22nd International Symposium on Plasma Chemistry
July 5-10, 2015; Antwerp, Belgium
Plasma interaction with phospholipid bilayer: molecular dynamics simulations
M. Yusupov, J. Van der Paal, C.C.W. Verlackt, N. Khosravian, E.C. Neyts and A. Bogaerts
Research group PLASMANT, Department of Chemistry, University of Antwerp, 2610 Antwerpen-Wilrijk, Belgium
Abstract: Atomic scale reactive molecular dynamics simulations based on the density
functional-tight binding method are performed to investigate the interactions of reactive
oxygen (i.e., OH, O 2 , O 3 and H 2 O 2 ) and reactive nitrogen (i.e., NO, NO 2 and ONOO-)
species with a phospholipid bilayer, which is the simple model system for the cell
membrane. Furthermore, the results of the reactive simulations are used in non-reactive
molecular dynamics simulations (based on the coarse-grained Martini model) to study the
further processes (e.g., membrane permeability and fluidity, as well as pore formation),
which require long timescale simulations.
Keywords: molecular dynamics, reactive oxygen and nitrogen species, phospholipid
1. Introduction
In recent years, the use of low temperature (or cold)
atmospheric pressure plasmas (CAPPs) for biological and
medical purposes, i.e., so-called “plasma medicine” is
gaining increasing interest in plasma research [1]. Plasma
medicine is a relatively new (i.e., it has been about 20
years since CAPP sources are used for healthcare
purposes [2]) and very much a multidisciplinary field on
the intersection of medicine, physics, chemistry, biology,
etc. Nowadays, the use of CAPPs for cancer treatment
(so-called “plasma oncology”) is one of the most exciting
sub-topics of plasma medicine [3]. The (mammalian)
cancers investigated up to now include breast cancer,
acute lymphoblastic leukemia, pancreatic cancer,
melanoma, lung cancer, prostate cancer, etc. [3].
It is known that CAPP sources produce reactive oxygen
and nitrogen species (ROS/RNS/RONS, e.g., OH, O 2 , O 3 ,
H 2 O 2 , NO, NO 2 , ONOO-), which are generally believed
to be the key to the success of plasma oncology [4].
Although many experimental works have already been
carried out so far, the underlying physical and chemical
processes, especially the interaction of reactive plasma
species (i.e., RONS) with biomolecule systems, are not
yet fully understood. In order to control the processes
occurring in the contact region of the plasma with the bioorganisms there is a need to deeply investigate the
interaction mechanisms of the plasma-generated species
with biomedically relevant structures.
While actual experiments are clearly indispensable,
computer simulations can also be very valuable as they
provide fundamental insight in the interaction processes,
allowing to investigate the underlying mechanisms in
detail, which is extremely difficult to achieve
experimentally. However, to date very limited attention
has been paid to detailed investigations of RONS
interacting with biomolecules.
Based on the above mentioned considerations, in this
work, we perform reactive molecular dynamics (MD)
simulations applying the density functional based tight
binding (DFTB) method to investigate the interaction
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mechanisms of RONS with a phospholipid bilayer (PLB),
which is the simple model system for the cell membrane.
Moreover, by using the outcome of the reactive MD
simulations, we perform non-reactive MD simulations
based on the coarse-grained Martini model to study the
subsequent processes, which require long simulation
times.
2. Description of the simulations
As mentioned above, in this work, we make use of two
different simulation techniques, i.e., DFTB and a coarsegrained model. For the reactive simulations, i.e., to
investigate bond breaking and formation processes by
RONS originating from the CAPP, the DFTB method is
used.
2.1. DFTB method
DFTB is a very efficient tool to perform quantum
mechanical calculations. It is an approximate density
functional theory (DFT) method [5], based on a Taylor
series expansion of the DFT total energy expression. The
computational speed of this method is similar to
semi-empirical methods like AM1 and PM3 [6]. We use
the so-called DFTB3 method [7], which is the extended
and improved version of previous DFTB approaches,
which is derived from a third order expansion of the DFT
total energy expression [7]. It accurately describes the H
binding energies, proton affinities and H transfer barriers
without losing its speed and robustness. In this work, the
parameter set, so-called “3OB”, is used, which was
specifically developed for DFTB3 [8]. Due to its
accuracy and availability to perform reactive simulations
on the atomic scale, the DFTB3 method (from now on
simply called DFTB) is computationally very expensive.
