The Interaction(s) of Pulsed
Electromagnetic Fields and Biology
Bioenergy Program Review
8 August 2012
Bennett L. Ibey
Radio Frequency Radiation Branch
Air Force Research Laboratory
DISTRIBUTION STATEMENT A. Approved for Public Release PA# 12-0079
Overall AF Research Goal
Fundamental Research Question:
What happens when high power electromagnetic pulses interact
with biological material?
Motivation:
•
•
•
Quantify Bioeffects (direct delivery/free field)
Develop Technology
Understand exposure impact to
military and civilian personnel
p2pnet.net
Simplify to understand basic mechanisms
random-blog.info
micro.magnet.fsu.edu
Interstitial Fluid
Cytoplasm
Nuclear
Membrane
nlm.nih.gov
Whole Body
Organs & Tissues
Cell
Progress from cell, to tissue, to whole organism
Plasma
Membrane
Membranes
2
Interaction between Cells and
Electric Fields
µs
Hours
mV
ELECTROTAXIS
kV
DEFORMATION
PORATION
Corovic et al. 2009
Overzealous Hypothesis: The mechanism
Riske et al. 2008 for these diverse interactions
spanning a large parameter space may be the same or at least the same to
the cell.
Question: What happens when cells are exposed to electrical pulses shorter
than
1Jus
at amplitudes exceeding
K. Antonova et al. 1MV
2010 EPL
http://www.inovio.com/
Sato M
et al. and
PNAS 2009
3
Cell Circuit Model
• Seminal Hypothesis: Pulse widths below ~100ns will selectively
permeabilize intracellular components and not impact the
plasma membrane
• Early publications supported this hypothesis through
demonstration of apoptotic initiation without uptake of
propidium ions (accepted electroporation marker)
Project Objective: Determine the mechanism(s) of interaction between
electromagnetic pulses and tissue below µs duration (with frequency
components in the MHz and GHz range).
14 August 2012
Schoenbach et al., 2001
4
Nanosecond Electrical Pulses – “Bringing
the EM Pulse to the tissue”
Nanosecond pulsing system for cellular exposure
• Born out of plasma generator technology
• Broad frequency composition
• Fast rise and fall times
• Very precise timing delays (<ns) using Stanford System
• Single or Multiple pulses (max. rep. rate 5Hz)
• Multiple pulse widths (10, 30, 60, 200, 400, 600)
Custom Pulse
Delivery System
Oscilloscope
High Voltage Power Supply
Dr. Shu Xiao
Old Dominion University
5
Nanosecond Electrical Pulses – “Bringing
the EM Pulse to the tissue”
0-500V across 120µm gap
0-30 KV/cm
6
Fundamental Observation
14 August 2012
7
Patch Clamp Results
Patch Clamp Findings
•
•
•
Single 60ns Exposure
•
Nanosecond pulses cause
immediate changes in plasma
membrane potential
Increased current at negative
command voltages suggests
movement of ions
Membrane Potential takes up to
15 minutes to recover
Effect seen in many cell types
and appears to be dose
dependent
Pakhomov et al. ABB. 2007
Pakhomov et al. BBRC 2009
Ibey et al. Bioelectromagnetics 2009
Ibey et al Bioelectrochemistry 2010
Ibey et al. SPIE 2009
Dr. Andrei Pakhomov
8
Fluorescent Microscopy Results
nsEP
Ibey et al., Bioelectrochemistry, 2010
Pakhomov et al, BBRC 2009
9
Molecular Dynamics Simulation of
Phospholipid Bilayer
Dr. P. Thomas Vernier
University of Southern
California
•Invited Speaker to AF Workshop
Bioelectromagnetics Conference
June 17th, 2012
•Invited Speaker PEMB
Workshop, 2011
•Committee Member, THz and
nsEP, SPIE 2013
10
Overarching Hypothesis
•
Exposure of cells to nanosecond electrical pulses results in the
formation of small pores (nanopores) in the plasma membrane as
opposed to the large pores formed from classic electroporation
Before Pulse
During Pulse
After Pulse
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Nanopores Get Bigger with Dose
Membrane Reorganization
nsEP
Vernier, P.T., et al. Biophys J, 2004.
