A reactive molecular dynamics study for plasma medicine applications
M. Yusupov, E. C. Neyts, J. Van der Paal, S. Aernouts, A. Bogaerts
Research group PLASMANT, Department of Chemistry, University of Antwerp, Antwerp, Belgium
Abstract: In this paper, we present atomic scale reactive molecular dynamics simulations
for the interactions of reactive oxygen species (i.e., O, OH, O 2, O3 and H2O2) with
biomolecules, more specifically with bacterial peptidoglycan, as well as with a
phospholipid bilayer. Furthermore, we have also investigated the behavior of these species
in water, as a simple model system for the liquid film surrounding biomolecules.
Keywords: molecular dynamics, reactive oxygen species, peptidoglycan, liquid film
1. Introduction
Although plasma medicine is gaining increasing
interest in recent years for many applications, the
underlying physical and chemical processes, especially
the interaction of plasma species with biological systems,
are not yet fully understood. These interaction
mechanisms are also not straightforward to study
experimentally. Therefore, we try to obtain a better
understanding of these interactions by means of computer
simulations. More specifically, we try to simulate the
interactions of reactive oxygen species with biomolecules,
by means of atomic scale reactive molecular dynamics
(MD) simulations. In this paper we will show results for
the interactions of O, OH, O2, O3 and H2O2 species with
bacterial peptidoglycan (PG), which is an important
component of the bacterial cell wall, as well as with a
phospholipid bilayer, as a more general model system for
the cell membrane. Furthermore, as all biochemically
important structures are surrounded by a liquid film, we
have also investigated the behavior of these reactive
oxygen species in water, as a simple model system for
this liquid film, in order to reveal whether these reactive
plasma species can indeed travel through a liquid film and
reach the biomolecules, or whether they form new
species. In this paper, we focus on reactive oxygen
species, as there is no accurate reactive force field
available yet for MD simulations of reactive nitrogen
species.
2. Description of the simulations
In MD simulations, the trajectories of all atoms in the
system are calculated by integrating the equations of
motion. The forces acting on all the atoms in the system
are derived from a suitable interatomic interaction
potential, also called “force field”. In our work, we make
use of the reactive force field ReaxFF [1]. The advantage
of this force field is that it can describe chemical
reactions, i.e., the breaking and formation of new bonds,
which is important for studying the interaction of plasma
species with biomolecules. This force field contains a lot
of parameters, which are optimized to obtain good general
agreement with quantum mechanical calculations for
reaction energies, barriers and structures. The total system
energy in the ReaxFF force field is the sum of several
partial energy terms that include lone pairs,
undercoordination, overcoordination, valence and torsion
angles, conjugation and hydrogen bonding. Moreover,
non-bonded interactions, namely Coulomb and van der
Waals energy terms, are also taken into account. These
interactions are calculated between every pair of atoms,
such that the ReaxFF potential is capable of describing
not only covalent bonds, but also ionic bonds and the
whole range of intermediate interactions. This force field
has now been successfully applied to describe nearly half
of the periodic table of the elements and their compounds,
including hydrocarbons, metals and metal-catalyzed
reactions, metal oxides, metal hydrides and silicon and
silicon dioxide. Recently, it has also been used for organic
molecules, such as glycine, as well as for complex
molecules, such as DNA. A detailed description of the
force field parameters used for the current simulations
study can be found in [2].
Fig. 1 Schematic representation of the PG structure
Because the MD simulations are on the atomic scale,
the simulated system forms only a small part of the real
system (e.g., cell membrane). Figure 1 shows the PG
structure assumed in our simulations. It is assembled from
repeating units, consisting of disaccharides, with
tetrapeptide stems, connected with one pentaglycine
interpeptide (bridge); see [3] for more details. With this
(simplified) structure, we are able to take into account all
possible atomic bonds occurring in the (3D) PG structure.
Figure 2 illustrates the phospholipid bilayer structure
considered in the simulations. It consists of only 5
phospholipid units to keep the calculation time
reasonable. In reality, the lipid layer of a cell membrane
contains of course many more phospholipid units, but we
make use of periodic boundary conditions in the x and y
direction, so that an entire “layer” of phospholipids is
formed, continuing in both x and y direction. In reality,
there is of course also a second lipid “layer” underneath
the layer shown in figure 2, with the fatty acids pointing
upwards, forming together a double layer. To take this
into account, we make use of a reflecting surface at the
bottom of this lipid layer, so that reactive species that
reach this bottom are reflected back to the lipid layer.
