Electroporation Sensitivity of Oxidized Phospholipid Bilayers
Zachary A. Levine1,2, Yu-Hsuan Wu3, Matthew J. Ziegler1,3, D. Peter Tieleman4, and
P. Thomas Vernier1,5
1
MOSIS, Information Sciences Institute, Viterbi School of Engineering (VSoE), University of Southern California (USC), Marina del Rey, USA
2 Department of Physics and Astronomy, College of Letters, Arts, and Sciences, USC, Los Angeles, USA
3 Mork Family Department of Chemical Engineering and Materials Science, VSoE, USC, Los Angeles, USA
4 Department of Biological Sciences, University of Calgary, Calgary, Canada
5 Ming Hsieh Department of Electrical Engineering, VSoE, USC, Los Angeles, USA,
Introduction
Molecular dynamics (MD) studies showing that oxidized lipids increase the frequency of water defects in phospholipid bilayers suggest that the presence of
oxidized lipids in a bilayer will also increase the sensitivity of the bilayer to electropermeabilization. We confirmed this hypothesis in MD simulations of PLPC
(1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphatidylcholine) bilayers containing oxidized PLPC (oxPLPC), showing that pore creation is affected by the
oxPLPC species, its concentration in the bilayer, and the electric field strength. We also demonstrated that these effects can be localized to a specific region
of the bilayer. Experimental observations with living cells are consistent with the simulations.
Methods
All simulations were performed using the GROMACS 3.3.1 package and all
initial oxPLPC systems were derived from previous work on oxidized lipids [1].
The systems were composed of PLPC plus oxPLPC lipids (with 12-oxo-cis-9dodecenoate (12-al) or 13-hydroperoxy-trans-11,cis-9-octadecadienoate (13tc) at the sn-2 position) in concentrations of 0%, 11%, 25%, or 50% of the
total system, with the oxPLPC lipids distributed equally in the two leaflets of
the bilayer. Each system contained 72 lipids and 2880 water molecules (40
waters per lipid) and was energy minimized and equilibrated for 80 ns. A
simulation with a larger area was created by doubling a 72-PLPC bilayer in x
and y and then individually replacing PLPC lipids with oxPLPC lipids in two
opposing quadrants, creating a quilted system where two quadrants are
heavily oxidized (50% oxPLPC) and the two remaining quadrants contain only
PLPC. This enables us to test whether electroporation occurs preferentially in
oxidized regions of a bilayer. Periodic boundary conditions were employed to
mitigate system size effects and the integration time step was 2 fs. Shortrange electrostatics and Lennard-Jones interactions were cut off at 1.0 nm.
Long-range electrostatics were calculated by the particle mesh Ewald (PME)
algorithm using fast Fourier transforms and conductive boundary conditions.
Electroporation times were calculated by identifying the first simulation step in
which any phosphorus atom in one leaflet approached within 1.2 nm of any
phosphorus atom in the other leaflet.
PLPC
Electroporation time for 50% oxidized systems
versus applied electric field
Field
(V/nm)
Trial
Number
13-tc oxPLPC
12-al oxPLPC
0.15
0.20
0.25
0.30
Blue – Nitrogen
Gold – Phosphorus
Red – Oxygen
Molecular Dynamics Results 2
Cyan – Carbon (Single Bond)
Black – Carbon (Double Bond)
12-al
Pore Time
(ns)
Top View of a Quilted PLPC Bilayer Containing Localized oxPLPC Clusters
13-tc
Mean Time
(ns)
Pore Time
(ns)
1
> 25
2
9
3
19
> 25
1
4
> 25
2
8
3
6
14
1
6
15
2
3
3
3
5
1
5
10
2
4
3
2
Mean Time
(ns)
> 25
10
> 25
> 25
6
14
> 25
4
10
> 25
4
7
7
5
The time to create a hydrophilic pore decreases as the electric field increases,
with 12-al oxPLPC more susceptible to electroporation than 13-tc. Each
simulation ran for a total of 25 ns.
Molecular Dynamics Results 1
Before an electric field is applied
Shown here is our large quilted system which contains PLPC (white) and selectively placed oxPLPC (blue). The system is a 4x4
array of sectors containing alternately pure PLPC and 50% oxPLPC (12-al). After an electric field is applied, there is a clear
correlation between pore location and local oxidation clusters.
