The role of lipid oxidation on electrical properties of planar lipid bilayers and its importance for understanding electroporation.

Electroporation is a useful tool for the manipulation with the cell membrane permeability. Underlying physicochemical processes taking place at the molecular level during electroporation are relatively well studied. However, various processes remain unknown, one of them is lipid oxidation, a chain reaction that causes degradation of lipids, and might explain the long-lasting membrane permeability after the electric field has ceased. The aim of our study was to observe the differences in the electrical properties of planar lipid bilayers, as in vitro cell membrane models, due to lipid oxidation. Phospholipids were chemically oxidized and oxidation products were analysed using mass spectrometry. Electrical properties, resistance R (Ω) and capacitance C (F) were measured using an LCR meter. Using a previously developed measuring device, a linear increasing signal was applied to a stable bilayer in order to measure its breakdown voltage Ubr (V) and lifetime tbr (µs). We observed an increase in conductance and capacitance of the oxidized planar lipid bilayers when compared to their non-oxidized counterparts. With increasing lipid oxidation, the core of the bilayer becomes more polar, and consequently more permeable. Our findings can explain the long-lasting permeability of the cell membrane after electroporation.

Effect of the cholesterol on electroporation of planar lipid bilayer.

Electroporation threshold depends on the membrane composition, with cholesterol being one of its key components already studied in the past, but the results were inconclusive. The aim of our study was to determine behaviour of planar lipid bilayers with varying cholesterol concentrations under electric field. This would give us a better insight into cholesterol effect on membrane properties during electroporation process, since cholesterol is one of the major components of biological membranes and plays a crucial role in membrane organisation, dynamics, and function. Planar lipid bilayers were prepared from phosphatidylcholine lipids with 0, 20, 30, 50 and 80 mol% cholesterol. Capacitance was measured using the discharge method. Results show no statistical difference of cBLM between the cholesterol concentrations. Breakdown voltage Ubr of planar lipid bilayers was measured by means of linear rising voltage with seven different slopes. Obtained results were fitted to a strength-duration curve, where parameter Ubrmin represents minimal breakdown voltage, and parameter τRC represents the inclination of the strength-duration curve. Adding cholesterol to planar lipid bilayer gradually increased its Ubrmin until 50 mol% cholesterol concentration. Afterwards at 80 mol% Ubrmin does not further increase, in fact it reduces by 20% of the Ubrmin at 50 mol% cholesterol concentration.

Calcium ion effect on phospholipid bilayers as cell membrane analogues.

Ion attachment can modify stability and structure of phospholipid bilayers. Of particular importance is the interaction of phospholipids with divalent cations, such as calcium ions playing an important role in numerous cellular processes. The aim of our study was to determine effects of calcium ions on phospholipid membranes employing two cell membrane analogues, liposomes and planar lipid bilayers, and for the first time the combination of two instrumental setups: gas-phase electrophoresis (nES GEMMA instrumentation) and electrical (capacitance and resistance) measurements. Liposomes and planar lipid bilayers consisted of phosphatidylcholine, cholesterol and phosphatidylethanolamine. Liposomes were prepared from dried lipid films via hydration while planar lipid bilayers were formed using a Mueller-Rudin method. Calcium ions were added to membranes from higher concentrated stock solutions. Changes in phospholipid bilayer properties due to calcium presence were observed for both studied cell membrane analogues. Changes in liposome size were observed, which might either be related to tighter packing of phospholipids in the bilayer or local distortions of the membrane. Likewise, a measurable change in planar lipid bilayer resistance and capacitance was observed in the presence of calcium ions, which can be due to an increased rigidity and tighter packing of the lipid molecules in the bilayer.

The good and the bad of cell membrane electroporation.

Electroporation is used to increase the permeability of the cell membrane through high-voltage electric pulses. Nowadays, it is widely used in different areas, such as medicine, biotechnology, and the food industry. Electroporation induces the formation of hydrophilic pores in the lipid bilayer of cell membranes, to allow the entry or exit of molecules that cannot otherwise cross this hydrophobic barrier. In this article, we critically review the basic principles of electroporation, along with the advantages and drawbacks of this method. We discuss the effects of electroporation on the key components of biological membranes, as well as the main applications of this procedure in medicine, such as electrochemotherapy, gene electrotransfer, and tissue ablation. Finally, we define the most relevant challenges of this promising area of research.

Water Pores in Planar Lipid Bilayers at Fast and Slow Rise of Transmembrane Voltage.