This mimics the reality because in practice, they would
just continue and arrive in the lower part of the lipid
bilayer, and at the same time, it reduces the calculation
time as it limits the number of atoms to be simulated.
Fig. 2 Schematic representation of the phospholipid layer,
as a model system for a cell membrane.
The impacts of the plasma species are performed by
randomly positioning them at a minimum distance of 10 Å
around the biological structure, so that there is initially no
interaction between the plasma species and the biological
structure. The initial energy of the impinging plasma
species corresponds to room temperature and their
velocity directions are chosen randomly. To study all
possible damaging mechanisms of the biological structure
and to obtain statistically valid results for bond-breaking
processes, we performed 50 runs for each plasma species.
Every simulation trajectory lasts 300 ps, corresponding to
3*106 iterations. This time is long enough to obtain a
chemically destroyed biological structure, at least if a
critical bond in the structure is broken (see below).
3. Results and discussion
3.1. Interaction with peptidoglycan
Our investigations on the H2O and O2 impacts reveal that
no bond-breaking events occur in important C−O, C−C,
and C−N bonds in PG. These molecules are found to
assemble around the PG, having weak attractive
nonbonded interactions with the structure (i.e., hydrogen
bridge formation). The impacts of OH, O, O 3, and H2O2
species do result in important bond breakings, which can
lead to the destruction of the bacterial cell wall. The
fraction of important bond dissociations upon impact of
these species, calculated from 50 independent simulations
for each incident species, are summarized in Table 1.
Table 1. Fraction of important bond dissociations (i.e., C–
N, C−O, and C−C bonds) and associated standard
deviations upon impact of O, O3, OH, and H2O2.
Incident
plasma
species
C-N bond
breaking
events, %
ether C-O
bond
breaking
events, %
C-C bond
breaking
events, %
O
26 ± 6
78 ± 6
38 ± 7
O3
8±4
56 ± 7
26 ± 6
OH
8±4
54 ± 7
14 ± 5
H2O2
0
44 ± 7
12 ± 5
Our calculations reveal that O3, OH, and especially O
atoms are more effective in bond cleavage than H 2O2
molecules. Furthermore, the ether C−O bonds in the
disaccharides are found to break up more easily, followed
by the C−C bonds and C−N bonds in the PG. In the case
of H2O2 molecules, no C−N bond-breaking events are
observed, indicating again that the H2O2 molecules are
somewhat less effective in bacterial cell wall destruction.
However, in contrast with the highly reactive O and OH
radicals, H2O2 (and O3) molecules are stable species in an
aqueous environment (see below) and are thus more likely
to interact directly with the PG. Hence, we expect that the
H2O2 molecules are also very important for bacteria
deactivation.
The mechanisms of the important bond breaking
processes in the PG were also studied in detail. It was
found that in all bond cleavage events the dissociation of
these bonds is initiated by hydrogen-abstraction.
However, a clear difference is observed in the
mechanisms upon impact of H2O2 molecules on one hand
and OH, O, and O3 species on the other hand. Indeed, in
the latter case a H atom is abstracted from the PG by the
plasma species (OH, O, or O3), whereas in the case of
H2O2 impacts, the H2O2 molecules first react with each
other, forming HO2 radicals, from which a H atom is
abstracted by an O atom in the PG structure. Abstraction
of a H atom from the HO2 radicals can then cause the
dissociation of the important bonds in the PG (i.e., cell
wall damage). This corresponds well with experimental
observations that hydroperoxyl (HO2) radicals are strong
bactericidal oxidants and can cause the inactivation of the
bacteria in an aqueous environment. More details about
these bond breaking mechanisms can be found in [3,4].
3.2. Interaction with the phospholipid bilayer
It should be noted that in reality, the phospholipid
double layer consists of two organic fatty acids,
connected to glycerol, and the third alcohol function of
this glycerol group is a polar group containing a
phosphate structure. However, as there exists no reactive
force field for ReaxFF that can handle interactions with P,
our model structure of the phospholipid bilayer under
study here does not contain a phosphate group. This is a
strong approximation, but we believe that we can still
obtain valuable insight in the interaction behavior of the
plasma species with the glycerol part and the fatty acids.
We considered two types of phospholipids. The first
one contains stearic acid (18:0) and linoleic acid (18:2),
whereas the second one contains two -linolenic acid
molecules, in order to obtain information on the effects of
the number of unsaturated bonds in the fatty acids.
