technology, or an infection fighter, ultrashort electric
fields have already proved a powerful research tool. And the mark they
ultimately make on medicine may be in allowing scientists
unprecedented access to the internal workings of cells.
Still, we hope for more practical—and potentially lucrative—
possibilities. While treatments for cancer and genetic diseases would
be revolutionary, somewhat more prosaic applications are in the
offing. We at Old Dominion University have recently used nanosecond
pulsed electric fields to destroy fat cells. Think of it as electric
liposuction. Hey, if it helps pay for the research needed to fight
dread diseases, we’re all for it.
ABOUT THE AUTHORS
Karl H. Schoenbach, an IEEE Fellow, holds the Frank Batten Endowed
Chair in Bioelectric Engineering at Old Dominion University, in
Norfolk, Va. There he directs the Frank Reidy Research Center for
Bioelectrics.
Richard Nuccitelli is a biophysicist at Frank Reidy who has studied
the role of ion currents and ion concentration changes in the
regulation of cell physiology for 30 years. He was the lead
investigator on the melanoma project.
Steven J. Beebe is a faculty member in the department of physiological
sciences and pediatrics at Eastern Virginia Medical School, in
Norfolk, and is on the staff at Frank Reidy. He has studied mechanisms
for signal transduction and apoptosis regulation for decades.
Acknowledgments
The authors would like to thank Peter F. Blackmore, E. Stephen
Buescher, Ravindra P. Joshi, Juergen F. Kolb, and R. James Swanson.
TO PROBE FURTHER
Proceedings of the IEEE devoted its July 2004 issue to pulsed power
technology and its applications. The issue includes a more detailed
look at bioelectrics: “Ultrashort Electrical Pulses Open a New Gateway
Into Biological Cells,” by Karl H. Schoenbach et al., pp. 1122–37.
For more on the effects of nanosecond pulses on cell biology, see
“Nanosecond Pulsed Electric Fields Modulate Cell Function Through
Intracellular Signal Transduction Mechanisms,” by Stephen J. Beebe et
al., Physiological Measurements, Vol. 25, 2004, pp. 1077–93.
For the latest on the authors’ experiments on melanoma, see
“Nanosecond Pulsed Electric Fields Cause Melanomas to Self-destruct,”
by Richard Nuccitelli et al.,Biochemical and Biophysical Research
Communications, 5 May 2006, pp. 351–60.
Cellular Circuit: A cell can be thought of as a circuit made up of
capacitors and resistors. Its membrane and those of its organelles,
such as the nucleus, act like capacitors. The briny liquid encased
within the membranes, the cytosol and nucleoplasm, is conductive and
so can be modeled as resistors.
Diminished: A skin tumor in a mouse before [top] and 16 days after
[bottom] treatmentwith nanoseconds-long pulses of voltage.
Images: Frank Reidy Research Center for Bioelectrics
Source www.papimi.gr
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