Basic understanding of the barrier properties of biological membranes can be obtained by studying model systems, such as planar lipid bilayers. Here, we study water pores in planar lipid bilayers in the presence of transmembrane voltage. Planar lipid bilayers were exposed to fast and slow linearly increasing voltage and current signals. We measured the capacitance, breakdown voltage, and rupture time of planar lipid bilayers composed of 1-pamitoyl 2-oleoyl phosphatidylcholine (POPC), 1-pamitoyl 2-oleoyl phosphatidylserine (POPS), and a mixture of both lipids in a 1:1 ratio. Based on the measurements, we evaluated the change in the capacitance of the planar lipid bilayer corresponding to water pores, the radius of water pores at membrane rupture, and the fraction of the area of the planar lipid bilayer occupied by water pores.planar lipid bilayer capacitance, which corresponds to water pores, water pore radius at the membrane rupture, and a fraction of the planar lipid bilayer area occupied by water pores. The estimated pore radii determining the rupture of the planar lipid bilayer upon fast build-up of transmembrane voltage are 0.101 nm, 0.110 nm, and 0.106 nm for membranes composed of POPC, POPS, and POPC:POPS, respectively. The fraction of the surface occupied by water pores at the moment of rupture of the planar lipid bilayer The fraction of an area that is occupied by water pores at the moment of planar lipid bilayer rupture is in the range of 0.1-1.8%.

Internal configuration and electric potential in planar negatively charged lipid head group region in contact with ionic solution.

The lipid bilayer composed of negatively charged lipid 1-palmitoyl-3-oleoyl-sn-glycero-3-phosphatidylserine (POPS) in contact with an aqueous solution of monovalent salt ions was studied theoretically by using the mean-field modified Langevin-Poisson-Boltzmann (MLPB) model. The MLPB results were tested by using molecular dynamic (MD) simulations. In the MLPB model the charge distribution of POPS head groups is theoretically described by the negatively charged surface which accounts for negatively charged phosphate groups, while the positively charged amino groups and negatively charged carboxylate groups are assumed to be fixed on the rod-like structures with rotational degree of freedom. The spatial variation of relative permittivity, which is not considered in the well-known Gouy-Chapman (GC) model or in MD simulations, is thoroughly derived within a strict statistical mechanical approach. Therefore, the spatial dependence and magnitude of electric potential within the lipid head group region and its close vicinity are considerably different in the MLPB model from the GC model. The influence of the bulk salt concentration and temperature on the number density profiles of counter-ions and co-ions in the lipid head group region and aqueous solution along with the probability density function for the lipid head group orientation angle was compared and found to be in qualitative agreement in the MLPB and MD models.

Specific electrical capacitance and voltage breakdown as a function of temperature for different planar lipid bilayers.

The breakdown voltage and specific electrical capacitance of planar lipid bilayers formed from lipids isolated from the membrane of archaeon Aeropyrum pernix K1 as a function of temperature were studied and compared with data obtained previously in MD simulation studies. Temperature dependence of breakdown voltage and specific electrical capacitance was measured also for dipalmitoylphosphatidylcholine (DPPC) bilayers and bilayers formed from mixture of diphytanoylphosphocholine (DPhPC) and DPPC in ratio 80:20. The breakdown voltage of archaeal lipids planar lipid bilayers is more or less constant until 50°C, while at higher temperatures a considerable drop is observed, which is in line with the results from MD simulations. The breakdown voltage of DPPC planar lipid bilayer at melting temperature is considerably higher than in the gel phase. Specific electrical capacitance of planar lipid bilayers formed from archaeal lipids is approximately constant for temperatures up to 40°C and then gradually decreases. The difference with MD simulation predictions is discussed. Specific electrical capacitance of DPPC planar lipid bilayers in fluid phase is 1.75 times larger than that of the gel phase and it follows intermediated phases before phase transition. Increase in specific electrical capacitance while approaching melting point of DPPC is visible also for DPhPC:DPPC mixture.

Electroporation threshold of POPC lipid bilayers with incorporated polyoxyethylene glycol (C12E8).

Electroporation relates to a phenomenon in which cell membranes are permeabilized after being exposed to high electric fields. On the molecular level, the mechanism is not yet fully elucidated, although a considerable body of experiments and molecular dynamic (MD) simulations were performed on model membranes. Here we present the results of a combined theoretical and experimental investigation of electroporation of palmitoy-oleoyl-phosphatidylcholine (POPC) bilayers with incorporated polyoxyethylene glycol (C12E8) surfactants. The experimental results show a slight increase of the capacitance and a 22% decrease of the voltage breakdown upon addition of C12E8 to pure POPC bilayers. These results were qualitatively confirmed by the MD simulations. They later revealed that the polyoxyethylene glycol molecules play a major role in the formation of hydrophilic pores in the bilayers above the electroporation threshold. The headgroup moieties of the latter are indeed embedded in the interior of the bilayer, which favors formation of water wires that protrude into its hydrophobic core. When the water wires extend across the whole bilayer, they form channels stabilized by the C12E8 head groups. These hydrophilic channels can transport ions across the membrane without the need of major lipid head-group rearrangements.