Electroporation time versus oxidized lipid concentration
Composite oxPLPC snapshots taken at 0.5 ns
intervals over a total time of 10 ns.
12-al oxPLPC
Facilitation of water entry into the bilayer interior
by oxPLPC aldehyde or hydroperoxy oxygens
all lipids shown
oxPLPC sn-2 tails shown
Mean
Phosphorus Plane
Pure
oxPLPC
lipids
have
a
tendency to bend their sn-2
tails towards the aqueous
interface.
This process does not
appear to be affected by
the presence of an external
electric field, though 13-tc
oxPLPC appears to bend
much more towards the
aqueous interface than
12-al oxPLPC.
12-al
1.3 ns
1.3 ns
13-tc
13-tc oxPLPC
Mean
Phosphorus Plane
oxPLPC
type
10.6 ns
14.6 ns
oxPLPC
%
Mean Bilayer
Thickness1
(nm)
Mean
Area/Lipid1
(nm2)
0%
3.62
0.65
11%
3.66
0.67
25%
3.49
0.69
50%
3.23
0.71
11%
3.59
0.67
25%
3.54
0.69
50%
3.33
0.72
Trial Pore Time
Number
(ns)
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
> 25
> 25
23
17
6
2
13
6
4
3
1
1
> 55
43
3
23
13
7
5
5
4
After an electric field is applied
Mean
Time
(ns)
Live Cell Results
Pulse-Induced YO-PRO-1 Uptake in Peroxidized Jurkat Cells
(30 ns, 3 MV/m, 50 Hz)
23
8
Normal, 0 pulses
Normal, 50 pulses
Peroxidized, 0 pulses
Peroxidized, 50 pulses
8
2
23
14
5
Pore creation time decreases as the concentration of oxidized lipids increases. A
field of 0.36 V/nm was used in all systems because it corresponded to a
‘minimum porating field’ for PLPC, a field we have previously defined as one
which electroporates a system in one of three trials within 25 ns [2].
Pulse-induced YO-PRO-1 uptake in peroxidized cells is significantly higher than in control cells
(1.8X in cells treated with 500 µM H2O2, 1 mM Fe2+, then 50 pulses).
Cells were treated with peroxidizing reagents for 10 minutes and pulsed immediately without washing.
Conclusions
Simulated bilayers containing oxidized lipids have an increased susceptibility to electroporation.
Average distance (nm) from tail oxygen or
PLPC C13 to the mean phosphorus plane
No Field
Field*
13-tc
0.32
0.34
12-al
0.60
0.73
PLPC
1.54
1.59
*At a strength of 0.36 V/nm
This increase in susceptibility is likely due to the facilitation of water transport into the bilayer
interior by hydroperoxy or aldehyde oxygens on the oxidized residues and may also be a
consequence of the fact that oxPLPC bilayers are thinner than pure PLPC bilayers.
[1] J. Wong-ekkabut, et al. Effect of Lipid Peroxidation on the Properties of Lipid Bilayers: A Molecular Dynamics Study. Biophys. J., 93: 4225–4236, 2007.
[2] M.J. Ziegler and P.T. Vernier. Interface Water Dynamics and Porating Electric Fields for Phospholipid Bilayers. J. Phys. Chem. B, 112(43), 13588–13596, 2008.
16.6 ns
Special thanks to the USC Center
for High Performance Computing
and Communications (HPCC) for
providing
the
computational
resources, and to MOSIS for
providing funding.
16.9 ns
Successive snapshots of electropore formation in an 11% 12-al
oxPLPC system. Average water penetration depth corresponds to
the average depth of oxygen atoms on the sn-2 tail. Only oxidized
sn-2 tails and water are shown.
MOSIS
The presence of an electric field does not appear to drastically change the average distance of
aldehyde or hydroperoxy oxygens to the aqueous interface.
Clusters of oxPLPC lipids attract a large number of individual waters into the bilayer interior,
creating localized regions of electroporation susceptibility.
MD results are consistent with experimental observations. Cells treated with peroxidizing agents
appear to electroporate more readily than untreated cells.