Rapid Solvent Exchange Method Research Articles

Interaction of βL- and γ-Crystallin with Phospholipid Membrane Using Atomic Force Microscopy.

Highly concentrated lens proteins, mostly β- and γ-crystallin, are responsible for maintaining the structure and refractivity of the eye lens. However, with aging and cataract formation, β- and γ-crystallin are associated with the lens membrane or other lens proteins forming high-molecular-weight proteins, which further associate with the lens membrane, leading to light scattering and cataract development. The mechanism by which β- and γ-crystallin are associated with the lens membrane is unknown. This work aims to study the interaction of β- and γ-crystallin with the phospholipid membrane with and without cholesterol (Chol) with the overall goal of understanding the role of phospholipid and Chol in β- and γ-crystallin association with the membrane. Small unilamellar vesicles made of Chol/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (Chol/POPC) membranes with varying Chol content were prepared using the rapid solvent exchange method followed by probe tip sonication and then dispensed on freshly cleaved mica disk to prepare a supported lipid membrane. The βL- and γ-crystallin from the cortex of the bovine lens was used to investigate the time-dependent association of βL- and γ-crystallin with the membrane by obtaining the topographical images using atomic force microscopy. Our study showed that βL-crystallin formed semi-transmembrane defects, whereas γ-crystallin formed transmembrane defects on the phospholipid membrane. The size of semi-transmembrane defects increases significantly with incubation time when βL-crystallin interacts with the membrane. In contrast, no significant increase in transmembrane defect size was observed in the case of γ-crystallin. Our result shows that Chol inhibits the formation of membrane defects when βL- and γ-crystallin interact with the Chol/POPC membrane, where the degree of inhibition depends upon the amount of Chol content in the membrane. At a Chol/POPC mixing ratio of 0.3, membrane defects were observed when both βL- and γ-crystallin interacted with the membrane. However, at a Chol/POPC mixing ratio of 1, no association of γ-crystallin with the membrane was observed, which resulted in a defect-free membrane, and the severity of the membrane defect was decreased when βL-crystallin interacted with the membrane. The semi-transmembrane or transmembrane defects formed by the interaction of βL- and γ-crystallin on phospholipid membrane might be responsible for light scattering and cataract formation. However, Chol suppressed the formation of such defects in the membrane, likely maintaining lens membrane homeostasis and protecting against cataract formation.

Interactions of beta-crystallin with a model of human eye lens-lipid membranes.

β-Crystallin helps maintain the eye lens's structure and refractivity. With age and cataract formation, α-, β-, and γ-crystallin may undergo various mutations and post-translational modifications, and consequently form higher molecular weight complexes, some of which bind to lens membranes. However, the binding of β-crystallin alone to the lens membrane is still unclear. This study aims to investigate the binding of β-crystallin to the model of the human lens-lipid (MHLL) membrane and the physical properties of the MHLL membrane after β-crystallin binding. Small unilamellar vesicles of MHLL membranes, resembling the human lens-lipid composition, were prepared using the rapid solvent exchange method and probe-tip sonication. The 1 mol% cholesterol analogue spin label (CSL) integrated into the membrane, which resides near the lens-lipid headgroup region, probes β-crystallin binding to the membrane using the electron paramagnetic resonance (EPR) spin-labeling method. Our results showed that β-crystallin binds to the MHLL membrane with a binding affinity (Ka) of 0.082 ± 0.006 μM−1 and saturates around 5% of the membrane surface occupied. Furthermore, β-crystallin binding to the MHLL membrane decreased the mobility parameter, making the headgroups of the membrane less mobile. However, β-crystallin binding to the MHLL membrane showed no significant change in maximum splitting, meaning it does not affect the order near the headgroup regions of the MHLL membrane. Moreover, the hydrophobicity of the MHLL membrane also did not change significantly with the binding of β-crystallin, meaning hydrophobicity near the headgroup regions does not significantly change with β-crystallin binding. Our results showed that β-crystallin binding with MHLL membranes changes the physical property of membranes, implying that β-crystallin binding to the lens membrane may change the integrity of the membrane and has the potential to promote cataract formation.

Multilamellar Liposomes as a Model for Biological Membranes: Saturation Recovery EPR Spin-Labeling Studies.

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.