Membrane Permeabilization
nsEP
Pakhomov, et al. BBRC 2009
Pakhomov, et al. BBRC 2009
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Mechanism of Poration
•Nanoporation is devoid of PI
influx but has been shown to
allow for influx of calcium ions,
thallium ions and PS expression
•Proposed MechanismElectrodeformation
F
PD
+
FITC -Annexin V
Key References:
-Ho, S.Y. and G.S. Mittal, Electroporation of Cell
Membranes: A Review. Critical Reviews in
Biotechnology, 1996. 16(4): p. 349 - 362.
-Teissie, J., M. Golzio, and M.P. Rols, Mechanisms
of cell membrane electropermeabilization: a
minireview of our present (lack of ?) knowledge.
Biochim Biophys Acta, 2005. 1724(3): p. 270-80.
-Zimmermann, U., et al., Fundamentals and
Application. IEEE Transactions on Plasma
Science, 2000.
13
Overarching Hypothesis –
Electrodeformation forms Nanopores
Classic Electrodeformation
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Research Outline
• Single Cell Research
– Nanopore formation
– Intracellular pathways
– Neurological Impact
– Nucleus Effects
– Membrane Recovery
• Multiple Cell Research
– Toxicity/Cell Death
– Genetic Response
• Free-Field Research
– Bipolar Pulses
– Exposure Modeling
– Cellular Exposure
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Electromechanical Transduction
To measure the subcellular bioeffects of
nsEPs on cells.
1. Visualize and characterize local mechanics
of porated areas using atomic force microscopy
(AFM).
2. Determine the contribution of mechanical
properties of cells to susceptibility to nsEPs.
3. Map changes in current density of cell
membrane during application of nsEPs using
scanning ion conductance microscopy.
4. Localize nsEP exposure using ultra-precise
positioning and field isolation using an AFM
probe tip.
Dr. Gary Thompson
(NRC-Postdoc)
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Application of nsEP by Nanoelectrode (Dr. Gary
Thompson)
CAD image of
AFM probe
XFDTD
model
Mechanical
nsEP
SEM
Before
Exposure
Thompson et al, SPIE 2011
1 V, 5 sec
10 V, 5 sec
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Investigating Temperature Effects on
nsPEF Efficiency using Extrinsic Dyes
• Impact of phospholipid polarity on nsPEF poration
–
–
–
–
–
PRODAN dye
Emission relative to membrane phase
Temperature directly impacts emission
Rapid lifetime (<2ns)
Does poration impact phospholipid polarity?
• System: Dual monochromator
spectrometer
– UV capable
– Continuous stirring
– Temperature control
– Enable high speed ratiometic
recording (1000 sample/sec)
http://www.kutztown.edu/acad/chem/instruments_html/fluorescence.htm
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Phospholipid Polarity and nsPEF
• Temperature Effects
Temp (20-40°C)
Results:
• Increased fluorescent
with time after nsEP
• Mimics that seem with
sustained 5 degree
temperature increase
Mrs. Samantha Franklin
Ph.D Student UTSA - Physics
• nsPEF Effects
Time (min)
Swelling!
Poration!