Table 2 presents the relative occurrence of different
types of reactions with both types of phospholipid
structures. It is clear that reaction with the glycerol group
is by far most abundant for the first phospholipid
structure. H-abstraction from a methylene group or an
allyllic C-atom in the fatty acids occurs as well, besides
the formation of an aldehyde. The latter was found to take
place only in the glycerol group, so that it did not have a
large effect on the structure of the cell membrane.
However, in the second, more unsaturated structure, the
formation of a ketone was also observed, and the latter
took place in the fatty acid part, meaning that the apolar,
hydrophobic part of the cell membrane could become
more polar, and hence hydrophilic. This could have large
consequences for the functioning of the cell membrane.
Furthermore, H-abstraction from a methylene group or an
allylic C-atom is also more abundant in this second
structure. Finally, our calculations reveal that the more
unsaturated phospholipid structure is far more reactive, as
the H-abstractions occurred already within a time-frame
of a few ps, whereas these reactions took a few tens of ps
for the more saturated phospholipid structure.
Table 3. Relative abundance (%) of different reactions for
two types of phospholipid structures, i.e., a more saturated
and a more unsaturated structure (see text), called
structure 1 and 2.
Reaction
Reaction with glycerol
group
H-abstraction from
methylene group
H-abstraction from allyllic
C-atom
Formation of aldehyde
Formation of ketone
Structure 1
75%
Structure 2
25.8%
8.3%
43.2%
5.6%
24.3%
11.1%
/
5.3%
1.4%
Figure 3 illustrates the reaction of OH atoms with the
glycerol group, leading to hydrolysis. This reaction is
initiated by H-abstraction, after which the C-C bond
between C2 and C3 breaks up, and the C-C bond between
C1 and C2 becomes a double bond. The reaction
mechanism is also illustrated in Figure 4.
Fig. 3 Schematic picture of one phospholipid unit
interacting with an OH radical at the glycerol group
(left), and zoomed pictures of the reaction evolution
(right: from top to bottom), leading to breaking of a C-C
bond (hydrolysis).
Fig. 4 Reaction mechanism of the hydrolysis of the
glycerol group.
3.3. Behavior in water
As mentioned above, all biomolecules are surrounded by
a liquid film. Therefore, an investigation of the behavior
of the reactive oxygen species in a liquid film is important
in order to determine whether these species can travel
through a liquid film and reach the biomolecules, or
possibly form new species.
Since the liquid film is mostly composed of water, we
assumed water in this work as a simple model system.
Reaction mechanisms of the reactive oxygen species
(i.e. O, OH, O2, HO2 and H2O2) showed that no bond
breaking of water molecules occurs in the cases of O2,
HO2 and H2O2. These species are found to have weak
attractive nonbonded interactions with water molecules
(i.e. hydrogen bridge formation). In the case of O atoms,
these species first react with a water molecule abstracting
hydrogen, so that two OH radicals are formed. These two
OH radicals then interact with new water molecules and
again new OH radicals are formed and so on. The same
reaction mechanism occurs in the case of OH radical
impacts. Calculated trajectories of the reactive oxygen
species show that O2 molecules cannot penetrate deeper
through the water slab (i.e. they reside close to the airwater interface, see fig. 5), while OH radicals are found to
penetrate deeper due to the reaction with water molecules.
Fig. 5 Trajectory of an O2 molecule travelling through the
air-water interface.
HO2 and H2O2 are also found to reside in the interface, at
least within the simulation time (i.e. 500 ps). However,
calculated free energy profiles show that these species can
in principle travel deeper in the water layer due to the
small difference in the free energies (i.e. ~0.5 kcal/mol).
4. Conclusion
We have studied the interactions of reactive oxygen
species (i.e., O, OH, O2, O3 and H2O2) with biomolecules,
by means of atomic scale reactive molecular dynamics
simulations. More specifically, we have investigated the
interaction with bacterial peptidoglycan, as well as with a
phospholipid double layer. Our calculations show that
these reactive oxygen species can break important bonds
in the biological structures, leading possibly to cell wall
damage. The calculations also give more information on
the interaction mechanisms. Furthermore, we have also
investigated the behavior of the reactive oxygen species in
water, as a simple model system for the liquid film
surrounding biomolecules. Our calculations predict that
OH radicals can travel through the water layer and reach
the biological structure.
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