Membrane elasticity modulated by cholesterol in model of porcine eye lens-lipid membrane

Experimental evidence shows that the eye lens loses its elasticity dramatically with age. It has also been reported that the cholesterol (Chol) content in the eye lens fiber cell plasma membrane increases significantly with age. High Chol content leads to the formation of cholesterol bilayer domains (CBDs) in the lens membrane. The role of high Chol associated with lens elasticity is unclear. The purpose of this research is to investigate the membrane elasticity of the model of porcine lens-lipid (MPLL) membrane with increasing Chol content to elucidate the role of high Chol in lens membrane elasticity. In this study, we used atomic force microscopy (AFM) to study the mechanical properties (breakthrough force and area compressibility modulus (KA)) of the MPLL membrane with increasing Chol content where KA is the measure of membrane elasticity. We varied Chol concentration in Chol/MPLL membrane from 0 to ∼71 mol%. Supported Chol/MPLL membranes were prepared by fusion of small unilamellar vesicles (SUVs) on top of a flat mica surface. SUVs of the Chol/MPLL lipid mixture were prepared with the rapid solvent exchange method followed by probe-tip sonication. For the Chol/MPLL mixing ratio of 0, AFM image showed the formation of two distinct phases of the membrane, i.e., liquid-disordered phase (ld) and solid-ordered phase (so) membrane. However, with Chol/MPLL mixing ratio of 0.5 and above, only liquid-ordered phase (lo) membrane was formed. Also, two distinct breakthrough forces corresponding to ld and so were observed for Chol/MPLL mixing ratio of 0, whereas only one breakthrough force was observed for membranes with Chol/MPLL mixing ratio of 0.5 and above. No significant difference in the membrane surface roughness was measured with increasing Chol content for these membranes; however, breakthrough force and KA for lo membrane increased when Chol/MPLL mixing ratio was increased from 0.5 to 1. Interestingly above the Chol/MPLL mixing ratio of 1, both breakthrough force and KA decreased, indicating the formation of CBDs. Furthermore, these results showed that membrane elasticity increases at high Chol content, suggesting that high Chol content in lens membrane might be responsible for maintaining lens membrane elasticity.

Alpha-Crystallin Association with the Model of Human and Animal Eye Lens-Lipid Membranes is Modulated by Surface Hydrophobicity of Membranes

Purpose This research aims to probe the interaction of α-crystallin with a model of human, porcine, and mouse lens-lipid membranes. Methods Cholesterol/model of human lens-lipid (Chol/MHLL), cholesterol/model of porcine lens-lipid (Chol/MPLL), and cholesterol/model of mouse lens-lipid (Chol/MMLL) membranes with 0–60 mol% Chol were prepared using the rapid solvent exchange method and probe-tip sonication. The hydrophobicity near the surface of model lens-lipid membranes and α-crystallin association with these membranes were investigated using the electron paramagnetic resonance spin-labeling approach. Results With increased Chol content, the hydrophobicity near the surface of Chol/MHLL, Chol/MPLL, and Chol/MMLL membranes, the maximum percentage of membrane surface occupied (MMSO) by α-crystallin, and the association constant (K a) decreased, showing that surface hydrophobicity of model lens-lipid membranes modulated the α-crystallin association with these membranes. The different MMSO and K a for different model lens-lipid membranes with different rates of decrease of MMSO and K a with increased Chol content and decreased hydrophobicity near the surface of these membranes suggested that the lipid composition also modulates α-crystallin association with membranes. Despite different lipid compositions, complete inhibition of α-crystallin association with model lens-lipid membranes was observed at saturating Chol content forming cholesterol bilayer domains (CBDs) with the lowest hydrophobicity near the surface of these membranes. The decreased mobility parameter with increased α-crystallin concentration suggested that membranes near the surface became less mobile due to α-crystallin association. The decreased mobility parameter and increased maximum splitting with increased Chol content suggested that membranes became less mobile and more ordered near the surface with increased Chol content. Conclusions This study suggested that the interaction of α-crystallin with model lens-lipid membranes is hydrophobic. Furthermore, our data indicated that Chol and CBDs reduce α-crystallin association with lens membrane, likely increase α-crystallin concentration in lens cytoplasm, and possibly favor the chaperone-like activity of α-crystallin maintaining lens cytoplasm homeostasis.