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High Speed Calcium Imaging Following
nsEP Exposure
Dr. Hope Beier
AFRL-RHDO
NG108 Cell
Tungsten
Electrodes
Beier et al. BBRC 2012 20
Activation of Intracellular Pathways by
nsPEF (Dr. Gleb Tolstykh)
• Tracking PIP2 hydrolysis with PLC-δPH-GFP
Oxotremorine
1x600ns, 18.2 kV/cm
Dr. Gleb Tolstykh
Neuroscientist
MD,PhD
UTHSCA
(NRC-Senior
Scientist)
21
Activation of Intracellular Pathways by
nsPEF (Dr. Gleb Tolstykh)
• Diacil glycerol (DAG) Tracking with EGFP-C1 domain of PKC
Oxotremorine
1x600ns, 18.2 kV/cm
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Protein Kinase C Activation
mApple-Actin
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Amplification of Ca2+ signal
Ratiometric Calcium
Imaging with Fura-2
nsEP
Dr. Andrei
Pakhomov
Removal of Ca2+
external Ca2+
uptake
Ca2+ release
from ER
Dr. Iurii
Semenov
Thapsagargin
increase of cytosolic Ca2+
Edelfosine
activate IP3R
IP3
Dr. Gleb
Tolstykh
activate PLC
Live Cell Imaging with
PLC-PH-GFP and C1-GFP
PIP2
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Impact of nsEP on Excitable Cells
(Mr. Roth)
Neurological Impact of nsEP
Hypothesis: That nsEP exposure to neurological tissue will cause inhibition of
action potentials for an extended duration by formation of nanopores
Overly Simplified Drawing
nsEP
nsEP
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Impact of nsEP on Excitable Cells
(Mr. Roth)
•
Calcium detection seems to be a sensitive method by
Probit analysis can be used to determine ED50 for
nanoporation
•
NG108 and PHN appear to have very similar ED 50 for
calcium uptake in both pulse width and pulse amplitude.
•
Future: Expose neurons in vitro with patch clamp
analysis to determine if nanoporation will reversibly
inhibit action potential transmission
•
Future: Explore channel blockers (TTX, Conotoxin,..) to
determine if what was seen in chromaffin cells holds true
for NG108 and HPN
Roth et al. Submitted to JBO, 2012
Mr. Caleb Roth
New SMART Student
Radiation Biology UTHSCA
26
nsEP Biostimulation
• Adrenal Chromaffin Cells:
– Shown to be sensitive to nsEP stimulation
– Release Epinephrine upon stimulation
– Possibility for standoff pulsed RF exposure to
cause sudden spike in blood stream adrenalin
– High speed imaging and patch clamp experiments
planned for TSRL in the fall
Dr. Gale Craviso
Department of Pharmacology
University of Reno, Nevada
Invited Speaker to AF Special Session at the
Bioelectromagnetics Conference June 17th, 2012
14 August 2012
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Cardoso’s “One Ring” image, which I think is the
best!
How does cell cycle
effect nanoporation?
Ms. Megan Mahlke
UTSA Biology Department
Captain Marjorie Kuipers
Ph.D Molecular Biophysics
28
H2B-GFP, PCNA-RFP CHO cell
10nm
https://wikispaces.psu.edu/display/Biol230WFall0
9/DNA+and+Chromosomes
• Histone-2B is a critical component
of the first level packaging of DNA
and highly conserved structure.
GFP-H2B &
RFP-PCNA
CHO cells
4nm
http://en.wikipedia.org/wiki/Proliferating_cell_nuclear_antigen
• Proliferation cell nuclear antigen (PCNA) is a
protein critical to DNA synthesis that
promotes replication
3D Image of molecular
overlap in nucleus in
normal cells (PCNA is
not localized to nucleus
during cellular division
(right)
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Effects of nsEP on H2B stability
600ns @ 18.2 KV/cm
20 Pulses
2mM Calcium
0 sec
30 sec
600ns @ 18.2 KV/cm
20 Pulses
2mM Calcium
0 sec
30 sec
• Photo-bleached line shows no migration of DNA within the nucleus despite
organization changes observed in chromatin
• Despite limited morphological changes a pronounced change in nuclear
morphology is evident.
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Effects of nsEP on PCNA Location
• PCNA leaking from
Nucleus after nsEP
exposure occuring in both
calcium rich and depleted
environments.
