The lipid composition of eye lens fiber cell membranes varies among species, and these differences increase as a function of lifespan. However, the way in which the lipids of the fiber cell membranes are organized in different animals has not yet been studied in detail. This study compares how the structure, properties, and organization of lipids change during the lifespan of the mouse, pig, and human. These species were chosen because of the wide range of lifespans and significant differences in lipid composition. Models were made of phospholipid compositions resembling those of the lens fiber cell membranes of a mouse, pig, and human aged of 3, 23, and 70 years, respectively. To separate the effects of phospholipids and cholesterol, membranes were investigated for samples without cholesterol, with a cholesterol content close to the cholesterol saturation limits, and with a cholesterol content close to the cholesterol solubility thresholds. Membrane physical properties were obtained using EPR spin labeling methods. No significant differences in the physical properties of the membranes of any of the models were detected for membranes without and those in the presence of a saturating cholesterol concentration. Thus, not only cholesterol but also the appropriate phospholipid composition is a significant factor in maintaining eye lens homeostasis.

Giant unilamellar vesicles (GUVs) are membrane models used to study membrane properties. Electroformation is one of the methods used to produce GUVs. During electroformation protocol, dry lipid film is formed. The drying of the lipid film induces the cholesterol (Chol) demixing artifact, in which Chol forms anhydrous crystals which do not participate in the formation of vesicles. This leads to a lower Chol concentration in the vesicle bilayers compared to the Chol concentration in the initial lipid solution. To address this problem, we propose a novel electroformation protocol that includes rapid solvent exchange (RSE), plasma cleaning, and spin-coating methods to produce GUVs. We tested the protocol, focusing on vesicles with a high Chol content using different spin-coating durations and vesicle type deposition. Additionally, we compared the novel protocol using completely dry lipid film. The optimal spin-coating duration for vesicles created from the phosphatidylcholine/Chol mixture was 30 s. Multilamellar vesicles (MLVs), large unilamellar vesicles (LUVs) obtained by the extrusion of MLVs through 100 nm membrane pores and LUVs obtained by extrusion of previously obtained LUVs through 50 nm membrane pores, were deposited on an electrode for 1.5/1 Chol/phosphatidylcholine (POPC) lipid mixture, and the results were compared. Electroformation using all three deposited vesicle types resulted in a high GUV yield, but the deposition of LUVs obtained by the extrusion of MLVs through 100 nm membrane pores provided the most reproducible results. Using the deposition of these LUVs, we produced high yield GUVs for six different Chol concentrations (from 0% to 71.4%). Using a protocol that included dry lipid film GUVs resulted in lower yields and induced the Chol demixing artifact, proving that the lipid film should never be subjected to drying when the Chol content is high.

Giant unilamellar vesicles (GUVs) are artificial membrane models which are of special interest to researchers because of their similarity in size to eukaryotic cells. The most commonly used method for GUVs production is electroformation. However, the traditional electroformation protocol involves a step in which the organic solvent is completely evaporated, leaving behind a dry lipid film. This leads to artifactual demixing of cholesterol (Chol) in the form of anhydrous crystals. These crystals do not participate in the formation of the lipid bilayer, resulting in a decrease of Chol concentration in the bilayer compared to the initial lipid solution. We propose a novel electroformation protocol which addresses this issue by combining the rapid solvent exchange, plasma cleaning and spin-coating techniques to produce GUVs from damp lipid films in a fast and reproducible manner. We have tested the protocol efficiency using 1/1 phosphatidylcholine/Chol and 1/1/1 phosphatidylcholine/sphingomyelin/Chol lipid mixtures and managed to produce a GUV population of an average diameter around 40 µm, with many GUVs being larger than 100 µm. Additionally, compared to protocols that include the dry film step, the sizes and quality of vesicles determined from fluorescence microscopy images were similar or better, confirming the benefits of our protocol in that regard as well.

The continuous wave EPR spin-labeling method was used to evaluate age-related changes in the amounts of phospholipids (PLs) and cholesterol (Chol) in domains present in intact, cortical, and nuclear fiber cell plasma membranes isolated separately from the left and right eye lenses of the same human donor. The relative amounts of boundary plus trapped PLs were evaluated with the PL analog 12-doxylstearic acid spin label (12-SASL) and the relative amounts of trapped Chol with the Chol analog androstane spin label (ASL). The donors ranged in age from 15 to 70 years. Both the left and right eye lenses from donors aged 60, 65, and 70 years had nuclear cataracts; additionally, the right eye lens only of the 60-year-old donor had a cortical cataract. In transparent lenses, the relative amounts of boundary plus trapped PLs increase monotonously with donor age, and, at all ages, this amount was greater in nuclear compared with cortical membranes. Moreover, in transparent lenses, the relative amount of trapped Chol increases with age in nuclear membranes. However, the EPR spectrum of ASL from cortical membranes of 15- to 60-year-old donors shows only the weakly immobilized component assigned to ASL in the bulk plus Chol bilayer domain. Only the cortical membranes of 61- to 70-year-old donors contain both weakly and strongly immobilized components. The strongly immobilized component is assigned to ASL in trapped lipids. We speculate that the age of 60 years may be considered as a “threshold” for appearance of trapped lipids in cortical membranes. The relative amounts of boundary plus trapped PLs in lenses with nuclear cataracts is lower than that predicted from the tendency of the age-dependent increase observed for transparent lenses. The differences in amounts of lipids in the indicated left and right eye domains of each donor are smaller than the differences in single donors of a similar age.