Cholesterol and cholesterol bilayer domains inhibit binding of alpha-crystallin to the membranes made of the major phospholipids of eye lens fiber cell plasma membranes

The concentration of α-crystallin decreases in the eye lens cytoplasm, with a corresponding increase in membrane-bound α-crystallin during cataract formation. The eye lens's fiber cell plasma membrane consists of extremely high cholesterol (Chol) content, forming cholesterol bilayer domains (CBDs) within the membrane. The role of high Chol content in the lens membrane is unclear. Here, we applied the continuous-wave electron paramagnetic resonance spin-labeling method to probe the role of Chol and CBDs on α-crystallin binding to membranes made of four major phospholipids (PLs) of the eye lens, i.e., phosphatidylcholine (PC), sphingomyelin (SM), phosphatidylserine (PS), and phosphatidylethanolamine (PE). Small unilamellar vesicles (SUVs) of PC, SM*, and PS with 0, 23, 33, 50, and 60 mol% Chol and PE* with 0, 9, and 33 mol% Chol were prepared using the rapid solvent exchange method followed by probe-tip sonication. The 1 mol% CSL spin-labels used during SUVs preparation distribute uniformly within the Chol/PL membrane, enabling the investigation of Chol and CBDs' role on α-crystallin binding to the membrane. For PC, SM*, and PS membranes, the binding affinity (Ka) and the maximum percentage of membrane surface occupied (MMSO) by α-crystallin decreased with an increase in Chol concentration. The Ka and MMSO became zero at 50 mol% Chol for PC and 60 mol% Chol for SM* membranes, representing that complete inhibition of α-crystallin binding was possible before the formation of CBDs within the PC membrane but only after the formation of CBDs within the SM* membrane. The Ka and MMSO did not reach zero even at 60 mol% Chol in the PS membrane, representing CBDs at this Chol concentration were not sufficient for complete inhibition of α-crystallin binding to the PS membrane. Both the Ka and MMSO were zero at 0, 9, and 33 mol% Chol in the PE* membrane, representing no binding of α-crystallin to the PE* membrane with and without Chol. The mobility parameter profiles decreased with an increase in α-crystallin binding to the membranes; however, the decrease was more pronounced for the membrane with lower Chol concentration. These results imply that the membranes become more immobilized near the headgroup regions with an increase in α-crystallin binding; however, the Chol antagonizes the capacity of α-crystallin to decrease the mobility near the headgroup regions of the membranes. The maximum splitting profiles remained the same with an increase in α-crystallin concentration, but there was an increase in the maximum splitting with an increase in the Chol concentration in the membranes. It implies that membrane order near the headgroup regions does not change with an increase in α-crystallin concentration but increases with an increase in Chol concentration in the membrane. Based on our data, we hypothesize that the Chol and CBDs decrease hydrophobicity (increase polarity) near the membrane surface, inhibiting the hydrophobic binding of α-crystallin to the membranes. Thus, our data suggest that Chol and CBDs play a positive physiological role by preventing α-crystallin binding to lens membranes and possibly protecting against cataract formation and progression.

Formation of cholesterol Bilayer Domains Precedes Formation of Cholesterol Crystals in Membranes Made of the Major Phospholipids of Human Eye Lens Fiber Cell Plasma Membranes

ABSTRACTPurpose/Aim: The goal of this study is to reveal how age-related changes in phospholipid (PL) composition in the fiber cell plasma membranes of the human eye lens affect the cholesterol (Chol) content at which Chol bilayer domains (CBDs) and Chol crystals start to form.Materials and Methods: Saturation-recovery electron paramagnetic resonance with spin-labeled cholesterol analogs and differential scanning calorimetry were used to determine the Chol contents at which CBDs and cholesterol crystals, respectively, start to form in in membranes made of the major PL constituents of the plasma membrane of the human eye lens fiber cells. To preserve compositional homogeneity throughout the membrane suspension, the lipid multilamellar dispersions investigated in this work were prepared using a rapid solvent exchange method. The cholesterol content changed from 0 to 75 mol%.Results: The saturation recovery electron paramagnetic resonance results show that CBDs start to form at 33, 50, 46, and 48 mol% Chol in the phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine, and sphingomyelin bilayers, respectively. The differential scanning calorimetry results show that Chol crystals start to form at 50, 66, 70, and 66 mol% Chol in the phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine, and sphingomyelin bilayers, respectively.Conclusions: These results, as well those of our previous studies, indicate that the formation of CBDs precedes the formation of Chol crystals in all of the studied systems, and the appearance of each depends on the type of PL forming the bilayer. These findings contribute to a better understanding of the molecular mechanisms involved in the regulation of Chol-dependent processes in eye lens fiber cell membranes.