• Noted lack of blebbing with
calcium in the outside
solution, but leakage is
maintained
• Third image is a cell
undergoing metaphase
cycle of mitosis when
PCNA is already outside
the nucleus and mobile.
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Membrane Recovery
Methods
• Morphology
• Fluorescence
• Calcium
• PI
• FM1-43
Dalzell et al., SPIE 2011
Calcium
Green
Calcium rich outside solution
Propidium
Iodide
Calcium-free outside solution
FM1-43
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Membrane Recovery – Lysosomal
Exocytosis?
RFP-LAMP1 CHO cell
Before
After
GFP-Tubulin CHO cell
Before
After
(McNeil et al. Nature 2005)
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Research Outline
• Single Cell Research
– Nanopore formation
– Intracellular pathways
– Neurological Impact
– Nucleus Effects
– Membrane Recovery
• Multiple Cell Research
– Toxicity/Cell Death
– Genetic Response
• Free-Field Research
– Bipolar Pulses
– Exposure Modeling
– Cellular Exposure
34
Dose Required for Cell Death at
Different Pulse Widths
Dr. Olga Pakhomova
Old Dominion University
Ibey et al. BBA General Subjects, 2010
35
Cellular Death Following nsPEF (Mr. Roth)
Cellular Lethality by 10ns pulses
Isolated Exposure
Ibey et al. PLoS ONE 2011
Combined Exposure
Hypothesis: Physiological differences amongst cell types determine vulnerability
to nsEP stress
36
Atomic Force Microscopy
Results
• Hypothesis: Mechanical rigidity of cells impacts sensitivity to nsEP
Dr. Gary Thompson
(NRC-Postdoc)
37
Electric Pulse Induced Cellular StressGenetic Changes
Dr. Gerald Wilmink
38
Comparison and Pathway
Analysis
•
•
•
Very Few Similar Genes
Different pathways activated
Suggests difference in cellular
response to nsEP that may be
rooted in physiological function
Jurkat - Cell Morphology
U937 - Cell Death
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Unearthing the Proteomic Response to
nsPEF Stimulation
•
Understanding the kinetics of
cell death due to nsPEF
• Mapping the proteomic
response using Luminex Assay
Technique
• Investigate the impact on
nsPEF on cellular metabolism
using Seahorse Methodology
Mr. Erick Moen
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Deliverables
Papers
•
Vincelette RL, Roth CC, Payne JA, Ibey BL, “Nonlinear trends in the dose response of CHO cells exposed to ultra-short electrical pulses (USEP)”
In Preparation, 2012.
•
Roth CC, Kuiper MA, Tolstykh G, Ibey BL, “Nanopore formation in exciteable cells” submitted to JBO, 2012
•
Nesin V, Pakhomov AG “Inhibition of voltage-gated Na(+) current by nanosecond pulsed electric field (nsPEF) is not mediated by Na(+) influx or
Ca(2+) signaling” Bioelectromagnetics 2012.
•
Nesin V, Bowman AM, Xiao S, Pakhomov AG. “Cell permeabilization and inhibition of voltage-gated Ca(2+) and Na(+) channel currents by
nanosecond pulsed electric field” Bioelectromagnetics. 2012
•
Beier HT, Roth CC, Tolstykh GP, Ibey BL. “Resolving the spatial kinetics of electric pulse-induced ion release” BBRC 2012
•
Ibey BL, Roth CC, Pakhomov AG, Bernhard J, Wilmink GJ, Pakhomova ON “Dose-dependent Thresholds of 10ns Electric Pulse induced Plasma
Membrane Disruption and Cytotoxicity in Multiple Cell Lines” PLoS OnE, 6(1), 2011.
•
Ibey BL, Pakhomov AG, Gregory BW, Khorokhorina VA, Roth CC, Rassokhin MA, Bernhard JA, Wilmink GJ, Pakhomova ON, “Comparative
cytotoxicity of intense nano- and microsecond-duration electric pulses in mammalian cells”, Biochimica Biophsyica Acta, Nov; 1800(11):1210-9,
2010.
