Indeed, recent experimental results showed that CAPs could enhance the effects of conventional chemotherapy even in resistant tumorous cells; the resistant cell population, if pre-treated with CAP, becomes sensitive to treatment with chemotherapy 5 , 6. Moreover, it was demonstrated that plasma treatment, both in vitro and in vivo , is able to attack a wide range of cancer cell lines without damaging their normal counterparts 1 , 2.
Thus, preliminary results seem very promising. Nevertheless, the application of CAPs for cancer treatment is still in its initial stage, and there is an enormous need for a better understanding of the underlying mechanisms. Several studies showed that CAPs elevate intracellular ROS levels, thereby inducing oxidative damage in cancer cells, which can lead to cell death, i. Normal cells, on the other hand, are able to defend themselves from this harmful effect of ROS by activating multiple anti-oxidative systems that reduce the increased oxidative stress and restore the balance 9 , It is suggested that the combination of nitrosative and oxidative stress induced by plasma can possibly avoid drug resistance of neoplastic cells 4.
To answer this question, there is a need for more fundamental investigations. Recently, Kaneko et al. Moreover, Hong et al. Hence, in the present work we will focus on the plasma membrane, surrounding the cell, as this is the first molecular structure of the cell that interacts with substances from outside, including plasma-generated RONS. In first instance, we need to elucidate how the plasma species interact with the head groups of the plasma membrane. In ref. On the other hand, OH radicals are very reactive and they can immediately react with any molecule most probably first with the head groups when reaching the membrane Thus, the interaction of ROS from the plasma with the head groups of the PLB forming part of the plasma membrane is the first step, which might subsequently cause per oxidation of the lipids.
The latter can then alter the structural and dynamic properties of the membrane, which can lead to an increase in the permeability, a change in the lipid order and bilayer thickness as well as in its fluidity 15 , 16 , 17 , 18 , 19 , 20 , 21 , 22 , There are already several experimental studies 15 , 16 , 17 , 18 , 24 , 25 and a few simulation papers 20 , 21 , 22 , 26 devoted to a detailed investigation of the effect of lipid oxidation on the properties of the lipid membrane. Tai et al. They found that OH radicals cause a significantly higher lateral fluidity of the membranes, while hydrogen peroxide has little effect An increase of the membrane disordering was observed in 16 , 17 , while in 18 , 24 , the opposite effect was reported, i.
On the other hand, several simulation studies, using non-reactive molecular dynamics MD , revealed an overall increase in the membrane permeability 20 , a change in the lipid mobility in a lipid bilayer 21 as well as pore creation and bilayer disintegration 26 upon introduction of oxidized lipids. Similar results were also recently reported by our group 22 , Moreover, we found that a higher cholesterol fraction in a bilayer with per oxidized lipids leads to an increase in the membrane order, and after a certain threshold i.
Note, however, that in all these studies devoted to lipid tail oxidation, the actual oxidation process of the lipids, and more specifically of the head groups of the PLB, as well as its subsequent effect on the properties of the membrane, was not yet investigated.
This forms exactly the subject of the present paper. Such a study is of great importance to find out a which of the RONS can react with the head groups and possibly destroy them and b what is the effect of the oxidized head groups on the structural and dynamic properties of the PLB. Thus, with this study, we aim to explain the onset of the lipid oxidation process. It should be mentioned here that our study is not only relevant for CAP treatment, but also to other therapies which produce ROS, such as chemotherapy, radiotherapy and photodynamic therapy.
This study requires a combination of different simulation approaches. It should be noted that RNS can also interact with these head groups. This is probably due to the limited simulation time see below , or maybe also because of the parameter set utilized in our DFTB simulations, which might not be accurate enough to describe the interactions of RNS with the head group.
Finally, we also perform experiments in order to validate our simulation results. The plasma membrane consists of a PLB i. In the present work, we consider only the PLB without cholesterol and without proteins as a simple model system for the eukaryotic plasma membrane, since the PLB determines the bilayer thickness.