Cholesterol (Chol) is an essential component of animal cell membranes and is most abundant in plasma membranes (PMs) where its concentration typically ranges from 10 to 30 mol%. However, in red blood cells and Schwann cells, PMs Chol content is as high as 50 mol%, and in the PMs of the eye lens fiber cells, it can reach up to 66 mol%. Being amphiphilic, Chol molecules are easily incorporated into the lipid bilayer where they affect the membrane lateral organization and transmembrane physical properties. In the aqueous phase, Chol cannot form free bilayers by itself. However, pure Chol bilayer domains (CBDs) can form in lipid bilayer membranes with the Chol content exceeding 50 mol%. The range of Chol concentrations surpassing 50 mol% is less frequent in biological membranes and is consequently less investigated. Nevertheless, it is significant for the normal functioning of the eye lens and understanding how Chol plaques form in atherosclerosis. The most commonly used membrane models are unilamellar and multilamellar vesicles (MLVs) and supported lipid bilayers (SLBs). CBDs have been observed directly using confocal microscopy, X-ray reflectometry and saturation recovery electron paramagnetic resonance (SR EPR). Indirect evidence of CBDs has also been reported by using atomic force microscopy (AFM) and fluorescence recovery after photobleaching (FRAP) experiments. The overall goal of this review is to demonstrate the advantages and limitations of the various membrane models and experimental techniques suitable for the detection and investigation of the lateral organization, function and physical properties of CBDs.

EPR spin labeling has been used extensively to study lipids in model membranes to understand their structures and dynamics in biological membranes. The lipid multilamellar liposomes, which are the most commonly used biological membrane model, were prepared using film deposition methods and investigated with the continuous wave EPR technique (T2-sensitive spin-labeling methods). These investigations provided knowledge about the orientation of lipids, their rotational and lateral diffusion, and their rate of flip-flop between bilayer leaflets, as well as profiles of membrane hydrophobicity, and are reviewed in many papers and book chapters. In the early 1980s, the saturation recovery EPR technique was introduced to membrane studies. Numerous T1-sensitive spin-label methods were developed to obtain detailed information about the three-dimensional dynamic membrane structure. T1-sensitive methods are advantageous over T2-sensitive methods because the T1 of spin labels (1–10 μs) is 10 to 1000 times longer than the T2, which allows for studies of membrane dynamics in a longer time–space scale. These investigations used multilamellar liposomes also prepared using the rapid solvent exchange method. Here, we review works in which saturation recovery EPR spin-labeling methods were applied to investigate the properties of multilamellar lipid liposomes, and we discuss their relationships to the properties of lipids in biological membranes.

Artificial vesicles are important tools in membrane research because they enable studying membrane properties in controlled conditions. Giant unilamellar vesicles (GUVs) are specially interesting due to their similarity in size to eukaryotic cells. We focus on optimization of GUV production from phosphatidylcholine/sphingomyelin/cholesterol mixtures using the electroformation method. This mixture has been extensively researched lately due to its relevance for the formation of lipid rafts. We measured the effect of voltage, frequency, lipid film thickness, and cholesterol (Chol) concentration on electroformation successfulness using spin-coating for reproducible lipid film deposition. Special attention is given to the effect of Chol concentrations above the phospholipid bilayer saturation threshold. Such high concentrations are of interest to groups studying the role of Chol in the fiber cell plasma membranes of the eye lens or development of atherosclerosis. Utilizing atomic force and fluorescence microscopy, we found the optimal lipid film thickness to be around 30 nm, and the best frequency–voltage combinations in the range of 2–6 V and 10–100 Hz. Increasing the Chol content, we observed a decrease in GUV yield and size. However, the effect was much less pronounced when the optimal lipid film thickness was used. The results underline the need for simultaneous optimization of both electrical parameters and thickness in order to produce high-quality GUVs for experimental research.