Structural properties of archaeal lipid bilayers: small-angle X-ray scattering and molecular dynamics simulation study.

Aeropyrum pernix is an aerobic hyperthermophilic archaeon that grows in harsh environmental conditions and as such possesses unique structural and metabolic features. Its membrane interfaces with the extreme environment and is the first line of defense from external factors. Therefore, lipids composing this membrane have special moieties that increase its stability. The membrane of A. pernix is composed predominantly of two polar lipids 2,3-di-O-sesterterpanyl-sn-glicerol-1-phospho-1'(2'-O-α-D-glucosyl)-myo-inositol (AGI) and 2,3-di-O-sesterterpanyl-sn-glicerol-1-phospho-myo-inositol (AI). Both have methyl branches in their lipid tails and ether linkages and carbohydrates in their headgroup. These moieties significantly affect the structure and dynamics of the bilayer. To provide a molecular level insight into these characteristics, we used here Molecular Dynamics (MD) simulations of lipid bilayers of composition similar to those of the archaeal membranes. First, we show that the electron density profiles along the normal to the bilayers derived from the simulations are in good agreement with the profiles obtained by the small-angle X-ray scattering (SAXS) technique, which provides confidence in the force fields used. Analyses of the simulation data show that the archaeal lipid bilayers are less hydrated than conventional phosphatidylcholine (PC) lipids and that their structure is not affected by the salt present in the surrounding solution. Furthermore, the lateral pressure in their hydrophobic core, due to the presence of the branched tails, is much higher than that at PC-based lipid bilayers. Both the methyl branched tails and the special headgroup moieties contribute to slow drastically the lateral diffusion of the lipids. Furthermore, we found that the lipid head groups associate via hydrogen bonding, which affects their reorientational dynamics. All together, our data provide links between the microscopic properties of these membranes and their overall stability in harsh environments.

Electroporation of archaeal lipid membranes using MD simulations.

Molecular dynamics (MD) simulations were used to investigate the electroporation of archaeal lipid bilayers when subjected to high transmembrane voltages induced by a charge imbalance, mimicking therefore millisecond electric pulse experiments. The structural characteristics of the bilayer, a 9:91 mol% 2,3-di-O-sesterterpanyl-sn-glicerol-1-phospho-myo-inositol (AI) and 2,3-di-O-sesterterpanyl-sn-glicerol-1-phospho-1'(2'-O-α-D-glucosyl)-myo-inositol (AGI) were compared to small angle X-ray scattering data. A rather good agreement of the electron density profiles at temperatures of 298 and 343 K was found assessing therefore the validity of the protocols and force fields used in simulations. Compared to dipalmitoyl-phosphatidylcholine (DPPC), the electroporation threshold for the bilayer was found to increase from ~2 V to 4.3 V at 323 K, and to 5.2 V at 298 K. Comparing the electroporation thresholds of the archaeal lipids to those of simple diphytanoyl-phosphatidylcholine (DPhPC) bilayers (2.5 V at 323 K) allowed one to trace back the stability of the membranes to the structure of their lipid head groups. Addition of DPPC in amounts of 50 mol% to the archaeal lipid bilayers decreases their stability and lowers the electroporation thresholds to 3.8 V and 4.1 V at respectively 323 and 298 K. The present study therefore shows how membrane compositions can be selected to cover a wide range of responses to electric stimuli. This provides new routes for the design of liposomes that can be efficiently used as drug delivery carriers, as the selection of their composition allows one to tune in their electroporation threshold for subsequent release of their load.

On the electroporation thresholds of lipid bilayers: molecular dynamics simulation investigations.

Electroporation relates to the cascade of events that follows the application of high electric fields and that leads to cell membrane permeabilization. Despite a wide range of applications, little is known about the electroporation threshold, which varies with membrane lipid composition. Here, using molecular dynamics simulations, we studied the response of dipalmitoyl-phosphatidylcholine, diphytanoyl-phosphocholine-ester and diphytanoyl-phosphocholine-ether lipid bilayers to an applied electric field. Comparing between lipids with acyl chains and methyl branched chains and between lipids with ether and ester linkages, which change drastically the membrane dipole potential, we found that in both cases the electroporation threshold differed substantially. We show, for the first time, that the electroporation threshold of a lipid bilayer depends not only on the "electrical" properties of the membrane, i.e., its dipole potential, but also on the properties of its component hydrophobic tails.