Specifically, we use a PLB consisting of phosphatidylcholine molecules that are one of the four main phospholipids found in mammalian plasma membranes Thus, in our simulations we use two different model systems, i. They have the same hydrophilic head group i. The structure above the red dashed lines in a is used for studying the reaction mechanisms. The water layers and lipid tails in c are shown in cyan and gray colors, respectively, and the P and N atoms are depicted with bigger beads, for the sake of clarity. The model systems presented in Fig. The model system of Fig. The membrane fluidity and the changes therein by plasma treatment are assessed by generalized polarization GP measurements using the fluorescent probe 6-Dodecanoyldimethylaminonaphthalene Laurdan for different treatment times.
The dynamics of the fluidity development of the liposomes is studied by time series after plasma treatment. Specifically, we use the so-called DFTB3 method Detailed information about this method as well as a parameter set used in this study is given in the Supplementary Information. As shown in Fig. It is clear that the impinging plasma species i. To study their behavior in the water layer, or in other words, to determine whether these species will react with water and possibly form new species, we perform some test runs by creating a single plasma species in a small box of water see the Supplementary Information for more details.
The total number of atoms in the system of Fig. Because of the high computational cost of the DFTB method, we have chosen to focus on the interaction with the head groups only. Thus, instead of following the full trajectory of the ROS traveling through the water layer which requires several tens of ps , we here consider a structure composed of a single PL molecule located in vacuum i. This structure is sufficient to elucidate which reactions can possibly occur in the head group of the PLB, and which reactions have a higher probability, and are thus interesting to study in more detail see below.
Only disadvantage of using this small structure is that we cannot consider the stabilizing effect of water covering the head group see the Results and Discussion section below. Details of the preparation of this model system are given in the Supplementary Information. Thus, to obtain statistically valid results for bond-breaking or bond-formation processes and to study all possible damaging mechanisms of the PL, we perform runs for each impinging species, i.
After the calculations with the small structure i. For this purpose we apply so-called united-atom MD simulations. This method allows to investigate larger systems up to , atoms and longer time-scales hundreds of ns 30 , 31 , which are clearly not accessible in an all atom-model like DFTB. In the united-atom approach, each hydrocarbyl group i. Thus, the united-atom representation is adopted for the apolar alkyl chains as well as for the hydrocarbyl groups in choline and glycerol, which reduces for instance the size of the DOPC molecule, containing atoms, to a system of 54 particles.
The polar H-atoms in the water molecules or in an alcohol-group remain, however, as separate atoms. In total, 9 different model systems are prepared for DOPC, and 5 for POPC, including intact and oxidized PLBs, with two different oxidation products at various concentrations see below , all containing lipids as well as water molecules surrounding them in the top and bottom layers.
On the other hand, the experimental results revealed only the OX2 product, together with products of lipid tail oxidation, which we call ALD, as more prominent oxidation products see below. Thus, in our non-reactive MD simulations, we describe the effects of these two oxidation products i. More details about the concentrations of the oxidation products as well as the preparation of these oxidized PLB systems are given in the Supplementary Information.
To validate the force field for this application, we compared our calculated values of the surface area per lipid and the bilayer thickness, which define typical properties of the bilayer, for the intact PLB, with results obtained from experiments and computational studies. It should be mentioned that for the oxidized PLs, new force field parameters are required for the newly formed bonds and associated angles and dihedrals.
Most of these parameters are not available in the applied force field, or in other force fields from literature.
Therefore, these parameters had to be determined first, by means of density functional theory DFT , as will be explained in the next section. The parameters of the ALD oxidation products are obtained from ref. We employ DFT calculations to obtain new parameters for the non-reactive force field by carrying out a fitting of some standard potential energy functions to the DFT energy. Note that for the newly formed bonds, angles and dihedrals, we try to use as many parameters as possible that already exist in the parameter set of the original force field i. In this way, we keep the parameter set as native as possible.
