Lecture 28,April 8, 2016 ECEN
5341,4341
Electroporation and Electric Shock
. Frank Barnes
1
.
ECEN 5031/4031
Electroporation Chapter 9 Vol. 2
1. Creating small holes in membranes with short
electrical pulses. Typical pulses are 1µs< τ<
50ms and the required membrane voltages
are between
0.2V < Um <1V
2.The membrane concentrates the electric field as
it has a low conductivity σ = 10-7 S/m and
low dielectric εr =2 constant compared to the
inside of a cell and the fluid around it.
σ = 1S/m, and εr=80
Electroporation
• 1. High electric fields short pulse duration to
punch holes in membranes.
• 2. This allows us to pass molecules into the
cell such as DNA and chemo therapeutic
agents into a cell
3
Applications
1.
2.
3.
Inserting DNA, proteins , drugs
Fusing two cells
Born energy is the energy to move from
the fluid to the center of the membrane
Na+ 2rs=0.4nm Membrane thickness d = 5nm
4
Born Energy
The forces are largely local so that
Where Km is the relative dielectric constant of the membrane and Kw
is that of water
Spontaneous crossing of large molecules negligible
For rise times faster than moving charges or   3ns or f>300 MHz distributed
E field
6
For slower rise times   10 sec most of the voltage is across the membranes
The field strength E = 0.1 to 1kV/cm and 10kV/cm for bacteria to get
0.2 to 1.5V in 1μs to 10ms.
Models Parallel plate capacitor   ReCm where Re = resistance of the
electrolyte and Cm = membrane capacity
The E field across the membrane depends on the length of the pulse
Em 
Um
0.1V
7


2
x
10
V /m
9
d m 5 x10
5
An Electroporation System
• Gene Pulser Xcell™ Electroporation Systems
6
Specifications
Gene Pulser Xcell™ Total System
Includes main unit, CE module, PC module, and
ShockPod™ cuvette chamber
Outputs
Waveform: Exponential or square
Voltage: 10–3,000 V
Capacitance
10–500 V,
25–3,275 µF in 25 µF increments
500–3,000 V, 10, 25, 50 µF
Resistance (parallel)
50–1,000 Ω in 50 Ω increments, plus infinity
Sample resistance
20 Ω minimum at 10–2,500 V
600 Ω minimum at 2,500–3,000 V
Square-wave timing
10–500 V: 0.05–10 ms in 0.05 ms increments, 10–100 ms
pulse in 1 ms increments, 1–10 pulses, 0.1–10 sec interval
500–3,000 V: 0.05–5 ms in 0.05 ms increments, 1–2
pulses, 5 sec minimum interval
7
Another Electroporation Device
• 1
8
Pulse Characteristics
• 1. Creating a small hole in the membrane.
• 2. Pulses 0.2V<V< 1V 1µs<τ<50ms
•
E
Membrane εm =2-4εo
Cell interior εi =50-80εo
9
Pulse Characteristics
• 1. Times greater than 1µs required to
concentrate the charges and fields.
• 2. Fields greater than 0.1KV/cm often greater
than 1KV/cm. Field varies with position on
the cell surface.
• 3. High fields and short pulses give the best
results
10
Typical Pulse
11
Fields for a Spherical Cell
12
Pore Formation 4 Stages
• 1. Charging the membrane
• 2. Constant Voltage Vm , small currents
• 3. Fluctuating current as transition to long
lived excited state,10minutes large σ, low V
• 4. Increase in pulse length leads to saturation
and irreversible damage.
log10 τm ~ Vm
13
A Molecular Dynamics Simulation
14
Some Theory for Pore Formation
15
Pore Formation Dynamics
16
Ion Transport Through a Pore
17
Some Theories for Electroporation
• 1. Hydrodynamic Flow
• 2. Compression
• 3. Pores that lead to membrane weakness that
is local.
• 4. Pore formation becomes significant from
thermal processes plus applied field when
• Um >~ 0.5 V
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Reversible Pore Formation
• 1. Vm≈ 1V τm≈ 400ns rp≈ 0.8nm
• 2. R drops by 109
• 3. Rapid discharge and the membrane
reforms.