Oxidation of Polyunsaturated Phospholipid Decreases the Cholesterol Content at which Cholesterol Bilayer Domains Start to form in Phospholipid-Cholesterol Membranes

Saturation-recovery electron paramagnetic resonance and differential scanning calorimetry were used to determine the cholesterol content at which pure cholesterol bilayer domains (CBDs) and cholesterol crystals begin to form in 1-palmitoyl-2-arachidonoylphosphochatidyline (PAPC) membranes. To preserve compositional homogeneity of the membrane suspension at a high cholesterol content, multilamellar liposome suspensions were prepared using the rapid solvent exchange method. The cholesterol content in membranes was changed from 0 mol% to 66 mol%.

Bottom up design of nanoparticles for anti-cancer diapeutics: “put the drug in the cancer’s food”

The story starts in Basel at CLINAM in 2013, when I asked Pieter about making nanoparticles and he advised me to “try this solvent-exchange method we have developed for making limit sized particles”. We are particularly interested in what are “limit size materials” because we want to test the feasibility of an idea: could we design, make, develop, and test the concept for treating metastatic cancer by, “Putting the Drug in the Cancer’s Food? “Limit size” is the size of the cancer‘s food, ? the common Low Density Lipoprotein, (LDL) ~20 nm diameter. In this contribution to Pieter’s LTAA we focus on the “bottom” (nucleation) and the “up” (growth) of “bottom-up design” as it applies to homogeneous nucleation of especially, hydrophobic drugs and the 8 physico-chemical stages and associated parameters that determine the initial size, and any subsequent coarsening, of a nanoparticle suspension. We show that, when made by the rapid solvent-exchange method, the same sized particles can be obtained without phospholipid. Furthermore, the obtained size follows the predictions of classic nucleation theory when the appropriate values for the parameters (surface tension and supersaturation) at nucleation are included. Calculations on dissolution time for nanoparticles reveal that a typical fewmicromolar-solubility, hydrophobic, anti-cancer drug (like Lapatinib, Niclosamide, Abiraterone, and Fulvestrant) of 500 nm diameter would take between 3?7 s to dissolve in an infinite sink like the blood stream; and a 50 nm particle would dissolve in less than a second! And so the nanoparticle design requires a highly water-insoluble drug, and a tight, encapsulating, impermeable lipid:cholesterol monolayer. While the “Y” junction can be used to mix an ethanolic solution with anti-solvent, we find that a “no-junction” can give equally good results. A series of nanoparticles (DiI-fluorescently labeled Triolein-cored and drug-cored nanoparticles of Orlistat) were then tested in well-characterized cell lines for uptake and efficacy as well as a PET-imageable nanoparticle in initial PET-imaging studies in animals for EPR uptake and tumor detection. We show that, while free-drug cannot be optimally administered in vivo, a nanoparticle formulation of orlistat could in principle represent a stable parenteral delivery system. The article ends with a brief discussion of what we see as the way forward in Individualized Medicine from the Diagnostic-Therapeutic (“Diapeutic”) side, requiring 18FDG detection of metastatic lesions, functional imaging of a protein target (e.g. Fatty Acid Synthase) using 11C acetate, then a PET (or other)-imageable nanoparticle to demonstrate EPR accumulation, and then the administration of the pure-drug nanoparticle taken in by the most aggressive cancer cells in the perivascular space, as they would their “food”.