Thus, we implement 16 new parameters to the force field. For the experiments, liposomes are treated with plasma-activated liquid. The plasma source used is a so-called kINPenSci The plasma jet is a needle type discharge in a dielectric tubing of 1. The grounded ring-shaped electrode is located at 2 mm distance from the dielectric nozzle exit. The plasma source is operated with argon as feed gas purity 5. An approximately 10 mm long visible plasma plume is driven out of the jet nozzle. The kINPenSci is based on the design of the commercially available kINPen, except that additional possibilities are available to measure electric signals of the excitation frequency.
It generates OH in the gas phase 39 and in the liquid phase By this procedure, the influence of variations of feed gas humidity can be minimized, which has a big impact on the produced plasma species and on the behavior of the plasma-treated cells Furthermore, a so-called gas-shield is used to prevent influences of changing natural ambient air on the effluent — gas interaction The used shielding gas mixture is 1. A stock solution of 2. The thermic cycle is repeated twice. Trasdingen, Switzerland. Positioning and moving of the jet is performed automatically by an XYZ-positioning stage Hylewicz CNC-Technik, Geldern, Germany above the liquid surface on a predefined meandered path to ensure a thorough and homogeneous treatment of the sample.
The petri dish is immediately closed after treatment. Membrane phase states gel- or liquid-phase can be detected by measuring the penetration of water molecules into the bilayer, which strongly correlates with the packing of the phospholipids. This can be done by using 6-Dodecanoyldimethylaminonaphthalene Laurdan , a lipophilic fluorescent probe that readily inserts into membranes. Laurdan is located at the sn -1 acyl chain The hydrophobic tail allows for integration parallel to the fatty acids of the lipids due to strong Van-der-Waals and hydrophobic interactions In this way, the hydrophilic and fluorescent naphthalene component aligns with the phospholipid glycerol backbone and points towards the aqueous surrounding.
The increase in the dipole moment caused by the excitation of the Laurdan molecule triggers a reorientation of the water dipoles in the immediate vicinity of the probe. This interaction requires energy, resulting in a dipolar relaxation, meaning that Laurdan loses energy in its excited state. This Stokes-shift correlates to head group hydration and mobility The detection of membrane phase transition is considered constant, as the fluorescent properties of Laurdan are independent of phospholipid head groups or lipid linkage Laurdan was purchased from Life Technologies Darmstadt, Germany and was dissolved in Two untreated samples are measured.
The treated sample is measured every three minutes over a course of three hours. After treatment, liposomes are collected from the aqueous phase by CHCl 3 extraction. The resulting lipid solution is diluted six-fold for direct infusion into a TripleTOF high resolution mass spectrometer MS by water-methanol-formic acid positive mode or water-methanol-ammonia negative mode.
Base peak and monoisotopic peaks are manually annotated and screened for potential covalent modifications related to the plasma treatment. Therefore, it is necessary to know which of the ROS can penetrate through the water layer and effectively reach the head group. We observed no bond breaking or formation events in the case of HO 2 and H 2 O 2 , so these species can freely move through the water layer. The OH radicals, on the other hand, can react with water, exchanging a hydrogen atom and forming again the same species i. The behavior of the ROS in a liquid layer was also studied in detail in our previous work by means of classical reactive MD simulations applying the ReaxFF potential 48 , and we found that OH, HO 2 and H 2 O 2 can travel deep into the water layer and eventually reach the surface of the biomolecule.
The same behavior is thus predicted here with the DFTB method. However, this is not so important, as the backward reaction found in the ReaxFF-MD results is so fast, and thus, both methods predict that the HO 2 radicals can penetrate through the water layer. Moreover, Moin et al.
Recently, Verlack et al. They concluded that the transport of OH radicals in water is not only governed by diffusion, but also through an equilibrium reaction of H-abstraction with water molecules, in line with our previous ReaxFF-MD study. They also studied the self-interaction of two OH radicals and observed some distinct reactions We can thus conclude that all of the ROS, i. As mentioned in the Introduction, we focus on the interaction of the ROS with the head groups of the PLB, and not with the lipid tails, as much less is known in literature about the products formed upon interaction of ROS with the head groups.
