Resealing of membrane pores is crucial for cell survival. Membrane surface charge and medium composition are studied as defining regulators of membrane stability. Pores are generated by electric field or detergents. Giant vesicles composed of zwitterionic and negatively charged lipids mixed at varying ratios are subjected to a strong electric pulse. Interestingly, charged vesicles appear prone to catastrophic collapse transforming them into tubular structures. The spectrum of destabilization responses includes the generation of long‐living submicroscopic pores and partial vesicle bursting. The origin of these phenomena is related to the membrane edge tension, which governs pore closure. This edge tension significantly decreases as a function of the fraction of charged lipids. Destabilization of charged vesicles upon pore formation is universal—it is also observed with other poration stimuli. Disruption propensity is enhanced for membranes made of lipids with higher degree of unsaturation. It can be reversed by screening membrane charge in the presence of calcium ions. The observed findings in light of theories of stability and curvature generation are interpreted and mechanisms acting in cells to prevent total membrane collapse upon poration are discussed. Enhanced membrane stability is crucial for the success of electroporation‐based technologies for cancer treatment and gene transfer.

Motivation A reliable characterization of the membrane pore edge tension of single giant unilamellar vesicles (GUVs) requires the measurement of micrometer sized pores in hundreds to thousands of images. When manually performed, this procedure has shown to be extremely time-consuming and to generate inconsistent results among different users and imaging systems. A user-friendly software for such analysis allowing quick processing and generation of reproducible data had not yet been reported. Results We have developed a software (PoET) for automatic pore edge tension measurements on GUVs. The required image processing steps and the characterization of the pore dynamics are performed automatically within the software and its use allowed for a 30-fold reduction in the analysis time. We demonstrate the applicability of the software by comparing the pore edge tension of GUVs of different membrane compositions and surface charges. The approach was applied to electroporated GUVs but is applicable to other means of pore formation. Availability and implementation The complete software is implemented in Python and available for Windows at https://dx.doi.org/10.17617/3.7h. Supplementary information Supplementary data are available at Bioinformatics Advances online.

Biological cells are contained by a fluid lipid bilayer (plasma membrane, PM) that allows for large deformations, often exceeding 50% of the apparent initial PM area. Isolated lipids self‐organize into membranes, but are prone to rupture at small (<2–4%) area strains, which limits progress for synthetic reconstitution of cellular features. Here, it is shown that by preserving PM structure and composition during isolation from cells, vesicles with cell‐like elasticity can be obtained. It is found that these plasma membrane vesicles store significant area in the form of nanotubes in their lumen. These act as lipid reservoirs and are recruited by mechanical tension applied to the outer vesicle membrane. Both in experiment and theory, it is shown that a “superelastic” response emerges from the interplay of lipid domains and membrane curvature. This finding allows for bottom‐up engineering of synthetic biomaterials that appear one magnitude softer and with threefold larger deformability than conventional lipid vesicles. These results open a path toward designing superelastic synthetic cells possessing the inherent mechanics of biological cells.

Resealing of membrane pores is crucial for cell survival. We studied the membrane surface charge and medium composition as defining regulators triggering bursting and collapse of giant unilamellar vesicles upon poration. The pores were generated by either electric field or a detergent. Vesicles composed of mixtures of zwitterionic and negatively charged lipids at varying molar ratios were subjected to a single strong electric pulse and their response was characterized for a given membrane and medium composition. We observe that charged vesicles are prone to catastrophic vesicle collapse transforming them into tubular structures. The spectrum of destabilization responses includes the generation of long-living submicroscopic pores and partial vesicle bursting. The origin of these phenomena is related to the membrane edge tension, which governs pore closure. This edge tension significantly decreases as a function of the molar fraction of charged lipids. Destabilization of charged vesicles upon pore formation is a universal process since it is also observed with other poration stimuli. Disruption propensity is enhanced for membranes made of lipids with higher degree of unsaturation. It can be reversed by screening membrane charge in the presence of calcium ions. We interpret the observed findings in light of theories of stability and curvature generation in charged membranes and discuss mechanisms acting in cells to prevent total membrane collapse upon poration. Enhanced membrane stability is crucial for the success of electroporation-based technologies for cancer treatment and gene transfer.Significance StatementLeaky membranes cannot support life. Cells have developed protective mechanisms to respond to pore formation in their membranes. We find that essential membrane components such as (asymmetrically distributed) charged lipids destabilize the membrane upon poration and lead to catastrophic collapse, shown here on giant unilamellar vesicles as model membranes porated either by electric fields or detergent. The charged lipids act via reducing the pore edge tension. The membrane destabilization can be reverted by calcium ions and is less pronounced for lipids with lower degree of unsaturation. The observed membrane stabilization can be employed for optimizing biotechnological developments employing electroporation for cancer treatment.