Oxidation of Cholesterol and Formation of Cholesterol Hydroperoxides Decreases the Cholesterol Concentration at which Formation of Cholesterol Bilayer Domains and Cholesterol Crystals is Initiated in Phospholipid Bilayers

Recently we demonstrated that formation of cholesterol bilayer domains (CBDs) precedes formation of cholesterol crystals (CHCs) in cholesterol/dimyristoylphosphatidylcholine (Chol/DMPC) membranes (Mainali et. al., J. Phys. Chem. B, 117(2013)8994-9003). In the presented studies, we show that the oxidation of Chol in Chol/DMPC membranes induces formation of CBDs and CHCs at significantly lower Chol concentrations than in membranes without oxidation. To preserve compositional homogeneity throughout the membrane suspension we prepared the multilamellar liposomes with a rapid solvent exchange method. The oxidation of Chol was induced using a photosensitizer, rose Bengal, and the extent of oxidation (formation of Chol hydroperoxides) was measured using iodometric assay. To avoid formation of phospholipid hydroperoxides, which would interfere with our assay, the saturated phospholipid (DMPC) was used to prepare liposomes. Saturation recovery electron paramagnetic resonance with spin-labeled cholesterol analogues (androstane and cholestane spin labels) was used to detect the formation of CBDs and differential scanning calorimetry was used to detect the formation of CHCs. We conclude that formation of Chol hydroperoxides induces the formation of CBDs at Chol concentrations where previously CBDs were not present in non-oxidized membranes as well as the collapse of the already formed CBDs to CHCs, presumably outside the lipid bilayer.

Properties of membranes derived from the total lipids extracted from clear and cataractous lenses of 61-70-year-old human donors.

Human lens-lipid membranes prepared from the total lipids extracted from clear and cataractous lens cortexes and nuclei of 61-70-year-old donors by use of a rapid solvent-exchange method were investigated. The measured cholesterol-to-phospholipid (Chol/PL) molar ratio in these membranes was 1.8 and 4.4 for cortex and nucleus of clear lenses, respectively, and 1.14 and 1.45 for cataractous lenses. Properties and organization of the lipid bilayer were investigated by use of electron paramagnetic resonance spin-labeling methods. Formation of Chol crystals was confirmed by use of differential scanning calorimetry. Pure cholesterol bilayer domains (CBDs) were formed in all the membranes investigated. It was shown that in clear lens membranes of the nucleus, Chol exists in three different environments: (1) dispersed in phospholipid bilayers (PCDs), (2) in CBDs, and (3) in Chol crystals. In clear lens membranes of the cortex, and in cortical and nuclear cataractous lens membranes, Chol crystals were not detected, because of the lower Chol content. Profiles of membrane properties (alkyl-chain order, fluidity, oxygen transport, and hydrophobicity) across the PCD were very similar for clear and cataractous membranes. Profiles ofthe oxygen transportparameter across the CBD were, however, different for cortical clear and cataractous membranes-the amount and size of CBDs was less in cataractous membranes. These results suggest that high Chol content, formation of CBDs, and formation of Chol crystals should not be regarded as major predispositions for the development of age-related cataracts.

Lysozyme binding to tethered bilayer lipid membranes prepared by rapid solvent exchange and vesicle fusion methods

Tethered bilayer lipid membranes (tBLMs) are important tools for studying protein-lipid interactions. The widely used methodology for the preparation of these membranes is the fusion of phospholipid vesicles from an aqueous medium onto an anchored phospholipid layer. The preparation of phospholipid vesicles is a long and tedious procedure. There is another simple method, rapid solvent exchange, for preparing lipid membranes. However, there is a lack of information on the effects of the preparation method of tBLMs on their interactions with proteins. Therefore, we present in this paper a comparative study on the binding of lysozyme onto tBLMs prepared by the abovementioned methods. The prepared tBLMs have either zwitterionic or anionic characteristics. The results show that lysozyme binding onto the prepared tBLMs is unaffected by the preparation method of the tBLMs, suggesting that the tedious fusion method might be replaced by the simple rapid solvent exchange method without altering the level of protein-lipid interactions.