Like in the water case, HO 2 and H 2 O 2 molecules do not react with the head group. They only have non-bonded interactions with the structure. Due to the severe computational cost of DFTB, we positioned the plasma species closer to the head groups. Again no bond breaking of the head groups upon interaction with these species i.
On the other hand, the OH radicals do react with the head groups of the PLB, leading to the cleavage or formation of some bonds.
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These reaction mechanisms will be discussed below in more detail. As mentioned in the section on Reactive DFTB MD simulations, we performed MD runs using a small structure, to gain some limited statistics, as well as to study the most probable reaction mechanisms of OH interacting with the head groups of the PLB.
We observed six reaction mechanisms, called OX1, OX2, …, OX6 see the Supplementary Information and found that all of them are initiated by H-abstraction from different parts of the head groups, but few of them give rise to further bond breaking. Therefore, we need to consider these reaction mechanisms with caution and validate them with experimental results, which is the case in this study see below in the Experimental validation section. We focus here only on two mechanisms which lead to the detachment of some parts of the PL head groups, and are thus important for the investigation of the longer-term behavior of the PLB see the Non-reactive MD results section.
Note that this reaction mechanism was observed most in our simulations i. Breaking mechanism of the C-N bond upon impact of an OH radical. A new OH radical can then react with this site, resulting in c the formation of an alcohol group. The OH radical first abstracts a H atom from the methyl group of choline see dashed circle in Fig. This leads to the breaking of a C-N bond see gray dashed line in Fig. Subsequently, a new OH radical can either react with this C radical see Fig.
To check which of these two reactions is energetically more favorable, we performed DFT calculations. The calculated reaction energies revealed that the formation of the alcohol group as depicted in Fig. This mechanism is schematically represented in Fig. Breaking mechanism of a C-O bond upon impact of an OH radical. The OH radical abstracts a H atom see black dashed circle in a leading to the formation of a water molecule. A new OH radical can then react with this site, forming an alcohol group c.
After the H-abstraction from CH 2 of glycerol see dashed circle in Fig. Subsequently, a CO 2 molecule detaches from this oleoyl chain, leaving behind a radical site see dashed circle in Fig. A new OH radical can then react with this radical site, forming an alcohol group. To summarize, we distinguish two reaction mechanisms of OH radicals interacting with the head groups, which lead to the destruction of the PL molecules and are therefore important for studying the subsequent longer-term effects of these oxidized PLs on the properties of the PLB.
We did not consider the other reaction mechanisms i. A more detailed discussion about the other reaction mechanisms can be found in the Supplementary Information see Fig. As mentioned above, we need to take aforementioned reaction mechanisms i. Therefore, we perform some experimental validation see next section in order to decide whether these oxidation products should be used in our further non-reactive MD simulations to study the longer-term behavior of the oxidized PLB.
Using mass spectrometry as an orthogonal technique, we searched for covalent changes to the molecular structure of both unsaturated DOPC and saturated DMPC after the plasma treatment. In positive mode, the molecular ion of DOPC was detected at The molecular ion of DMPC was detected at Prominent ions beside the base peaks and their isotopes were complexes with alkali and earth alkali ions.
No principal changes in the mass spectra of the respective lipids were observed, indicating that the impact of the reactive species was limited, e.
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High resolution mass spectrometry peak intensities of a nonanoic acid Two routes of lipid modifications were experimentally observed: oxidation of the lipid side chain which we call ALD, see Fig. Side chain oxidation was only observed when using unsaturated DOPC lipids and manifested in the presence of two products, a short fatty acid nonanoic acid, According to the proposed mechanism from MD simulations 20 , 22 , 26 , the initial product of ALD is the corresponding nonanal see short chain in Fig.