• 4. Relaxation times for a fluid τf=εf/σf
– for saline τ f τ≈ 0.5ns much less than the charging
time Vm
50µs
0.4
19
Energy
• 1. Energy of a charge in the medium
W 
1 2
E V
2
The essential barrier function of cell membranes
can be represented by a thin sheet of lipid. To
move a charge through the membrane
W 
e2
[
1
8o rs  m

1
w
]  100kT
20
Forces
• 1. E Fields on the Membranes are a function
of time. Increase and then Decrease
• 2. Force the opening in the pores that expand
with time.
• 3. E fields drive currents and carry along
neutral molecules.
21
Membrane Recovery
• 1. Reported times vary from nanoseconds to
minutes or hours.
• 2. Strongly depends on Temperature
• 3. Depends on the size of the pore also on
what molecules or ions are being
transported.
22
Cell Stress And Survival
• 1. Cell survival and stress are mainly to exchange of molecules
with the environment. Chemical or ion imbalances.
• 2. Cells can be killed without significant heating.
• 3. There is a fuzzy threshold for the transport of molecules into or
out of the cells and not a large margin to cell death.
• 4. Transport is not very selective with respect to molecules or
ions.
• 5. Survival seems to go with the ratio of the external volume to
the internal volume of the cell. In vitro Vex/Vin is large and
favors cell death In vivo it is the reverse with Vex/Vin≈0.15
• 6. This mean that in vivo cell damage for the same pulses are less
likely.
23
Tissue Electroporation and In Vivo
Delivery
• 1 A purposeful electroporation of tissue in vivo
and in vitro has been motivated by therapeutic
interventions such as tumor treatment by
delivery of anticancer drugs ,gene therapy by
delivery of DNA, and other genetic material and
delivery of various sized molecules into and
across the skin
• 2. Also tissue electroporation may be relevant to
• neuromuscular incapacitation (stunning) pulses
24
Voltage Concentration in Tissue
• 1. Voltage concentration in tissue needs to be
across the membranes.
• 2. For preferential electroporation, two features
should be sought:
• (I) tissue barriers comprised mainly of lipids
• (ii) mechanical deformability (compliance) of
membranes comprise of the particular lipids so
that the electrostatically favored entry of water
into a deformable phospholipid-based membrane
results in the creation of aqueous pathways.
•
25
Current Flows in Tissue.
• 1. At low fields most of the current flows around
the cell membranes.
• 2. After electroporation much of the current
flows through the cell.
• 3 Tumor tissue is an important example of tissue
for which many cells have intercellular aqueous
pathways. Even without electroporation there is a
significant physiological resistance to entry of
anticancer drugs because of limited blood
perfusion, elevated interstitial pressure, and
relatively large distances to blood vessels
26
Application to Tumors.
• 1. Without electroporation there is a significant
physiological resistance to entry of anticancer
drugs because of limited blood perfusion, elevated
interstitial pressure, and relatively large distances
to blood vessels
2. Local tissue electroporation should create aqueous
pathways that assist drug movement and that may
also relieve pressure, but the fourth power
dependence of volumetric flow on pathway size
implies that significant water flow may be more
difficult than diffusion and drift of small drugs.
27
Applications
• 1. Electroporation has value largely in cancer treatment
for drugs that to not go through the membranes
naturally. Bleomycin is an example where
electroporation helps.
• 2. Transdermal Drug delivery through skin.
– The stratum corneum is the main barrier plus sweat ducts
and hair follicles.
• 3. The double cell lining of sweat ducts should
experience electroporation at about U =2 to 4 V,
but the approximately 100 bilayers of the SC need
U =50 to 100V for pulses with duration of 100 ms
to 1 ms, i.e., about 0.5 to 1V per lipid bilayer
barrier
barrier
28
Skin
• Experiments of this type with human skin show that if
exponential pulses with Voltage across SC,0 50 to 300V
and time constant, t, pulse 1 ms are applied every 5 s
for 1 h, then there is an enhancement by up to a factor
of 104 in the flux of charged molecules of up to about 1
kDa Companion electrical impedance measurements
show a rapid (25 ms) decrease in skin resistance and
both molecular flux and electrical measurements show
that either reversible or irreversible behavior occurs,
depending on the transdermal pulse amplitude,
Voltage SC, 0. Several in vivo experiments show that
transdermal delivery can be achieved with minimal
damage.
29
Gene Therapy
• 1. Gene therapy also requires movement of
large molecules through the cell walls.
• 2. You only need succeed with some of the
cells to be effective. Smaller longer pulses.