Phospholipid-Cholesterol Bilayers, Cholesterol Bilayer Domains, and Cholesterol Crystals were Detected in Lipid Dispersion Prepared from Lipids Extracted from Lens Nucleus of Old Human Donors

Human lens lipid membranes prepared using a rapid solvent exchange method from the total lipids extracted from the clear lens cortex and nucleus of 61- to 70-year-old donors were investigated. The measured cholesterol-to-phospholipid (Chol/PL) molar ratio in these preparations was extremely high, showing values of 1.8 and 4.4 for cortex and nucleus, respectively. We expect that at this elevated Chol content, the entire membrane will become saturated with Chol (as in the case of the cortical membrane) and the excess of Chol will form Chol crystals, presumably outside the membrane (as in the case of the nuclear membrane). Properties and organization of the lipid bilayer were investigated using electron paramagnetic resonance spin-labeling methods. Formation of Chol crystals was confirmed using the differential scanning calorimetry. We showed that in the lipid dispersion prepared from nuclear lipids Chol exists in three distinguished environments: (1) Chol dispersed in PL bilayer, (2) Chol in non-crystalline membrane domains (cholesterol bilayer domains, CBDs), and (3) Chol in crystals. In cortical membranes, because of the lower Chol content, Chol crystals were not detected. Amounts of Chol in CBDs were almost the same in cortical and nuclear membranes which indicates that Chol content in both membranes is close or exceeds the Chol solubility thresholds in these membranes. Profiles of cortical and nuclear membrane properties (alkyl-chain order, fluidity, oxygen transport parameter, and hydrophobicity) were very similar to each other and to those reported for cortical and nuclear lens lipid membranes of 41- to 60-year-old donors reported earlier. This confirms our earlier statement that saturation with Chol determines properties of the PL bilayer with the minor effect of the PL composition.

Formation of Cholesterol Bilayer Domains Precedes Formation of Cholesterol Crystals in Cholesterol/Dimyristoylphosphatidylcholine Membranes: EPR and DSC Studies

Saturation-recovery EPR along with DSC were used to determine the cholesterol content at which pure cholesterol bilayer domains (CBDs) and cholesterol crystals begin to form in dimyristoylphosphatidylcholine (DMPC) membranes. To preserve compositional homogeneity throughout the membrane suspension, lipid multilamellar dispersions were prepared using a rapid solvent exchange method. The cholesterol content increased from 0 to 75 mol %. With spin-labeled cholesterol analogues, it was shown that the CBDs begin to form at ~50 mol % cholesterol. It was confirmed by DSC that the cholesterol solubility threshold for DMPC membranes is detected at ~66 mol % cholesterol. At levels above this cholesterol content, monohydrate cholesterol crystals start to form. The major finding is that the formation of CBDs precedes formation of cholesterol crystals. The region of the phase diagram for cholesterol contents between 50 and 66 mol % is described as a structured one-phase region in which CBDs have to be supported by the surrounding DMPC bilayer saturated with cholesterol. Thus, the phase boundary located at 66 mol % cholesterol separates the structured one-phase region (liquid-ordered phase of DMPC with CBDs) from the two-phase region where the structured liquid-ordered phase of DMPC coexists with cholesterol crystals. It is likely that CBDs are precursors of monohydrate cholesterol crystals.

Properties of membranes derived from the total lipids extracted from the human lens cortex and nucleus

Human lens lipid membranes prepared using a rapid solvent exchange method from the total lipids extracted from the clear lens cortex and nucleus of 41- to 60-year-old donors were investigated using electron paramagnetic resonance spin-labeling. Profiles of the phospholipid alkyl-chain order, fluidity, oxygen transport parameter, and hydrophobicity were assessed across coexisting membrane domains. Membranes prepared from the lens cortex and nucleus were found to contain two distinct lipid environments, the bulk phospholipid–cholesterol domain and the cholesterol bilayer domain (CBD). The alkyl chains of phospholipids were strongly ordered at all depths, indicating that the amplitude of the wobbling motion of alkyl chains was small. However, profiles of the membrane fluidity, which explicitly contain time (expressed as the spin-lattice relaxation rate) and depend on the rotational motion of spin labels, show relatively high fluidity of alkyl chains close to the membrane center. Profiles of the oxygen transport parameter and hydrophobicity have a rectangular shape and also indicate a high fluidity and hydrophobicity of the membrane center. The amount of CBD was greater in nuclear membranes than in cortical membranes. The presence of the CBD in lens lipid membranes, which at 37°C showed a permeability coefficient for oxygen about 60% smaller than across a water layer of the same thickness, would be expected to raise the barrier for oxygen transport across the fiber cell membrane. Properties of human membranes are compared with those obtained for membranes made of lipids extracted from cortex and nucleus of porcine and bovine eye lenses.