Via secondary oxidation, the detectable nonanoic acid arises. For saturated DMPC, missing an attackable allyl position, similar products were not detected. Beside the side chain, covalent changes in lipid structure can occur at the polar head group, because the high electron density and the presence of heteroatoms allow numerous chemical reactions. OX1 reflects the attack of hydroxyl radicals at the choline residue, leading to the loss of the amino group. However, the corresponding product, phosphatidylglycol Either OX1 is not occurring after plasma treatment, or the products withdraw from detection for their physicochemical properties lower polarity, bad ionization efficacy , or concentration.
In contrast, the OX2 mechanism reflects an elimination of a fatty acid chain at the glycerol moiety, leaving a propendiol group. The respective products propendiol-phosphatidylcholines were detected in positive mode for DOPC Due to limitations of fragmentation modes and signal intensity, the structure could not be completely established but the molecular composition was determined with a sub-ppm error.
Presumably, two propendiols stabilize by eliminating the double bonds and expelling a further fatty acid side chain, creating a molecule bearing two phosphatidylcholine head groups, one fatty acid, and a glycerol — propandiol ether structure. Signals for both OX2 and ALD oxidations were observed with intensities almost independent from the water admixture to the working gas, although this enhances the flux of H 2 O 2 and OH radicals.
Schematic representations of the native DOPC molecule a , together with its oxidation products b , c and d. All the hydrocarbyl groups i. Note that although the experiments and simulations cannot be directly compared due to the vastly different time scales, they certainly provide a complementary view, and the experiments seem to validate our calculation results on OX2 formation.
As is clear from the previous sections, the reactive DFTB MD results reveal two important oxidation products of the head groups i. A schematic picture of these oxidation products is given in Fig. For this purpose we analyze some important properties of the bilayers:. The bilayer thickness , calculated by averaging all distances z -components between the phosphate groups of the two opposite layers of the PLB.
The deuterium order parameter, S CD , which is a measure for the order of the lipid tails in the bilayer. The degree of ordering of the lipid tails can also be influenced when the head groups are oxidized see below. More detailed information about S CD can be found in ref. The surface area per lipid, the bilayer thickness and the deuterium order parameter are plotted in Fig. Surface area per lipid a and d , thickness of the bilayer b and e and average deuterium order parameter c and f , as a function of the concentration of the oxidized PLs, for two types of oxidation products, for the DOPC PLB.
Based on the experimental results given in the next section, we consider two cases. This case can be related to the direct treatment of the DOPC vesicle with the plasma source see the discussion in the next section. Secondly, we examine the properties of the PLB applying both oxidation products i. The latter concentration i. This case can correspond to the post treatment of the DOPC vesicle with plasma. It is clear from Fig. We believe that the reason for these two opposite phenomena is the following.
In the case of OX2, the detached lipid tails see Fig. This stiffening effect leads to a slight increase in the order of the tails see Fig. As the bilayer thickness slightly increases, the probability of per oxidation of the lipid tails will slightly decrease. Thus, the bilayer becomes slightly more rigid upon OX2 oxidation. Moreover, shortened lipid tails see Fig.
At the same time, this leads to a decrease of the bilayer thickness see Figs. The long-term effect of the latter was recently studied in refs 22 and 23 , and it was observed that this can even lead to pore formation, in accordance with previous MD results 20 , The color legend is identical to Fig.
Moreover, we have also carried out simulations for a fully saturated lipid, more specifically for a DPPC i. The results are given in the Supplementary Information. It is clear that similar changes in the surface area per lipid and the bilayer thickness as a function of the concentration of the OX2 oxidation are obtained, but the relative changes are more pronounced than in case of DOPC and POPC see Fig.
Thus, our calculations predict an overall increase in area per lipid and a drop of the bilayer thickness upon equal oxidation of the head groups and lipid tails, thereby decreasing the lipid order, which eventually leads to an increase of the bilayer fluidity. The change in fluidity was also studied by experiments to validate our simulation results and similar results were observed see next section.