• 3. Concerns about damage.
• 4. Surfactants can improve membrane
recovery.
30
Electroporation of Organelles
• 1 Short high field pulses (ns) Um = 1.2V
•
E= 106V/m
• 2. Many small pores in outer membrane as
well as in the organelle
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Lecture 29 April 11,2016
• Read chapters 9, 10, and 12.
• Two paper reviews due this week.
• A possible topic of interest would be the use
of small electrical signals to control various
biological processes. See what you can find.
32
Electric Shocks Trauma
• 1. 21% of the burns in 2000-2001
• 2. Electrocution 5th leading cause of death for
occupational injuries
• 3. More than 90% of these injuries for utility
workers occur in men, mostly between the
ages of 20 and 34, with 4 to 8 y of experience
on the job [4].
• 4. For survivors, the injury pattern is very
complex, with a high disability rate due to
accompanying neurologic damages and loss
of limbs.
33
Low Voltage Shocks
1. Low voltage shocks < 1000 V mostly minor neural
damage.
• 2. Low-voltage shocks are more likely to produce
a prolonged, ‘‘no-let-go’’ contact with the power
source. This ‘‘no-let-go’’ phenomenon is caused
by an involuntary, current-induced, muscle
spasm. For 60 Hz electrical current the ‘‘no-letgo’’ threshold for axial current passage through
the forearm is 16 mA for males and 11 mA for
females
34
Lightning
• There are roughly 200 human deaths annually in
the United States due to lightning strikes and
there are three times those many who survive.
The range of lightning injury extent is quite
broad, depending upon the magnitude of
exposure and the condition of the victim. Usually
lightning hits result in surface burns, complex
neurological damage similar to blunt head
trauma, peripheral neurologic injury, and cardiac
damage
35
Current Flow +RF
• 1 Current flow is along the paths of low
resistance. In the extra cellular medium and in
long cells as they have a small fractional
volume of membrane.
• 2. At RF and Microwaves membranes are less
of barrier and the absorption coefficient and
dielectric constants. determine the
distributions of currents.
36
AC vs DC
• Alternating current (AC) is more dangerous than
direct current (DC), and 60-cycle current is more
dangerous than high-frequency current. AC is
said to be four to five times more dangerous than
DC because AC causes more severe muscular
contraction. In addition, AC stimulates sweating
that lowers the skin resistance. Humans and
animals are most susceptible to frequencies at 50
to 60 hertz because the internal frequency of the
nerve signals controlling the heart is
approximately 60 hertz. (Electric Shock
Precautions)
37
Physiological Effects of Shock
Table 1. Physiological Effects of Shock
Electric Current (1sec.
Contact)
Physiological Effect
Voltage required to produce the current
with assumed body resistance:
100,000 ohms
1,000 ohms
1 mA
Threshold of feeling, tingling
sensation.
100 V
1V
5 mA
Accepted as maximum harmless
current.
500 V
5V
Beginning of sustained muscular
contraction ("Can't let go” current).
1000 V
10 V
Ventricular fibrillation, fatal if
continued. Respiratory function
continues.
10,000 V
100 V
Sustained ventricular contraction
followed by normal heart rhythm
(defibrillation). Temporary respiratory
paralysis and possibly burns.
600,000 V
6000 V
10-20 mA
100-300 mA
6A
38
Safety Guide Lines
• 1
The National Electrical Code (NEC) in the U.S. considers 5 mA
(0.005Amps) to be a safe upper limit for children and adults;
hence the 5 mA Ground Fault Interrupter (GFI) circuit breaker
requirement for wet locations. (The Physical Effects of
Electricity) The values in Table 1 should be used as a guide
instead of absolute data points. For instance, 99% of the
female populations have a “let go” limit above 6 mA with an
average of 10.5 mA. 99% of the male populations have a “let
go” above 9 mA, with an average of 15.5 mA. (The Physical
Effects of Electricity)
Ventricular fibrillation can occur at current levels as low as 30
mA for a two year old child and 60 mA for adults. Most adults
will go into ventricular fibrillation at hand to hand currents
below 100 mA (0.1 Amp). (The Physical Effects of Electricity)
39
40
Will the 120 volt common household voltage
produce a dangerous shock? It depends!