Therefore, a small part of the real system (i.e., cell
membrane) is used as a model system, compared to the
simulation system used in the non-reactive simulations
(see below). This is done to keep the simulation time
reasonable.
Fig. 1 shows the PLB structure (i.e., a simple model
system for the cell membrane) assumed in our
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simulations.
As mentioned above, in the DFTB
simulations, a smaller model system (see red rectangle in
Fig. 1) is used, which consists of 8 phospholipids (PLs),
including the water layer on top and below (containing in
total about 1900 atoms). Here we use the model system
of the PLB composed of phosphatidylcholine molecules,
i.e., one of the four main PLs found in mammalian cells
and most abundant in the cell membrane [9]. PLs have a
hydrophilic head group which is linked to glycerol,
containing two hydrophobic fatty acid tails (i.e., aliphatic
chains), normally consisting of 14 to 24 carbon atoms,
and one or more tails contain (multiple) double bounds.
These double bonds are responsible for a kink in the
chains, giving the bilayer its fluidic nature.
Fig. 1. Schematic representation of the PLB structure.
The red rectangle shows the model system used in the
DFTB simulations. Water molecules are shown in
greyish green color. The red, grey, blue, orange and small
white spheres represent O, C, N, P and H atoms,
respectively.
The basic function of the PLB is to protect the cell from
substances that are applied from outside (including
plasma-generated reactive species, i.e., RONS).
Therefore, it is of great importance to investigate how
plasma species interact with the cell membrane, how they
can penetrate and form e.g., lipid peroxidation products.
This will shed light on the effects of reactive plasma
species on the protective nature of the cell membrane and
as a result on the possible responses within the cell.
The PLB structure, in DFTB simulations, is placed in a
box with dimensions ∼17 Å × 18.5 Å × 72 Å. Because
the structure is too small (compared to the real system),
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we make use of periodic boundary conditions (PBCs) in
all three directions (i.e., in x, y and z direction), so that an
entire “layer” of PLs is formed, continuing in both x and y
direction. In other words, PBCs helps to mimic the
infinity of the system in x and y direction, whereas in z
direction the water density is kept constant (see Fig. 1).
Prior to the particle impacts, the structure is optimized
using the conjugate gradient method and then equilibrated
at room temperature (i.e., 300 K) applying an
isothermal-–isobaric ensemble (i.e., NPT dynamics) for
5 ps (i.e., 2x104 steps, the time step is 0.25 fs). The latter
is performed to equilibrate the temperature of the system
and to obtain a structure at zero stress. The interaction of
the plasma species is investigated by randomly creating
them inside the water layer, so that they can penetrate
through this layer and eventually react with the head
groups of the PLB. To study the (per)oxidation processes
in the lipid tails, we create the plasma species close to
these chains. The latter is done to keep the computational
time reasonable. Each DFTB simulation is repeated
20 times, to allow different reaction types to occur and to
gather some (limited) statistics.
2.2. Coarse-grained model
After performing DFTB, we use the results obtained
from these simulations to study the further processes. It is
performed by using a coarse-grained model applying the
Martini force field, developed by the research group of
Marrink and Tieleman [10]. By using a coarse-grained
Martini model we can expand the spatial- and timescale
of the processes we want to investigate. This model uses
a four-to-one mapping which means that, on average, four
heavy atoms (with their associated hydrogen atoms) are
grouped into one coarse grained bead.
Using the modified (after interaction with RONS) PLB
structure obtained from DFTB, we map this system into
the Martini model. The larger scale which we can
investigate by using this model allows us to study certain
properties of the modified cell membrane (e.g., membrane
permeability or pore formation) which are not accessible
in an all atom-model (i.e., in DFTB). The properties of
the modified membrane can then be compared to those of
a native membrane.
