Regulation of the intermittent release of giant unilamellar vesicles under osmotic pressure

Osmotic pressure can break the fluid balance between intracellular and extracellular solutions. In hypo-osmotic solution, water molecules, which transfer into the cell and burst, are driven by the concentrations difference of solute across the semi-permeable membrane. The complicated dynamic processes of the intermittent burst have been previously observed. However, the underlying physical mechanism has yet to be thoroughly explored and analyzed. Here, the intermittent release of inclusion in giant unilamellar vesicles was investigated quantitatively, applying the combination of experimental and theoretical methods in the hypo-osmotic medium. Experimentally, we adopted highly sensitive EMCCD to acquire intermittent dynamic images. Notably, the component of the vesicle phospholipids affected the stretch velocity, and the prepared solution of the vesicle adjusted the release time. Theoretically, we chose equations numerical simulations to quantify the dynamic process in phases and explored the influence of physical parameters such as bilayer permeability and solution viscosity on the process. It was concluded that the time taken to achieve the balance of giant unilamellar vesicles was highly dependent on the structure of the lipid molecular. The pore lifetime was strongly related with the internal solution environment of giant unilamellar vesicles. The vesicle prepared in viscous solution accessed visualized long-lived pore. Furthermore, the line tension was measured quantitatively by the release velocity of inclusion, which was in the same order of magnitude as the theoretical simulation. In all, the experimental values well matched the theoretical values. Our investigation clarified the physical regulatory mechanism of intermittent pore formation and inclusion release, which had an important reference for the development of novel technologies such as gene therapy based on transmembrane transport as well as controlled drug delivery based on liposomes.

Experimental platform for the functional investigation of membrane proteins in giant unilamellar vesicles

Giant unilamellar vesicles (GUVs) are micrometer-sized model membrane systems that can be viewed directly under the microscope. They serve as scaffolds for the bottom-up creation of synthetic cells, targeted drug delivery and have been used in many in vitro studies of membrane related phenomena. GUVs are also of interest for the functional investigation of membrane proteins that carry out many key cellular functions. A major hurdle to a wider application of GUVs in this field is the diversity of existing protocols that are optimized for individual proteins. Here, we compare PVA assisted and electroformation techniques for GUV formation under physiologically relevant conditions, and analyze the effect of immobilization on vesicle structure and membrane tightness towards small substrates and protons. There, differences in terms of yield, size, and leakage of GUVs produced by PVA assisted swelling and electroformation were found, dependent on salt and buffer composition. Using fusion of oppositely charged membranes to reconstitute a model membrane protein, we find that empty vesicles and proteoliposomes show similar fusion behavior, which allows for a rapid estimation of protein incorporation using fluorescent lipids.

Amyloid-beta peptide (25–35) triggers a reorganization of lipid membranes driven by temperature changes

The amyloid-beta peptide (Aβ) is considered a key factor in Alzheimer's disease (AD) ever since the discovery of the disease. The understanding of its damaging influence has however shifted recently from large fibrils observed in the inter-cellular environment to the small oligomers interacting with a cell membrane. We studied the effect of temperature on the latter interactions by evaluating the structural characteristics of zwitterionic phosphatidylcholine (PC) membranes with incorporated Aβ25–35 peptide. By means of small angle neutron scattering (SANS), we have observed for the first time a spontaneous reformation of extruded unilamellar vesicles (EULVs) to discoidal bicelle-like structures (BLSs) and small unilamellar vesicles (SULVs). These changes in the membrane self-organization happen during the thermodynamic phase transitions of lipids and only in the presence of the peptide. We interpret the dramatic changes in the membrane's overall shape with parallel changes in its thickness as the Aβ25–35 triggered membrane damage and a consequent reorganization of its structure. The suggested process is consistent with an action of separate peptides or small size peptide oligomers rather than the result of large Aβ fibrils.

Giant Unilamellar Vesicle Electroformation: What to Use, What to Avoid, and How to Quantify the Results

Since its inception more than thirty years ago, electroformation has become the most commonly used method for growing giant unilamellar vesicles (GUVs). Although the method seems quite straightforward at first, researchers must consider the interplay of a large number of parameters, different lipid compositions, and internal solutions in order to avoid artifactual results or reproducibility problems. These issues motivated us to write a short review of the most recent methodological developments and possible pitfalls. Additionally, since traditional manual analysis can lead to biased results, we have included a discussion on methods for automatic analysis of GUVs. Finally, we discuss possible improvements in the preparation of GUVs containing high cholesterol contents in order to avoid the formation of artifactual cholesterol crystals. We intend this review to be a reference for those trying to decide what parameters to use as well as an overview providing insight into problems not yet addressed or solved.