DNA translocation to giant unilamellar vesicles during electroporation is independent of DNA size.

Delivery of naked DNA molecules into living cells via physical disruption of the membrane under electric pulses has potential biomedical applications ranging from gene electro-transfer, electro-chemotherapy, to gene therapy, yet the mechanisms involved in DNA transport remain vague. To investigate the mechanism of DNA translocation across the cell membrane, giant unilamellar vesicles (GUVs) were electroporated in the presence of DNA molecules keeping the size of the DNA molecules as a variable parameter. We experimentally determined the translocation efficiency for each size of the DNA molecule, to compare the results with the existing and conflicting theories of the translocation mechanism i.e. stochastic threading and bulk electrophoresis. We observed that the translocation efficiency is independent of DNA size (ranging from 25-20 000 bp, bp = base pairs), implying that DNA molecules translocate freely across the electro-pores in the lipid membrane in their native polymer conformation, as opposed to unravelling and threading through the electro-pore. Bulk electrophoretic mobility determines the relationship between translocation efficiency and the size of the DNA molecule. This research provides experimental evidence of the mechanistic understanding of DNA translocation across lipid membranes which is essential for devising efficient and predictable protocols for electric field mediated naked DNA delivery.

Response of an actin network in vesicles under electric pulses.

We study the role of a biomimetic actin network during the application of electric pulses that induce electroporation or electropermeabilization, using giant unilamellar vesicles (GUVs) as a model system. The actin cortex, a subjacently attached interconnected network of actin filaments, regulates the shape and mechanical properties of the plasma membrane of mammalian cells, and is a major factor influencing the mechanical response of the cell to external physical cues. We demonstrate that the presence of an actin shell inhibits the formation of macropores in the electroporated GUVs. Additionally, experiments on the uptake of dye molecules after electroporation show that the actin network slows down the resealing process of the permeabilized membrane. We further analyze the stability of the actin network inside the GUVs exposed to high electric pulses. We find disruption of the actin layer that is likely due to the electrophoretic forces acting on the actin filaments during the permeabilization of the GUVs. Our findings on the GUVs containing a biomimetic network provide a step towards understanding the discrepancies between the electroporation mechanism of a living cell and its simplified model of the empty GUV.

The role of gel-phase domains in electroporation of vesicles.

Transient permeabilisation of the cell membrane is a critical step to introduce drugs or DNA into living cells, yet challenging for both biological research and therapeutic applications. To achieve this, electroporation (or electropermeabilisation) has become a widely used method due to its simplicity to deliver almost any biomolecule to any cell type. Although this method demonstrates promise in the field of drug/gene delivery, the underlying physical mechanisms of the response of the heterogeneous cell membrane to strong electric pulses is still unknown. In this study, we have investigated the role of gel-phase lipids in the electroporation of binary giant unilamellar vesicles (GUVs), composed from DPPC (gel-phase) and DPhPC (fluid-phase) lipids (molar ratio 8:2 and 2:8). We have observed that the exposure to electric pulses leads to expel of fluid-phase lipids and concomitant decrease in GUV size, whereas the gel-phase domains become buckled. Based on experiments on pure fluid-phase and gel-phase GUVs, we have found that fluid-phase lipids can be expelled by electrical forces and the highly viscous gel-phase lipids cannot. Moreover, our analyses suggest that pore formation occurs primarily in fluid-phase domains and that the pore size is similar in all GUVs containing fluid-phase lipids, irrespective of the gel-phase percentage.

Lipid vesicles in pulsed electric fields: Fundamental principles of the membrane response and its biomedical applications.

The present review focuses on the effects of pulsed electric fields on lipid vesicles ranging from giant unilamellar vesicles (GUVs) to small unilamellar vesicles (SUVs), from both fundamental and applicative perspectives. Lipid vesicles are the most popular model membrane systems for studying biophysical and biological processes in living cells. Furthermore, as vesicles are made from biocompatible and biodegradable materials, they provide a strategy to create safe and functionalized drug delivery systems in health-care applications. Exposure of lipid vesicles to pulsed electric fields is a common physical method to transiently increase the permeability of the lipid membrane. This method, termed electroporation, has shown many advantages for delivering exogenous molecules including drugs and genetic material into vesicles and living cells. In addition, electroporation can be applied to induce fusion between vesicles and/or cells. First, we discuss in detail how research on cell-size GUVs as model cell systems has provided novel insight into the basic mechanisms of cell electroporation and associated phenomena. Afterwards, we continue with a thorough overview how electroporation and electrofusion have been used as versatile methods to manipulate vesicles of all sizes in different biomedical applications. We conclude by summarizing the open questions in the field of electroporation and possible future directions for vesicles in the biomedical field.

Negatively Charged Lipid Membranes Catalyze Supramolecular Hydrogel Formation.

In this contribution we show that biological membranes can catalyze the formation of supramolecular hydrogel networks. Negatively charged lipid membranes can generate a local proton gradient, accelerating the acid-catalyzed formation of hydrazone-based supramolecular gelators near the membrane. Synthetic lipid membranes can be used to tune the physical properties of the resulting multicomponent gels as a function of lipid concentration. Moreover, the catalytic activity of lipid membranes and the formation of gel networks around these supramolecular structures are controlled by the charge and phase behavior of the lipid molecules. Finally, we show that the insights obtained from synthetic membranes can be translated to biological membranes, enabling the formation of gel fibers on living HeLa cells.

High-resolution excitation and absorption spectroscopy of gas-phase p-coumaric acid: unveiling an elusive chromophore.

We report on the first successful high-resolution spectroscopic studies on isolated para-coumaric acid, the chromophore of the photoactive yellow protein which has become a model system for studying biological light-induced signal transduction. Employing various double-resonance multiphoton ionization techniques in combination with mass-resolved ion detection and the results of quantum chemical calculations, we identify three conformations the molecule can adopt under our experimental conditions. The vibrational activity in the excitation spectra allows us to conclude that in the Franck-Condon region accessed from the ground state S(1) is the V'(pipi*) state. Interestingly, we find considerable out-of-plane vibrational activity, indicating that the molecule adopts a nonplanar geometry in S(1). The ionization requirements show that after excitation rapid internal conversion takes place to a lower-lying npi* state. Such a state has been postulated by ab initio calculations on para-coumaric acid and derivatives, but until the present study no direct evidence had been found for its presence.