Monovalent ions and water dipoles in contact with dipolar zwitterionic lipid headgroups-theory and MD simulations.

The lipid bilayer is a basic building block of biological membranes and can be pictured as a barrier separating two compartments filled with electrolyte solution. Artificial planar lipid bilayers are therefore commonly used as model systems to study the physical and electrical properties of the cell membranes in contact with electrolyte solution. Among them the glycerol-based polar phospholipids which have dipolar, but electrically neutral head groups, are most frequently used in formation of artificial lipid bilayers. In this work the electrical properties of the lipid layer composed of zwitterionic lipids with non-zero dipole moments are studied theoretically. In the model, the zwitterionic lipid bilayer is assumed to be in contact with aqueous solution of monovalent salt ions. The orientational ordering of water, resulting in spatial variation of permittivity, is explicitly taken into account. It is shown that due to saturation effect in orientational ordering of water dipoles the relative permittivity in the zwitterionic headgroup region is decreased, while the corresponding electric potential becomes strongly negative. Some of the predictions of the presented mean-field theoretical consideration are critically evaluated using the results of molecular dynamics (MD) simulation.

Molecular-level characterization of lipid membrane electroporation using linearly rising current.

We present experimental and theoretical results of electroporation of small patches of planar lipid bilayers by means of linearly rising current. The experiments were conducted on ~120-µm-diameter patches of planar phospholipid bilayers. The steadily increasing voltage across the bilayer imposed by linearly increasing current led to electroporation of the membrane for voltages above a few hundred millivolts. This method shows new molecular mechanisms of electroporation. We recorded small voltage drops preceding the breakdown of the bilayer due to irreversible electroporation. These voltage drops were often followed by a voltage re-rise within a fraction of a second. Modeling the observed phenomenon by equivalent electric circuits showed that these events relate to opening and closing of conducting pores through the bilayer. Molecular dynamics simulations performed under similar conditions indicate that each event is likely to correspond to the opening and closing of a single pore of about 5 nm in diameter, the conductance of which ranges in the 100-nS scale. This combined experimental and theoretical investigation provides a better quantitative characterization of the size, conductance and lifetime of pores created during lipid bilayer electroporation. Such a molecular insight should enable better control and tuning of electroporation parameters for a wide range of biomedical and biotechnological applications.

System for measuring planar lipid bilayer properties.

We present a system for measuring planar lipid bilayer properties. The system is composed of a control unit, an output stage, an LCR meter, pumps for filling reservoirs, a bath with temperature regulation and a measurement chamber with four electrodes. The planar lipid bilayer is automatically formed using a folding method on apertures of different sizes. The automatization is assured by two syringes, which are clamped in actuators. Actuators are driven and controlled by a control unit via RS-232 communication. The temperature of the planar lipid bilayer can be regulated between 15 and 55 °C. The regulation is assured by insertion of the measurement chamber into the temperature-regulated bath. Different shapes of voltage- or current-clamp signals can be applied to the planar lipid bilayer. By measuring the response of the planar lipid bilayer to the applied signal, the capacitance and breakdown voltage of the planar lipid bilayer can be determined. The cutoff frequencies of the system output stage for voltage- and current-clamp methods are 11 and 17 kHz, respectively.

Planar lipid bilayers: observing pore creation and extinction.

From an electrical point of view a planar lipid bilayer can be considered as a non-perfect capacitor; it can be presented as an ideal capacitor in parallel with resistor. In this study the whole measuring system including planar lipid bilayer was modeled by an equivalent electric circuit in Spiceopus software. Such a model gives additional information of experimentally obtained results. In this way we analyze measurements of transmembrane voltage that appears on planar lipid bilayer as consequence of linear rising current. Small voltage drops were obtained before the planar lipid bilayer breakdown. The model showed that effective current on planar lipid bilayer is actually much smaller than the current applied with current generator and should be used in calculations of a conductance related to voltage drops.

A system for the determination of planar lipid bilayer breakdown voltage and its applications.

In this paper, we focus on measurement principles used in electroporation studies on planar lipid bilayers. In particular, we point out the voltage-clamp measurement principle that has great importance when the breakdown voltage of a planar lipid bilayer is under consideration; however, it is also appropriate for the determination of other planar lipid bilayer electrical properties such as resistance and capacitance. A new experimental system that is based on the voltage-clamp measurement principle is described. With the use of a generator that can generate arbitrary-type signals, many specific shapes of a voltage signal could be generated, and therefore, the experimental system is appropriate for a broad spectrum of measurements.