Molecular oxygen (O2) is the perfect probe molecule for membrane studies carried out using the saturation recovery EPR technique. O2 is a small, paramagnetic, hydrophobic enough molecule that easily partitions into a membrane's different phases and domains. In membrane studies, the saturation recovery EPR method requires two paramagnetic probes: a lipid-analog nitroxide spin label and an oxygen molecule. The experimentally derived parameters of this method are the spin-lattice relaxation times (T 1s) of spin labels and rates of bimolecular collisions between O2 and the nitroxide fragment. Thanks to the long T 1 of lipid spin labels (from 1 to 10 μs), the approach is very sensitive to changes of the local (around the nitroxide fragment) O2 diffusion-concentration product. Small variations in the lipid packing affect O2 solubility and O2 diffusion, which can be detected by the shortening of T 1 of spin labels. Using O2 as a probe molecule and a different lipid spin label inserted into specific phases of the membrane and membrane domains allows data about the lateral arrangement of lipid membranes to be obtained. Moreover, using a lipid spin label with the nitroxide fragment attached to its head group or a hydrocarbon chain at different positions also enables data about molecular dynamics and structure at different membrane depths to be obtained. Thus, the method can be used to investigate not only the lateral organization of the membrane (i.e., the presence of membrane domains and phases), but also the depth-dependent membrane structure and dynamics, and, hence, the membrane properties in three dimensions.

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.

Giant unilamellar vesicles (GUVs) are used extensively as models that mimic cell membranes. The cholesterol (Chol) content in the fiber cell plasma membranes of the eye lens is extremely high, exceeding the solubility threshold in the lenses of old humans. Thus, a methodological paper pertaining to preparations of model lipid bilayer membranes with high Chol content would significantly help the study of properties of these membranes. Lipid solutions containing 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC) and Chol were fluorescently labeled with phospholipid analog 1,1 '-dioctadecyl-3,3,3 ',3 '-tetramethylindocarbocyanine perchlorate (DiIC(18)(3)) and spin-coated to produce thin lipid films. GUVs were formed from these films using the electroformation method and the results were obtained using fluorescent microscopy. Electroformation outcomes were examined for different electrical parameters and different Chol concentrations. A wide range of field frequency-field strength (ff-fs) combinations was explored: 10-10,000 Hz and 0.625-9.375 V/mm peak-to-peak. Optimal values for GUVs preparation were found to be 10-100 Hz and 1.25-6.25 V/mm, with largest vesicles occurring for 10 Hz and 3.75 V/mm. Chol:POPC mixing ratios (expressed as a molar ratio) ranged from 0 to 3.5. We show that increasing the Chol concentration decreases the GUVs size, but this effect can be reduced by choosing the appropriate ff-fs combination.

We are pleased and honored to present this special issue for CBBI on recent advances in selected biophysical methods to study biological systems, oxidative phenomena involved in the pathology of human diseases, free radical metabolism and bioenergetic changes in cardiovascular diseases and cancer, and advances in drug, active surfaces and biosensor development.The papers herein resulted from the most recent 2017 Second Adriatic Symposium on Biophysical Approaches in Biomedical Studies held in Split, Croatia, that encompasses work from outstanding researchers in the field.This is the second in a series of Symposia to be held in Split to share the latest technological developments in various biomedical applications from Croatian and international experts, stimulate innovative research, train young scientists, postdoctoral fellows, graduate students, and enable future scientific collaborations among meeting participants.Invited speakers have been drawn from the leaders in interdisciplinary fields.Scientists from academia, industry and government laboratories from all over the world participated, coming from fields of biophysics, biology, chemistry, biomedicine, and others.Our participants perform are researchers using analytical and biophysical techniques including EPR, NMR, fluorescence and other spectroscopies, microscopy, and nanoscale technologies.

The cholesterol (Chol) content in the fiber cell plasma membranes of the eye lens is extremely high, exceeding the solubility threshold in the lenses of old humans. This high Chol content forms pure Chol bilayer domains (CBDs) and Chol crystals in model membranes and membranes formed from the total lipid extracts from human lenses. CBDs have been detected using electron paramagnetic resonance (EPR) spin-labeling approaches. Here, we confirm the presence of CBDs in giant unilamellar vesicles prepared using the electroformation method from Chol/1-palmitoyl-2-oleoylphosphocholine and Chol/distearoylphosphatidylcholine mixtures. Confocal microscopy experiments using phospholipid (PL) analog (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine-5,5'-disulfonic acid) and cholesterol analog fluorescent probes (23-(dipyrrometheneboron difluoride)-24-norcholesterol) were performed, allowing us to make three major conclusions: (1) In all membranes with a Chol/PL mixing ratio (expressed as a molar ratio) >2, pure CBDs were formed within the bulk PL bilayer saturated with Chol. (2) CBDs were present as the pure Chol bilayer and not as separate patches of Chol monolayers in each leaflet of the PL bilayer. (3) CBDs, presented as single large domains, were always located at the top of giant unilamellar vesicles, independent of the change in sample orientation (right-side-up/upside-down). Results obtained with confocal microscopy and fluorescent Chol and PL analogs, combined with those obtained using EPR and spin-labeled Chol and PL analogs, contribute to the understanding of the organization of lipids in the fiber cell plasma membranes of the human eye lens.

