Effects of cholesterol on the anionic magnetite nanoparticle-induced deformation and poration of giant lipid vesicles.

We have investigated the effects of cholesterol on the deformation and poration of giant unilamellar vesicles (GUVs) induced by anionic magnetite nanoparticles (NPs). Negatively charged lipid, neutral lipid, and cholesterol were used to prepare the charged GUVs (surface charge density of membranes - 0.16 C m-2), while only neutral lipid and cholesterol were used to prepare the neutral GUVs. Cholesterol content varied from 0 to 40 mole% for preparing the biologically relevant membranes. The degree of deformation has been characterized by compactness, the value of which remains at 1.0 for spherical GUVs. The value of compactness increases with time for both membranes, but this increase depends on cholesterol content. The average compactness decreases with cholesterol content, and at 60 min, the values are 1.280 ± 0.002 and 1.131 ± 0.010 for 0 and 40 mole% cholesterol containing charged GUVs. The average compactness is relatively lower for neutral GUVs for the corresponding cholesterol. Membrane poration has been investigated by the leakage of calcein, which indicates a two-state transition model. The fraction of deformation is higher for charged GUVs than for neutral ones, while the fraction of poration shows the opposite result. Both the fractions decrease with cholesterol content.

Influence of sugar concentration on the vesicle compactness, deformation and membrane poration induced by anionic nanoparticles.

Sugar plays a vital role in the structural and functional characteristics of cells. Hence, the interaction of NPs with cell membranes in the presence of sugar concentrations is important for medicinal and pharmacological innovations. This study integrated three tools: giant unilamellar vesicles (GUVs), anionic magnetite nanoparticles (NPs), and sugar concentrations, to understand a simplified mechanism for interactions between the vesicle membranes and NPs under various sugar concentrations. We focused on changing the sugar concentration in aqueous solution; more precisely, sucrose inside the GUVs and glucose outside with equal osmolarity. 1,2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (sodium salt) (DOPG) and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) were used to prepare the charged membranes of 40mole%DOPG/60mole%DOPC-GUVs, whereas only DOPC was used to prepare the neutral membranes. Phase contrast fluorescence microscopy shows that the adherence of 18 nm magnetite NPs with anionic charge depends on the sugar concentration. The alterations of GUVs induced by the NPs are characterized in terms of i) vesicle compactness, ii) deformation, and iii) membrane poration. The presence of sugar provides additional structural stability to the GUVs and reduces the effects of the NPs with respect to these parameters; more precisely, the higher the sugar concentration, the smaller the alteration induced by the NPs. The differences in NPs effects are explained by the change in the type of interaction between sugar molecules and lipid membranes, namely enthalpy and entropy-driven interaction, respectively. In addition, such alterations are influenced by the surface charge density of the lipid bilayer. The surface pressure of membranes due to the adsorption of NPs is responsible for inducing the poration in membranes. The differences in deformation and poration in charged and neutral GUVs under various sugar concentrations are discussed based on the structure of the head of lipid molecules.

Effects of sugar concentration on the electroporation, size distribution and average size of charged giant unilamellar vesicles.

We investigated the effects of sugar concentration on the electroporation, size distribution and average size of giant unilamellar vesicles (GUVs). GUVs were prepared from 40 mol% of 1,2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DOPG) and 60 mol% of 1, 2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) lipids. Pulsed electric field was applied to the 40%DOPG/60%DOPC-GUVs and it induced lateral electric tension (σc) in the membranes of vesicles. The σc-induced probability of rupture (Ppore) and the rate constant of rupture (kp) of GUVs under the sugar concentration, c = 40, 100 and 300 mM, were determined. Both the Ppore and kp increased with the increase of σc, but higher tension was required to generate the same values of Ppore and kp with increasing c. We also investigated average sizes of GUVs from the size distribution of vesicles under various sugar concentrations. With the increase of c, the peak of the size distribution histograms shifted to the region of smaller vesicles. The average size decreased 1.6-fold when c increased from 10 to 300 mM. These investigations help to understand various biomedical, biophysical, and biochemical processes in vesicles and cells. Electroporation, size distribution and average size of charged GUVs were investigated under various sugar concentrations. The sugar concentration influences the electroporation of vesicles and the average size of GUVs.