•
•
•
•
•
•
•
•
•
•
•
•
•
•
If your body resistance is 100,000 ohms, then the current which would flow would
be:
I =
120 volts = .0012 A = 1.2 mA
100,000 Ω
This is just about at the threshold of perception, so it would only produce a tingle.
If one had just played a couple of sets of tennis, and is sweaty and barefoot, then
the resistance to ground might be as low as 1000 ohms. Then the current would be:
I =
120 volts
1,000 Ω
= .12A = 120 mA
This is a lethal shock, capable of producing ventricular fibrillation and death! The
severity of shock from a given source will depend upon its path through the body.
(Nave & Nave)
41
Body Resistance
Table 2. Typical Human Body Resistance to Electrical Current
Body Area
Resistance (ohms)
Dry Skin
100,000 to 600,000
Wet Skin
1,000
Internal body (hand to foot)
Ear to Ear
400 to 600
~100
Table 2 shows some of the typical human body resistances to electrical
current. Barring broken skin, body-circuit resistance, even in contact with
liquid, will probably be not less than 500 ohms. However, the current flow at
this resistance and 120 volts is 240 mA—over twice what is required to cause
death. (Biological Effects of Electric Shock)
42
Mechanism vs Frequency
43
Current Flow
44
Tissue Damage
Thresholds 1KV/cm to 60 V/cm for long nerve or muscle cells
45
Electrical Damage
• A burn from electrocution is much different than a burn from
scalding or fire. Fleshy tissue is destroyed at 122° F and vascular
tissue serving the nerves suffers damage at considerably less.
Victims of industrial high-voltage accidents will present to the
emergency room with obvious thermal destruction at the skin
contact points. The extremities may be slightly swollen and
otherwise without visible surface damage. Yet beneath the
involved skin, the skeletal muscle will often exist in a state of severe
unrelenting spasm or rigor. There will be frequently marked
sensory and motor nerve malfunction. Within the first week after
injury, many victims will undergo sequential surgical procedures to
remove damaged nonviable skeletal muscle, resulting in a weak,
stiff extremity that is often anesthetic because of nerve damage,
and cold because of poor circulation. Under these circumstances,
the patient is better off by undergoing amputation and then
receiving a prosthetic extremity. (R. Lee 223-230)
46
Muscle Damage
• In general, muscle and nerve appear to be the tissues
with the greatest vulnerability to injury by electrical
current. There is a characteristic skeletal muscle tissue
injury pattern in victims of high-voltage electrical shock
which is relatively unique to shock victims. Muscle
adjacent to the bone and joints is recognized clinically
to be the most vulnerable to electrical trauma. In
addition, muscle cells located in the central region of
the muscle may also be vulnerable and nerves seem to
have a lower threshold for damage than muscle.
• (R. Lee 223-230)
•
47
MRI Image
48
A Relatively New Use of Shocks
• 1. To prevent over stimulation of the immune
system that lead to inflammation for
alleviating the pain, swelling and tissue
damage that is a consequence of many
diseases.
• Kevin J. Tracey, Scientific American (March
2015), 28-35 Published online: 17 February
2015 | doi:10.1038/scientificamerican0315-28
49
Reflex Signals
• 1. Reflex signals are automatic responses to an
event. Like heart rate to need for oxygen when
running.
• 2. They are coordinated nerve firing that
release drugs that carry out the desired
function.
• 3. There is an advantage to having the nerve
cells control the release of the desired drugs
or molecules only where you need them.
50
A reflex system controls the immune
system.
• 1. A signal from the brain through the vagus
nerve can block the generation of tumor
necrosis factor, or TNF, which in turn can turn
off the inflammatory process.
• 2. You can do this with a small electrical signal
that stimulates the vagus nerve.
51
More Details
• Fibers deep within the spleen release a signaling
molecule, norepinephrine, that then binds to
immune system cells called T lymphocytes.
Norepinephrine attaches to receptors on T cells,
which trigger production of another
neurotransmitter, acetylcholine, that binds to
receptors on immune cells called macrophages,
which produce TNF in the spleen. Acetylcholine
docking onto the receptor—abbreviated α7
nAChR—causes macrophages to shut down TNF
production by inhibiting two molecular pathways.
52
Some Speculation
• 1. This kind of work using electrical
stimulation of specific nerves could lead to
some new result in many areas of medicine.
• 2. You might be able to build on acupuncture
with respect to what nerves control different
processes and where you might stimulate
them.
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