Selected Proceedings
•
Thompson GL, Payne JA, Roth CC, Wilmink GJ, Ibey BL, “Local plasma membrane permeabilization of living cells by nanosecond electric pulses
using atomic force microscopy,” SPIE BiOS Photonics West, - Energy-based Treatment of Tissue and Assessment VI, San Francisco, CA,
2011
•
Dalzell DR, Roth CC, Bernhard JA, Payne JA, Wilmink GJ, Ibey BL, “Lysosomal exocytosis in response to subtle membrane damage following
nanosecond pulse exposure,” SPIE BiOS Photonics West, 2011- Energy-based Treatment of Tissue and Assessment VI, San Francisco,
CA, 2011
•
Roth CC, Payne JA, Wilmink GJ, Ibey BL, “Nanopore Formation in Neuroblastoma Cells Following Ultrashort Electric Pulse Exposure” SPIE
BiOS Photonics West— Photons and Neurons, San Francisco, CA, 2011
•
Ibey BL, Roth CC, Bernhard JA, Pakhomov AG, Wilmink GJ, Pakhomova O, “Determination of Cellular Injury and Death thresholds following
exposure to high voltage 10ns electrical pulses.” SPIE BiOS Photonics West, - Energy-based Treatment of Tissue and Assessment VI, San
Francisco, CA, 2011
•
Pakhomova ON, Ibey BL, Gregory BW, Khorokhorina VN, Pakhomov AG “Evaluation of Cytotoxic Efficiency of Nano- and Microsecond Electric
Pulses.” 6th International Workshop on Biological Effects of Electromagnetic Fields, Akyarlar, Bodrum, Turkey, October 2010
41
Team Building
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Mr. Caleb Roth, Project Start
Dr. Gerald Wilmink, Project Start
Dr. Andrei Pakhomov (ODU), Project Start
Dr. Olga Pakhomova (ODU), 2010
Dr. Rebecca Vincelette (NRC), Project Start-2010
Lt Dalzell, Jan 2010-2012
Dr. Hope Beier (RHDO), Jan 2010
Captain Kuipers, Jan 2011
Mrs. Samantha Franklin (Consortium), May 2011
Dr. Gary Thompson (NRC), May 2011
Dr. Gleb Tolstykh (NRC), Oct 2011
Ms. Megan Malhke (Consortium), Nov 201
Mr. Erick Moen (USC – Student)
Dr. P. Thomas Vernier (USC), Future Collaborator
Dr. Gale Craviso (Univ of Reno),
42
Acknowledgements
Collaborators/Co-Investigators:
Dr. Hope Beier
Mr. Caleb Roth
Mrs. Samantha Franklin (Consortium)
Mrs. Megan Malhke (Consortium)
SrA Joshua A. Bernhard
Mr. Jason Payne
Dr. Jerry Wilmink
Dr. Ibtissam Echchgadda
Dr. Marjorie Kuipers
Dr. Gary Thompson (NRC)
Dr. Gleb Tolshyk (NRC)
Air Force Research Laboratory
Directed Energy Bioeffects Division
Frank Reidy Research
Center for Bioelectrics
Dr. Mauris DeSilva (NAMRU-SA)
Dr. Andrei Pakhomov
Dr. Olga Pakhomova
Dr. Xiao Shu
CBE
OLD DOMIN IONUN IVERS ITY,NORF OLK,VA
Supported by:
- Air Force Office of Research LRIR 09RH09COR
- NRC Postdoctoral Fellowship, and Director’s Funds 711th Human Performance Wing
- Old Dominion supported by NIH R01CA125482 from the National Cancer Institute
- Air Force Research Laboratory under U.S. Air Force Contract (FA8650-07-D-6800)
awarded to General Dynamics Information Technology, Brooks City-Base, San Antonio, TX.
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