Effects of cholesterol on the size distribution and bending modulus of lipid vesicles.

The influence of cholesterol fraction in the membranes of giant unilamellar vesicles (GUVs) on their size distributions and bending moduli has been investigated. The membranes of GUVs were synthesized by a mixture of two elements: electrically neutral lipid 1, 2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and cholesterol and also a mixture of three elements: electrically charged lipid 1,2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DOPG), DOPC and cholesterol. The size distributions of GUVs have been presented by a set of histograms. The classical lognormal distribution is well fitted to the histograms, from where the average size of vesicle is obtained. The increase of cholesterol content in the membranes of GUVs increases the average size of vesicles in the population. Using the framework of Helmholtz free energy of the system, the theory developed by us is extended to explain the experimental results. The theory determines the influence of cholesterol on the bending modulus of membranes from the fitting of the proper histograms. The increase of cholesterol in GUVs increases both the average size of vesicles in population and the bending modulus of membranes.

A new purification technique to obtain specific size distribution of giant lipid vesicles using dual filtration.

A new purification technique is developed for obtaining distribution of giant unilamellar vesicles (GUVs) within a specific range of sizes using dual filtration. The GUVs were prepared using well known natural swelling method. For filtration, different combinations of polycarbonate membranes were implemented in filter holders. In our experiment, the combinations of membranes were selected with corresponding pore sizes-(i) 12 and 10 µm, (ii) 12 and 8 µm, and (iii) 10 and 8 µm. By these filtration arrangements, obtained GUVs size distribution were in the ranges of 6-26 µm, 5-38 µm and 5-30 µm, respectively. In comparison, the size distribution range was much higher for single filtration technique, for example, 6-59 µm GUVs found for a membrane with 12 µm pores. Using this technique, the water-soluble fluorescent probe, calcein, can be removed from the suspension of GUVs successfully. The size distributions were analyzed with lognormal distribution. The skewness became smaller (narrow size distribution) when a dual filtration was used instead of single filtration. The mode of the size distribution obtained in dual filtration was also smaller to that of single filtration. By continuing this process of purification for a second time, the GUVs size distribution became even narrower. After using an extra filtration with dual filtration, two different size distributions of GUVs were obtained at a time. This experimental observation suggests that different size specific distributions of GUVs can be obtained easily, even if GUVs are prepared by different other methods.

Effects of osmotic pressure on the irreversible electroporation in giant lipid vesicles.

Irreversible electroporation (IRE) is a nonthermal tumor/cell ablation technique in which a series of high-voltage short pulses are used. As a new approach, we aimed to investigate the rupture of giant unilamellar vesicles (GUVs) using the IRE technique under different osmotic pressures (Π), and estimated the membrane tension due to Π. Two categories of GUVs were used in this study. One was prepared with a mixture of dioleoylphosphatidylglycerol (DOPG), dioleoylphosphatidylcholine (DOPC) and cholesterol (chol) for obtaining more biological relevance while other with a mixture of DOPG and DOPC, with specific molar ratios. We determined the rate constant (kp) of rupture of DOPG/DOPC/chol (46/39/15)-GUVs and DOPG/DOPC (40/60)-GUVs induced by constant electric tension (σc) under different Π. The σc dependent kp values were fitted with a theoretical equation, and the corresponding membrane tension (σoseq) at swelling equilibrium under Π was estimated. The estimated membrane tension agreed well with the theoretical calculation within the experimental error. Interestingly, the values of σoseq were almost same for both types of synthesized GUVs under same osmotic pressure. We also examined the sucrose leakage, due to large osmotic pressure-induced pore formation, from the inside of DOPG/DOPC/chol(46/39/15)-GUVs. The estimated membrane tension due to large Π at which sucrose leaked out was very similar to the electric tension at which GUVs were ruptured without Π. We explained the σc and Π induced pore formation in the lipid membranes of GUVs.