Lipid composition determines membrane properties, and cholesterol plays a major role in this determination as it regulates membrane fluidity and permeability, as well as induces the formation of coexisting phases and domains in the membrane. Biological membranes display a very diverse lipid composition, the lateral organization of which plays a crucial role in regulating a variety of membrane functions. We hypothesize that, during biological evolution, membranes with a particular cholesterol content were selected to perform certain functions in the cells of eukaryotic organisms. In this review, we discuss the major membrane properties induced by cholesterol, and their relationship to certain membrane functions.

PURPOSE:This research was undertaken to document the changes in the organization and properties of human lens lipid membranes that occur with age.METHODS:Human lens lipid membranes prepared from the total lipids extracted from clear lens cortices and nuclei of donors from age groups 0-20 and 21-40 years were investigated. An electron paramagnetic resonance technique and nitroxide spin labels (analogues of phospholipids and cholesterol) were used.RESULTS:Two distinct lipid domains, the phospholipid-cholesterol domain (PCD) and the pure cholesterol bilayer domain (CBD), were detected in all investigated membranes. Profiles of the acyl chain order, fluidity, hydrophobicity, and oxygen transport parameter across discriminated coexisting lipid domains were assessed. Independent of the age-related changes in phospholipid composition, the physical properties of the PCD remained the same for all age groups and were practically identical for cortical and nuclear membranes. However, the properties of pure CBDs changed significantly with the age of the donor and were related to the size of the CBD, which increased with the age of the donor and was greater in nuclear than in cortical membranes. A more detailed analysis revealed that the size of the CBD was determined mainly by the cholesterol content in the membrane.CONCLUSIONS:This paper presents data from four age groups: 0-20, 21-40, 41-60, and 61-70 years. Data from age groups 41-60 and 61-70 years were published previously. Combining the previously published data with those data obtained in the present work allowed us to show the changes in the organization of cortical and nuclear lens lipid membranes as functions of age and cholesterol. It seems that the balance between age-related changes in membrane phospholipid composition and cholesterol content plays an integral role in the regulation of cholesterol-dependent processes in fiber cell membranes and in the maintenance of fiber cell membrane homeostasis.

The most unique biochemical characteristic of the eye lens fiber cell plasma membrane is its extremely high cholesterol content, the need for which is still unclear. It is evident, however, that the disturbance of Chol homeostasis may result in damages associated with cataracts. Electron paramagnetic resonance methods allow discrimination of two types of lipid domains in model membranes overloaded with Chol, namely, phospholipid-cholesterol domains and pure Chol bilayer domains. These domains are also detected in human lens lipid membranes prepared from the total lipids extracted from lens cortices and nuclei of donors from different age groups. Independent of the age-related changes in phospholipid composition, the physical properties of phospholipid-Chol domains remain the same for all age groups and are practically identical for cortical and nuclear membranes. The presence of Chol bilayer domains in these membranes provides a buffering capacity for cholesterol concentration in the surrounding phospholipid-Chol domains, keeping it at a constant saturating level and thus keeping the physical properties of the membrane consistent with and independent of changes in phospholipid composition. It seems that the presence of Chol bilayer domains plays an integral role in the regulation of cholesterol-dependent processes in fiber cell plasm membranes and in the maintenance of fiber cell membrane homeostasis.

Reported results have documented changes that occur with age in the properties and the organization of lens lipid membranes. Results were obtained for membranes derived from the total lipids extracted from the cortex and nucleus of the clear lenses of donors from age groups 0 to 20, 21 to 40, 41 to 60, and 61 to 70 years. The physical properties, including profiles of the alkyl chain order, fluidity, hydrophobicity, and oxygen transport parameter, were investigated using EPR spin-labeling methods, which also provided an opportunity to discriminate coexisting lipid domains and to evaluate the domain's size. All investigated membranes were found to contain two distinct lipid environments: phospholipid-cholesterol domains (PCDs) and pure cholesterol bilayer domains (CBDs). The size of CBDs increased with the donor age and was always greater in nuclear than in cortical membranes. More detailed analysis revealed that the size of CBDs was determined mainly by the cholesterol content in the investigated membranes. Independently of the age-related changes in the phospholipid composition, the physical properties of the PCD remained the same for all age groups and were practically identical for cortical and nuclear membranes. These data additionally confirm that profiles of membrane physical properties do not depend on the phospholipid composition when membranes are saturated with cholesterol.