The Effect of the Osmotically Active Compound Concentration Difference on the Passive Water and Proton Fluxes across a Lipid Bilayer.

The molecular details of the passive water flux across the hydrophobic membrane interior are still a matter of debate. One of the postulated mechanisms is the spontaneous, water-filled pore opening, which facilitates the hydrophilic connection between aqueous phases separated by the membrane. In the paper, we provide experimental evidence showing that the spontaneous lipid pore formation correlates with the membrane mechanics; hence, it depends on the composition of the lipid bilayer and the concentration of the osmotically active compound. Using liposomes as an experimental membrane model, osmotically induced water efflux was measured with the stopped-flow technique. Shapes of kinetic curves obtained at low osmotic pressure differences are interpreted in terms of two events: the lipid pore opening and water flow across the aqueous channel. The biological significance of the dependence of the lipid pore formation on the concentration difference of an osmotically active compound was illustrated by the demonstration that osmotically driven water flow can be accompanied by the dissipation of the pH gradient. The application of the Helfrich model to describe the probability of lipid pore opening was validated by demonstrating that the probability of pore opening correlates with the membrane bending rigidity. The correlation was determined by experimentally derived bending rigidity coefficients and probabilities of lipid pores opening.

Statistical Analysis of Bending Rigidity Coefficient Determined Using Fluorescence-Based Flicker-Noise Spectroscopy.

Bending rigidity coefficient describes propensity of a lipid bilayer to deform. In order to measure the parameter experimentally using flickering noise spectroscopy, the microscopic imaging is required, which necessitates the application of giant unilamellar vesicles (GUV) lipid bilayer model. The major difficulty associated with the application of the model is the statistical character of GUV population with respect to their size and the homogeneity of lipid bilayer composition, if a mixture of lipids is used. In the paper, the bending rigidity coefficient was measured using the fluorescence-enhanced flicker-noise spectroscopy. In the paper, the bending rigidity coefficient was determined for large populations of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine and 1,2-dioleoyl-sn-glycero-3-phosphocholine vesicles. The quantity of obtained experimental data allows to perform statistical analysis aiming at the identification of the distribution, which is the most appropriate for the calculation of the value of the membrane bending rigidity coefficient. It has been demonstrated that the bending rigidity coefficient is characterized by an asymmetrical distribution, which is well approximated with the gamma distribution. Since there are no biophysical reasons for that we propose to use the difference between normal and gamma fits as a measure of the homogeneity of vesicle population. In addition, the effect of a fluorescent label and types of instrumental setups on determined values has been tested. Obtained results show that the value of the bending rigidity coefficient does not depend on the type of a fluorescent label nor on the type of microscope used.

The Elucidation of the Molecular Mechanism of the Extrusion Process.

Extrusion is a popular method for producing homogenous population of unilamellar liposomes. The technique relies on forcing a lipid suspension through cylindrical pores in a polycarbonate membrane. The quantification of the extrusion and/or recalibration processes make possible the acquisition of experimental data, which can be correlated with the mechanical properties of the lipid bilayer. In this work, the force needed for the extrusion process was correlated with the mechanical properties of a lipid bilayer derived from other experiments. Measurements were performed using a home-made dedicated device capable of maintaining a stable volumetric flux of a liposome suspension through well-defined pores and to continuously measure the extrusion force. Based on the obtained results, the correlation between the lipid bilayer bending rigidity and extrusion force was derived. Specifically, it was found that the bending rigidity of liposomes formed from well-defined lipid mixtures agrees with data obtained by others using flicker-noise spectroscopy or micromanipulation. The other issue addressed in the presented studies was the identification of molecular mechanisms leading to the formation of unilamellar vesicles in the extrusion process. Finally, it was demonstrated that during the extrusion, lipids are not exchanged between vesicles, i.e., vesicles can divide but no membrane fusion or lipid exchange between bilayers was detected.

The effect of lipid phase on liposome stability upon exposure to the mechanical stress.

Mechanical properties of a lipid bilayer are parameters determined mainly for giant unilamellar vesicles (GUVs). It is not clear if values obtained on the GUV model can be directly translated to submicron large unilamellar vesicles (LUVs). This ambiguity is a major obstacle in exploring the effect of lipid bilayer mechanics on membrane associated processes and effectiveness of liposome-based targeted drug delivery systems. In presented work extrusion, which is a common method to prepare LUVs, was used to study liposomes preparation and stability upon exposure to mechanical stress. The effect of parameters of the extrusion process (temperature, membrane pore size, extrusion force and volumetric flux) on the properties of liposome suspension (average liposome size, polydispersity index and lipid recovery ratio) was determined for model liposomes composed of DPPC lipid. The state of the DPPC lipid bilayer depends on temperature, therefore, the effect of lipid bilayer mechanics on the extrusion process can be quantitated without altering membrane composition. The extrusion process was carried out with the automated extruder delivering quantitative data on the extrusion force and volumetric flux. Obtained results have been interpreted in terms of mechanical properties of the lipid bilayer. Determined mechanical properties of the lipid bilayer and its dependence on temperature are in good agreement with the literature results determined for GUVs. This shows that mechanical properties of the lipid bilayer does not depend on the liposome size in the range from 100 nm to hundreds of microns.

Effects of electroformation protocol parameters on quality of homogeneous GUV populations.

Giant unilamellar vesicles (GUVs) have become one of extensively studied biological bilayer models especially when investigating topological and mechanical properties of cell membranes. They are also used to visualize membrane-related phenomena. However, the method of preparation and the effects of parameters of preparation on the vesicular structure are extensively varied. Therefore, it is important to understand how the process of formation of GUVs influences the outcome population, as it can influence the outcome of the experiment that is planned. Therefore, in this study, we investigated the effects of protocol parameters of electroformation on properties of homogeneous population of POPC GUVs. The parameters investigated in this study are duration of electroformation, usage of electrodes and frequency of applied AC field and its voltage. The properties investigated, which can be used to describe GUV populations are average diameter of vesicle, the amount of lipid molecules in population, and structure of vesicles. According to our results, prolonged time (greater than 4 h) does not influence outcome; however, parameters of applied electrical field (voltage and frequency) did significantly influence the properties of obtained POPC GUV populations.