An investigation into the critical tension of electroporation in anionic lipid vesicles.

Irreversible electroporation (IRE) is a technique for the disruption of localized cells or vesicles by a series of short and high-frequency electric pulses which has been used for tissue ablation and treatment in certain diseases. It is well reported that IRE induces lateral tension in the membranes of giant unilamellar vesicles (GUVs). The GUVs are prepared by a mixture of anionic lipid dioleoylphosphatidylglycerol (DOPG) and neutral lipid dioleoylphosphatidylcholine (DOPC) using the natural swelling method. Here the influence of DOPG mole fraction, XDOPG, on the critical tension of electroporation in GUVs has been investigated in sodium chloride-containing PIPES buffer. The critical tension decreases from 9.0 ± 0.3 to 6.0 ± 0.2 mN/m with the increase of XDOPG from 0.0 to 0.60 in the membranes of GUVs. Hence an increase in XDOPG greatly decreases the mechanical stability of membranes. We develop a theoretical equation that fits the XDOPG dependent normalized critical tension, and obtain a binding constant for the lipid-ion interaction of 0.75 M-1. The decrease in the energy barrier for formation of the nano-size nascent or prepore state, due to the increase in XDOPG, is the main factor explaining the decrease in critical tension of electroporation in vesicles.

Correction to: Kinetics of irreversible pore formation under constant electrical tension in giant unilamellar vesicles.

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Electrostatic effects on the electrical tension-induced irreversible pore formation in giant unilamellar vesicles.

Irreversible electroporation (IRE) is a new technique in which a series of short pulses with high frequency electrical energy is applied on the targeted regions of cells or vesicles for their destruction or rupture formation. IRE induces lateral tension in the membranes of vesicles. We have investigated the electrostatic interaction effects on the constant electrical tension-induced rate constant of irreversible pore formation in the membranes of giant unilamellar vesicles (GUVs). The electrostatic interaction has been varied by changing the salt concentration in buffer and the surface charge density of membranes. The membranes of GUVs are synthesized by a mixture of negatively charged lipid dioleoylphosphatidylglycerol (DOPG) and neutral lipid dioleoylphosphatidylcholine (DOPC) using the natural swelling method. The rate constant of pore formation increases with the decrease of salt concentration in buffer along with the increase of surface charge density of membranes. The tension dependent probability of pore formation and the rate constant of pore formation are fitted to the theoretical equation, and obtained the line tension of membranes. The decrease in energy barrier of a prepore due to electrostatic interaction is the key factor causing an increase of rate constant of pore formation.

Kinetics of irreversible pore formation under constant electrical tension in giant unilamellar vesicles.

Stretching in the plasma membranes of cells and lipid membranes of vesicles plays important roles in various physiological and physicochemical phenomena. Irreversible electroporation (IRE) is a minimally invasive non-thermal tumor ablation technique where a series of short electrical energy pulses with high frequency is applied to destabilize the cell membranes. IRE also induces lateral tension due to stretching in the membranes of giant unilamellar vesicles (GUVs). Here, the kinetics of irreversible pore formation under constant electrical tension in GUVs has been investigated. The GUVs are prepared by a mixture of dioleoylphosphatidylglycerol and dioleoylphosphatidylcholine using the natural swelling method. An IRE signal of frequency 1.1 kHz is applied to the GUVs through a gold-coated electrode system. Stochastic pore formation is observed for several 'single GUVs' at a particular constant tension. The time course of the fraction of intact GUVs among all the examined GUVs is fitted with a single-exponential decay function from which the rate constant of pore formation in the vesicle, kp, is calculated. The value of kp increases with an increase of membrane tension. An increase in the proportion of negatively charged lipids in a membrane gives a higher kp. Theoretical equations are fitted to the tension-dependent kp and to the probability of pore formation, which allows us to obtain the line tension of the membranes. The decrease in the energy barrier for formation of the nano-size nascent or prepore state, due to the increase in electrical tension, is the main factor explaining the increase of kp.