It should be mentioned here that despite the fact that the effect of OX2 is less pronounced, its oxidation byproduct, namely the phospholipid structure with a single lipid tail see oxidized structure with acyl sn1 chain in Fig. Indeed, the chemical structure of this byproduct is very similar to the structure of so-called alkylphospholipids APLs, e. Unlike most anticancer drugs, APLs do not target DNA, but they insert in the plasma membrane and subsequently they induce a wide range of biological effects, eventually leading to cell death.
The effectiveness of APLs is mediated by changes i. Therefore, the oxidation byproduct of OX2 generated by the CAP induced ROS might destabilize the lipid domains in the cell membrane, which may ultimately result in cell death. For more information about APLs and their apoptotic effects, we refer to some reviews 52 , 53 , 54 and references therein. To validate the simulation results, we investigated the influence of plasma-treatment on the fluidity of lipid bilayers as model membranes.
DOPC liposomes were prepared with integration of the fluorescent probe 6-Dodecanoyldimethylaminonaphthalene Laurdan. This lipophilic molecule inserts parallel to the lipid acyl chains and detects alterations in membrane fluidity as a consequence of a change in its dipole moment when water penetrates the membrane bilayer First, GP values were calculated for different temperatures see Fig. Samples were treated for indicated durations with the kINPen source. Experiments were performed in such way that no phase changes may occur, only changes in fluidity behavior of the disturbed lipid phase.
If temperature is kept constant, GP values can be used to assess lipid fluidity changes triggered by chemicals Afterwards, GP values decrease over time. In summary, plasma treatment of DOPC liposomes revealed an initial and short-term higher rigidity of the lipid bilayer, but overall and in the long-term, a fluidization effect.
So overall, our simulations predict a higher fluidity, assuming that the disorder correlates with fluidity. During or shortly after plasma treatment, a higher rigidity was observed. This is probably due to the initial OX2 oxidation. In a second step, presumably the ALD oxidation of lipid tails takes place, increasing the disorder. Indeed, the MS results showed that cross-linked structures are formed that are thought to be the consequence of the OX2 oxidation, which might allow water and ROS to penetrate deep into the lipid region from the sides of these cross-linked lipids, eventually causing lipid tail oxidation, i.
Our long-term simulations already revealed that even OX2 and ALD oxidation occur together at the same time, and this overall leads to a decrease of the lipid order, thereby increasing the cell membrane fluidity. Nevertheless, it has to be noted that the experiments intrinsically observe a different time scale than the calculated data. The experimental results will not be able to observe the short term results from the simulations.
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Although our simulations only show the head group and lipid tail oxidations, these will later on lead to more oxidations of other lipid tails, as a chain reaction see the post plasma treatment times in Fig. The effect of our simulations, although obtained for short time-scales, can thus be correlated to a longer-term effect, i. It should also be mentioned that according to the density profiles of ROS across the PLB obtained by Cordeiro 13 , the OH radicals preferably stay closer to the carbonylester groups, where OX2 can take place.
As mentioned above, this might then explain the higher ordering seen in the experiments upon direct plasma treatment. The other oxidation product obtained from the reactive simulations i. Indeed, in vacuum, the polar groups i. We should also mention here that the simulation results presented in this study are the effects of primary or single impacts of OH radicals on the structure. In reality, a consecutive number of OH radicals can impinge the PLB and react with the byproducts created from primary interactions, leading to the further destruction of the system.
Moreover, plasma is not solely a source of OH radicals but also of other ROS as well as charged species. Future work is thus needed to further correlate experiments with the simulations. Oxygen consumption of this tissue was determined in absence of substrate as well as in the presence of oxalacetate and succinate, respectively.
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The accuracy of the two methods was equal though small differences in absolute values were found. Rapidity and simplicity of performance is considerably increased with the spectrophotometric procedure. Volume 90 , Issue 3. The full text of this article hosted at iucr. If you do not receive an email within 10 minutes, your email address may not be registered, and you may need to create a new Wiley Online Library account. If the address matches an existing account you will receive an email with instructions to retrieve your username. Acta Physiologica Scandinavica Volume 90, Issue 3.
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