The development of biomaterials to enable application of antimicrobial peptides represents a strategy of high and current interest. In this study, a bioparticle was produced by the complexation between an antimicrobial polypeptide and the biocompatible and biodegradable polysaccharides chitosan-N-arginine and alginate, giving rise to a colloidal polyelectrolytic complex of pH-responsive properties. The inclusion of the polypeptide in the bioparticle structure largely increases the binding sites of complexation during the bioparticles production, leading to its effective incorporation. After lyophilization, detailed evaluation of colloidal structure of redispersed bioparticles evidenced nano or microparticles with size, polydispersity and zeta potential dependent on pH and ionic strength, and the dependence was not withdrawn with the polypeptide inclusion. Significant increase of pore edge tension in giant vesicles evidenced effective interaction of the polypeptide-bioparticle with lipid model membrane. Antibacterial activity against Aeromonas dhakensis was effective at 0.1% and equal for the isolated polypeptide and the same complexed in bioparticle, which opens perspectives to the composite material as an applicable antibacterial system.

Introduction: Cardiovascular diseases represent a major cause of death worldwide and one of their greatest complications is the development of cardiac arrhythmias, in which ventricular fibrillation (VF) stands out as the most severe one. The only therapy that reverses VF is defibrillation. However defibrillatory shock is capable of killing heart cells and it is known that the orientation of the cell major axis with respect to the electrical field (E) direction is a determining factor for cellular excitation and injury, which is leading to the development of new defibrillation protocols. The aim of this work is to fill the gap in information about cell lethality for intermediate cell orientation angles. Methods: Ventricular myocytes were extracted from adult male Wistar rats and the cells were plated in a chamber for perfusion and stimulation with bipolar voltage pulses to determine the stimulation threshold (E T). Then, monopolar stimulus was applied and amplitude was increased until cell lethal injury. This protocol was performed on four experimental groups: cells oriented at 0°, 30°, 60° and 90°, with respect to E direction. Results: 87 cells were analyzed and an increase in amplitude of E associated with 50% lethality (E50) was verified as the direction of E application and cell major axis orientation departed. Conclusion: Taken the same probability of lethality, our data suggest a nonlinear increase of E amplitude from 0° to 90° similar to that of E T. These in-between data had not yet been shown and are important for service-based future defibrillation protocols.

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Multidirectional defibrillation protocols have shown better efficiency than monodirectional; still, no testing was performed to assess cell lethality. We investigated lethality of multidirectional defibrillator-like shocks on isolated cardiomyocytes. Cells were isolated from adult male Wistar rats and plated into a perfusion chamber. Electrical field stimulation threshold (E) was obtained, and cells were paced with suprathreshold bipolar electrical field (E) pulses. Either one monodirectional high-intensity electrical field (HEF) pulse aligned at 0° (group Mono0) or 60° (group Mono60) to cell major axis or a multidirectional sequence of three HEF pulses aligned at 0°, 60°, and 120° each was applied. If cell recovered from shock, pacing was resumed, and a higher amplitude HEF, proportional to E, was applied. The sequence was repeated until cell death. Lethality curves were built by means of survival analysis from sub-lethal and lethal E. Non-linear fit was performed, and E values corresponding to 50% probability of lethality (E50) were compared. Multidirectional groups presented lethality curves similar to Mono0. Mono60 displayed the highest E50. The novel data endorse the idea of multidirectional stimuli being safer because their effects on lethality of individual cells were equal to a single monodirectional stimulus, while their defibrillatory threshold is lower. Graphical abstract Monodirectional and multidirectional lethality protocol comparison on isolated rat cardiomyocytes. The heart image is a derivative of "3D Heart in zBrush" ( https://vimeo.com/65568770 ) by Laloxl, used under CC BY 3.0 ( https://creativecommons.org/licenses/by/3.0/legalcode )/image extracted from original video.