A New Phase Boundary in Phosphatidylcholine/Cholesterol Bilayers (In the Dimyristoylphosphatidylcholine/Cholesterol Bilayer): EPR and DSC Studies

A new phase boundary, at cholesterol/dimyristoylphosphatidylcholine (Chol/DMPC) mixing ratio of ∼1, was observed by saturation-recovery electron paramagnetic resonance (EPR) spin-labeling method in a multilamellar suspension of DMPC and Chol prepared using a rapid solvent exchange method. With spin-labeled cholesterol analogues (androstane spin label [ASL] and cholestane spin label [CSL]) it was shown that the upper limit of the cholesterol concentration in the liquid-ordered phase of the DMPC membrane is ∼50 mol%, above which the excess of cholesterol forms the pure cholesterol bilayer domain (CBD). Thus, the phase boundary at a Chol/DMPC mixing ratio of ∼1 separates the region with a single liquid-ordered phase from the region with a coexisting liquid-ordered phase and the CBD. Because the pure cholesterol cannot form the free bilayer in the buffer, the CBD has to be supported by the surrounding phospholipid bilayer saturated with cholesterol. The next phase boundary is defined by the cholesterol solubility threshold (CST) that indicates the amount of cholesterol which can saturate the DMPC bilayer and form the CBD. The excess of cholesterol above the CST forms monohydrate cholesterol crystals which precipitate outside of the lipid bilayer. It was shown by differential scanning calorimetry (DSC) that the CST, which separates the region with a coexisting liquid-ordered phase saturated with cholesterol and the CBD from the region in which cholesterol crystals are formed, is located at the Chol/DMPC mixing ratio of ∼2 (∼66 mol% cholesterol). The extended phase diagram for the DMPC/cholesterol membrane, covering the region where the membrane is saturated and oversaturated with cholesterol, is proposed.

Phase Boundaries in Phosphatidylcholine Membranes Saturated and Oversaturated with Cholesterol

Conventional and saturation-recovery EPR along with differential scanning calorimetry (DSC) were used to determine the cholesterol/phosphatidylcholine (Chol/PC) mixing ratio at which cholesterol bilayer domains (CBDs) and cholesterol crystals (CR) start to form in dimyristoyl-PC (DMPC) and 1-palmitoyl-2-oleoy-PC (POPC) membranes. The Chol/PC mixing ratio was changed from 0 to 3. Lipid multilamellar dispersions investigated in this work were prepared using the rapid solvent exchange method to preserve compositional homogeneity throughout the suspension. EPR spin-labeling methods were used with spin-labeled cholesterol analogues (cholestane spin label [CSL] and androstane spin label [ASL]) as well as with a spin-labeled phospholipid (1-palmitoyl-2-(5-doxylstearoyl)phosphatidylcholine (5-PC)) to locate the phase boundary for CBD formation. DSC was used to locate the phase boundary for the formation of CRs. Results showed that in both DMPC and POPC membranes the CBD is already formed at a Chol/PC mixing ratio of 1, while CRs are formed only when the Chol/PC mixing ratio exceeds the value of 2. Previous results and data presented here indicate that when the Chol/PC mixing ratio increases, the PC bilayer becomes saturated with cholesterol first. This phospholipid bilayer, saturated with cholesterol, possesses unique physical properties. Increase in cholesterol content beyond the Chol/PC mixing ratio of 1 causes formation of the CBD, and further increase, beyond the Chol/PC mixing ratio of 2, causes formation of CRs (presumably outside of the membrane). Thus, the phase boundary at a Chol/PC mixing ratio of 1 separates the region with a single liquid-ordered phase and the region with a coexisting liquid-ordered phase and CBD. The cholesterol solubility threshold at a Chol/PC mixing ratio of 2 separates the region with a coexisting liquid-ordered phase and CBD from the region in which CRs are formed.

A more efficient device for preparing model-membrane liposomes by the rapid solvent exchange method

We modified the original design for a rapid solvent exchange (RSE) device with the intent of making the RSE method (i) more efficient and (ii) easier to adopt and implement. Our modifications improved solvent-removal kinetics by a factor of 2, while reducing sample-prep time by a factor of 3. In this paper, we develop the kinetic model that informed the device revision and we address several RSE parameters that have not yet been discussed in the literature. We also provide detailed mechanical drawings and present solvent-removal efficiency data that confirm the improved performance of our device.