Fig. 2 illustrates the structure of the PLB used in this
model, which is prepared as follows. First, a box with
dimensions 40 Å × 40 Å × 90 Å is filled with 38 PLs and
16 cholesterol molecules. This box is then minimized for
40 ps by using the steepest descent energy minimization
algorithm. After that 350 water coarse grained beads are
added randomly (representing 1400 water molecules).
This structure is again minimized as described above.
Finally a self-assembly MD simulation run is performed
for 27 ns (with time steps of 30 fs) at a temperature of
300 K applying the Berendsen thermostat.
3. Results and discussion
As mentioned in previous section, the interactions of
plasma species (i.e., RONS, see above) with the PLB
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at even 5 percent of oxidized lipids and the pores show
more stability at higher percentage of oxidation. In
addition to changes in the structural properties of the
bilayer, the dynamic properties of the lipids were changed
by the oxidation process. To evaluate changes in the
dynamics of the bilayer, the lateral diffusion coefficient
from the mean-square displacements of the lipids as a
function of time were calculated. The typical result
indicates that diffusion appears in the hydroperoxide
lipids.
Fig. 2. Schematic representation of the PLB used in the
coarse-grained model. It is surrounded by water, with
cholesterol imbedded in it. The grey beads represent
water molecules, the yellow beads represent cholesterol.
The PLB consists of a NC3 head group (dark blue beads),
a phosphate group (green beads), a diacylglycerol linker
(red beads) and two lipid tails (light blue beads).
were investigated using the DFTB method.
Typical results were obtained by applying this method.
Our investigation showed that ROS, namely OH radicals
can break the important bonds in the hydrophilic head
group as well as in the lipid tails (i.e., aliphatic chains),
modifying/oxidizing the structure.
Note that when
travelling through the water layer, the OH radicals
continuously react with water molecules, abstracting a H
atom from water and forming a new OH radical. It was
found that after oxidation of the lipid tails by RONS, O 2
molecules can react with radical sites of the tails and form
peroxyl radicals (i.e., lipid peroxidation processes can
occur). We also found that the reaction of RONS with
fatty acid chains can also lead to the breaking of C-C/H
bonds, which can result in the formation of various
molecules, including aldehydes and alcohols.
The modified PLB structure is used in the coarsegrained model for further analysis. It was found that the
interaction of RONS with the lipid bilayer leads to
conformational changes in the structure. To investigate
the structural properties of the oxidized bilayer, the
average area per lipid and the bilayer thickness in all the
simulations has been calculated. Also, the permeability of
water through oxidized and non-oxidized PLB was
studied. It has been observed that water pores are created
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4. Conclusion
We have studied the interactions of reactive oxygen and
nitrogen species (i.e., OH, O 2 , O 3 , H 2 O 2 , NO, NO 2 ,
ONOO-) with a PLB and their consequences, by means of
atomic scale reactive and non-reactive molecular
dynamics simulations.
More specifically, we have
investigated the interaction of reactive plasma species
with the hydrophilic head group as well as with lipid tails
of the phospholipid double layer. Our calculations show
that these reactive species can break important bonds in
the biological structure, leading possibly to cell
membrane damage. The calculations also give more
information on the interaction mechanisms. Furthermore,
we have also investigated the long time behavior of the
modified PLB structure (due to oxidative and nitrosative
stress), applying a long timescale simulation technique,
i.e., a coarse-grained model to investigate the processes,
such as the membrane permeability and fluidity, as well
as the pore formation.
5. Acknowledgments
The authors acknowledge FWO-Flanders for financial
support. This work was carried out using the Turing HPC
infrastructure at the CalcUA core facility of the
Universiteit Antwerpen, a division of the Flemish
Supercomputer Center VSC, funded by the Hercules
Foundation, the Flemish Government (department EWI),
and the Universiteit Antwerpen.
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